RU2798845C2 - Long-acting polypeptides and methods of their production and administration - Google Patents

Abstract

FIELD: pharmaceuticals.

SUBSTANCE: invention provides CTP-modified human growth hormone polypeptides, pharmaceutical compositions and pharmaceutical compositions containing them, as well as methods of their preparation and use.

EFFECT: obtaining long-acting polypeptides and methods of their production and administration.

30 cl, 39 dwg, 23 tbl, 11 ex

Description

FIELD OF TECHNOLOGY

[001] The present invention provides CTP-modified human growth hormone polypeptides, pharmaceutical compositions and pharmaceutical compositions containing these polypeptides, as well as methods for their preparation and use.

BACKGROUND OF THE INVENTION

[002] Polypeptides are susceptible to denaturation or enzymatic degradation in the blood, liver, or kidneys. Accordingly, polypeptides typically have a short half-life of a few hours. Due to their low stability, peptide drugs are usually delivered at a constant rate in order to maintain an effective plasma concentration of the active peptide. In addition, since peptide drugs are usually administered by infusion, frequent injections of peptide drugs cause significant discomfort to the subject.

[003] Unfavorable pharmacokinetic parameters, such as a short serum half-life, may hinder the pharmaceutical development of many drug candidates that are promising in other respects. The serum half-life is an empirical characteristic of the molecule and must be determined experimentally for each new potential drug. For example, in the case of lower molecular weight polypeptide drugs, physiological clearance mechanisms, such as renal filtration, may make maintenance of therapeutic drug levels impossible due to the cost or frequency of the required dosing regimen. In contrast, a long serum half-life is undesirable if the drug or its metabolites have toxic side effects.

[004] Therefore, there is a need for technologies that will extend the half-life of therapeutic polypeptides while maintaining their high pharmacological efficacy. Said desired peptide drugs must also meet the requirements for increased serum stability, high potency, and low likelihood of inducing an undesirable immune response when administered to a subject. The present invention addresses this need by providing CTP-modified peptides having a long half-life while maintaining high pharmacological efficacy, while having improved serum stability, high potency, and a low likelihood of inducing unwanted immune responses in a subject.

BRIEF DESCRIPTION OF THE INVENTION

[005] According to one implementation variant, the present invention provides a pharmaceutical composition containing a buffer, a tonicity agent, and a CTP-modified polypeptide consisting of growth hormone and one carboxy-terminal peptide (CTP) of human chorionic gonadotropin attached to the amino-terminus of the specified growth hormone, as well as two human chorionic gonadotropin CTPs attached to the carboxy-terminus of said growth hormone. In one embodiment of the present invention, the growth hormone is human growth hormone (HGH). In one embodiment of the present invention, the tonicity agent is sodium chloride. In one embodiment of the present invention, the tonicity agent is sodium chloride at a concentration of 147 mM. According to one embodiment of the present invention, the formulation is a liquid formulation. According to one implementation variant of the present invention, the pH of the specified composition is approximately 6.2-6.4.

[006] According to one implementation variant, the present invention provides a composition for administration once a week to a subject having growth hormone deficiency. According to another embodiment of the present invention, the subject is an adult subject. According to another embodiment of the present invention, the subject is an adult subject having growth hormone deficiency. According to another embodiment of the present invention, the subject is a child. According to another embodiment of the present invention, the subject is a child with growth hormone deficiency. In another embodiment, the present invention provides a method for preparing a pharmaceutical composition for administration once a week to a subject deficient in growth hormone, said method comprising the following steps:

a). modifying growth hormone by attaching one carboxy-terminal peptide (CTP) of human chorionic gonadotropin attached to the amino-terminus of said growth hormone, as well as two CTPs of human chorionic gonadotropin attached to the carboxy-terminus of said growth hormone;

b). mixing said modified growth hormone obtained in step (a) with said buffer and said tonicity agent at a pH of 6.2-6.4; And

c). pre-filling the syringe with the specified composition.

[007] According to one implementation variant, the present invention provides a pharmaceutical composition for administration once a week to a subject having growth hormone deficiency, containing a CTP-modified polypeptide, and the specified CTP-modified polypeptide consists of growth hormone and one carboxy-terminal peptide (CTP ) human chorionic gonadotropin attached to the amino terminus of said growth hormone, as well as two human chorionic gonadotropin CTPs attached to the carboxy terminus of said growth hormone. In one embodiment of the present invention, the growth hormone is human growth hormone. According to another embodiment of the present invention, the subject is an adult subject. According to another embodiment of the present invention, the subject is an adult subject having growth hormone deficiency. According to another embodiment of the present invention, the subject is a child. According to another embodiment of the present invention, the subject is a child with growth hormone deficiency.

[008] According to one implementation variant, the present invention provides a dosage form for administration once a week, containing a pharmaceutical composition according to the present invention or a pharmaceutical composition according to the present invention.

[009] In one embodiment, the present invention provides a method for filling a syringe with a composition of the present invention, comprising the steps of:

a). manufacturing a dosage form of said CTP-modified human GH for administration once a week, containing a predetermined amount of CTP-modified human GH; And

b). filling the syringe with the specified composition.

[0010] Other features and advantages of the present invention will be apparent from the following detailed description, examples and drawings. It should be understood, however, that the detailed description and specific examples describing preferred embodiments of the present invention are provided by way of illustration only, since various changes and modifications that are within the spirit and scope of the present invention will be apparent to those skilled in the art at based on the detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

[0011] The following drawings form part of this specification and are included to further demonstrate certain aspects of the present invention, the principles of which will be better understood with reference to one or more of these drawings in conjunction with a detailed description of the specific embodiments proposed in the present invention. The patent or application file contains at least one color drawing. Copies of this patent or patent application publication containing the color drawing(s) will be made available by the Patent Office upon request and upon payment of the required fee.

[0012] FIG. 1 shows the results of a Western blot study that characterizes the molecular weight and identity of MOD-4020 (SEQ ID NO: 36), MOD-4021 (SEQ ID NO: 37), MOD-4022 (SEQ ID NO: 38), MOD- 4023 (SEQ ID NO: 39) and MOD-4024 (SEQ ID NO: 40). The gel after electrophoresis in SDS-PAGE was subjected to blotting and stained using monoclonal antibodies to human growth hormone. The photograph shows that, similarly to the commercial preparation of human GH and wild-type human GH, MOD-7020-4 variants are recognized by antibodies to human GH.

[0013] FIG. 2 is a bar graph showing weight gain in pituitary-removed rats following administration of the GH-CTP polypeptides of the present invention.

[0014] FIG. 3 shows two schemes: (1) a map of the plasmid pCI-dhfr CTP-human GH-CTP-CTP and (2) the structural formula of the protein CTP-human GH-CTP-CTP.

[0015] FIG. 4 are graphs showing mean plasma concentrations of human CTP-GH-CTP-CTP or GH (pg/mL) after intravenous (IV) or subcutaneous (SC) administration of a single dose of CTP-human GH-CTP -CTP or GH in rats (n=3-6 per dose/route).

[0016] FIG. 5 are graphs showing the mean incremental weight gain after subcutaneous administration of single doses of human CTP-GH-CTP-CTP (0.4, 0.8, and 4 mg/kg) in pituitary-removed rats compared with that after daily injections of growth hormone (0.1 mg/kg/day) (n=10 for a single dose).

[0017] FIG. 6 is a graph showing that area under the concentration-time curve after a single injection of human CTP-GH-CTP-CTP correlates with body weight gain in rats.

[0018] FIG. 7 is a graph showing incremental weight gain after s.c. doses of human CTP-GH-CTP-CTP (0.4, 0.8, and 4 mg/kg) at 4-day intervals in pituitary-removed rats. compared with that after daily GH injections (0.1 mg/kg/day) (n=10 per dose).

[0019] FIG. 8 is a graph showing serum concentrations of human GH in pituitary-removed rats after s.c. injection of human CTP-GH-CTP-CTP and commercial human GH. A single dose of human CTP-GH-CTP-CTP 0.6 or 1.8 mg/kg and biotropin 0.35 or 1.05 mg/kg was administered by subcutaneous injection to pituitary-removed rats to determine the PK/PD profile. ). The concentration of human GH in serum after injection was measured using specific kits for enzyme-linked immunosorbent assay (ELISA).

[0020] FIG. 9 is a graph showing serum levels of IGF-1 in pituitary-removed rats following s.c. injection of human CTP-GH-CTP-CTP and commercial human GH. A single dose of human CTP-GH-CTP-CTP 0.6 or 1.8 mg/kg and biotropin 0.35 or 1.05 mg/kg was administered by subcutaneous injection to pituitary-removed rats to determine the PK/PD profile. Post-injection serum IGF-1 concentrations were measured using specific ELISA kits (Roche Diagnostics).

[0021] In FIG. 10 is a diagram of the phase II clinical trial design.

[0022] FIG. 11 shows the standard deviation index (SDS) for IGF-1 levels after the 4th weekly dose - all cohorts.

[0023] FIG. 12 shows the mean change from baseline in plasma IGF-1 concentration following subcutaneous administration of MOD-4023 to growth hormone deficient adult patients (stage I, after 4th injection).

[0024] FIG. 13 shows mean IGF-1 levels (determined 4 days post-dose) during a 4-month follow-up clinical study (52 patients).

[0025] In FIG. 14 is a schematic of the phase II clinical study of MOD-4023.

[0026] FIG. 15 is a graph showing: (A) the mean FA profile of MOD-4023 administered weekly and (B); the average FC profile of human GH administered daily.

[0027] FIG. 16 is a graph showing the mean SDS profile for IGF-1 levels during the 6-month pediatric phase II clinical trial MOD-4023.

[0028] FIG. 17 is a graph showing the IGF-1-SB-3 profile during week 2 at each final dose for the pediatric phase II clinical trial MOD-4023.

[0029] FIG. 18 is a graph showing the mean IGF-1-SB-3 profile for the 6-month pediatric phase II clinical trial MOD-4023.

[0030] FIG. 19 is a graph showing the mean annual growth rate results for all patients who completed the 6-month treatment period in the 6-month pediatric phase II clinical study MOD-4023, aimed at assessing growth rate.

[0031] In FIG. 20 is a graph showing: (A) the results of the pediatric phase II growth rate SDS determination of the MOD-4023 preliminary growth rate clinical study and (B) the results of the growth rate SDS determination over 6 months.

[0032] FIG. 21 is a graph showing the PD profile (IGF-1 SDS) at each final dose for the pediatric phase II clinical trial MOD-4023.

[0033] FIG. 22 is a graph showing SDS values for relative change in growth rate (ΔCP).

[0034] FIG. 23 is a graph showing the change in IGF-1 concentration from baseline at the final dose in the pediatric phase II clinical trial MOD-4023.

[0035] FIG. 24 is a table showing the characteristics of non-GMP and GMP batches prepared in 10 mM citrate, 147 mM NaCl at pH=6.

[0036] FIG. 25A is a graph showing the stability results of clone 2 MOD-4023 by RP-HPLC at -20°C.

[0037] FIG. 25B is a graph showing the stability results of clone 2 MOD-4023 by RP-HPLC at 5°C.

[0038] FIG. 26 is a graph showing the stability results of clone 2 MOD-4023 by RP-HPLC at 25°C.

[0039] FIG. 27A is a graph showing the results of the stability evaluation of clone 28 (Xcellerex) at -20°C (RP-HPLC).

[0040] FIG. 27B is a graph showing the results of the stability evaluation of clone 28 (Xcellerex) at 5°C (RP-HPLC).

[0041] In FIG. 28A is a graph showing the results of the stability evaluation of clone 28 (Xcellerex) at 25°C (RP-HPLC).

[0042] FIG. 28B is a graph showing the results of comparing the stability profiles of clones 2 and 28 at 5°C (RP-HPLC).

[0043] FIG. 29 is a table showing the transfer of MOD-4023 production from Xcellerex (XC) to Rentschler (RB).

[0044] FIG. 30A is a graph showing the differences in RP-HPLC stability scores between Ha Rentschler (RB) and Xcellerex (XCLX) - the main peak.

[0045] FIG. 30B is a table showing the results of the stability evaluation of the main peak of GMP1 (RB).

[0046] FIG. 30C is a table showing the stability evaluation results of the main XCLX peak (tested in RB).

[0047] FIG. 31A is a graph showing the differences in RP-HPLC stability scores between Rentschler (RB) and Xcellerex (XCLX) - Peak 3.

[0048] FIG. 31B is a table showing the results of GMP1 Peak 3 (RB) stability evaluation.

[0049] FIG. 31C is a table showing the stability evaluation results of XCLX peak 3 (tested in RB).

[0050] FIG. 32A is a graph showing the differences in RP-HPLC stability scores between Rentschler (RB) and Xcellerex (XCLX) - Peak 5.

[0051] FIG. 32B is a table showing the stability results of GMP1 Peak 5 (RB).

[0052] FIG. 32C is a table showing the stability evaluation results of XCLX peak 5 (tested in RB).

[0053] FIG. 33A is a graph showing RP-HPLC stability results after 3 months storage at 25°C, GMP Xcellerex lot. Peak 7 was not observed in the XC material.

[0054] FIG. 33B is a graph showing the results of stability evaluation by RP-HPLC. A batch of GMP-1 at Rentschler. Peak 7, which was not observed in the XC material, appeared after 2 weeks of storage at 25°C.

[0055] FIG. 34 is a graph showing the results of GMP-1 stability evaluation by RP-HPLC after 3 months of storage at 25°C. The arrow points to a new peak (7), which was not observed in the XC material.

[0056] FIG. 35A shows the IEF profile of batches of MOD-4023. In the range of IEP values from 3.5 to 4.2, a similar nature of the bands was observed. In one lot of XCLX, at the border of high IEP, there are isoforms with a less weak band intensity. The RB batch contains isoforms with weaker band intensity at the border of low IEP.

[0057] FIG. 35B shows the results of a stability evaluation (IEF) from RB and XCLX (3 months at 25°C). More diffuse bands are present in the XCLX sample.

[0058] FIG. 36A-D show the results of evaluating the effect of high temperatures on % of peaks (clone 2) (FIG. 36A - main peak; FIG. 36D). The formation of both peaks (3 and 5) is temperature dependent and is accelerated at high temperature. (Fig. 36B and Fig. 36C)

[0059] FIG. 37A-D show the results of evaluating the effect of pH on % peaks (clone 2) (FIG. 37A - main peak; FIG. 37D). Peak 3: No change in % peak after incubation for 5 days at pH=4 and for no more than 2 hours at pH=12 (FIG. 37B). Peak 5: No change in % peak after incubation for no more than 6 hours at pH=4. However, after 6 hours, a sharp increase in % peak was observed. When incubated for no more than 2 hours at pH=12, the peak disappears (FIG. 37C).

[0060] FIG. 38 shows the results of studies with forced degradation in Rentschler (clone 28). Overlay of enlarged areas of native (above) and stressed (below) active ingredient MOD-4023. A stressed sample of active ingredient MOD-4023 (CTP-Human GH-CTP-CTP) was prepared (65°C for approximately three days) to study the related form 5 in active ingredient MOD-4023 because the peak is below the sample limit of quantitation, unstressed.

[0061] In FIG. 39 shows the results of the evaluation of the effect of pH on related forms, which was evaluated using RP-HPLC. Test samples: RB - 40 mg/ml, pH=5.9; RB - 10 mg/ml, pH=6.2; XCLX - 40 mg/ml, pH=6.2.

DETAILED DESCRIPTION OF THE INVENTION

[0062] This application claims priority under U.S. Patent Application Publication No. US-2014-0113860-A1, filed Oct. 21, 2013, and U.S. Patent Application No. 14/309,496, filed June 19, 2014. These patent applications are incorporated in their entirety in this application by reference.

[0063] According to one implementation variant, the present invention provides a pharmaceutical composition containing a buffer, a tonicity agent, and a CTP-modified polypeptide consisting of growth hormone and one carboxy-terminal peptide (CTP) of human chorionic gonadotropin attached to the amino-terminus of the specified growth hormone, as well as two human chorionic gonadotropin CTPs attached to the carboxy-terminus of said growth hormone.

[0064] According to one implementation variant, the present invention provides a composition for administration once a week to a subject having growth hormone deficiency. According to another embodiment of the present invention, the subject is an adult subject. According to another embodiment of the present invention, the subject is an adult subject having growth hormone deficiency. According to another embodiment of the present invention, the subject is a child. According to another embodiment of the present invention, the subject is a child with growth hormone deficiency. In another embodiment, the present invention provides a method for preparing a pharmaceutical composition for administration once a week to a subject deficient in growth hormone, said method comprising the following steps:

a). modifying a growth hormone by attaching one carboxy-terminal peptide (CTP) of human chorionic gonadotropin attached to the amino terminus of said growth hormone and two CTPs of human chorionic gonadotropin attached to the carboxy-terminus of said growth hormone;

b). mixing said modified growth hormone obtained in step (a) with said buffer and said tonicity agent at pH=6.2-6.4; And

c). pre-filling the syringe with the specified composition.

[0065] In one embodiment, the present invention provides a method for filling a syringe with a composition of the present invention, comprising the steps of:

a). manufacturing a dosage form of said CTP-modified human growth hormone (GHH) for administration once a week, containing a predetermined amount of CTP-modified human growth hormone; And

b). filling the syringe with the specified composition.

[0066] According to one implementation variant, the present invention describes long-acting polypeptides, as well as methods for their preparation and use. According to another embodiment of the present invention, the long acting polypeptides comprise a carboxy-terminal peptide (CTP) of human chorionic gonadotropin (hCG). According to another embodiment of the present invention, CTP acts as an agent that protects proteins or peptides derived from them from degradation. According to another implementation variant of the present invention, CTP increases the duration of the half-life of proteins or peptides derived from them. In some embodiments, CTP enhances the efficacy of proteins or peptides derived therefrom.

[0067] According to another embodiment of the present invention, the terms "CTP peptide", "carboxy-terminal peptide", "CTP sequence" and "C-terminal human chorionic gonadotropin peptide" are used interchangeably herein. According to another embodiment of the present invention, the carboxy-terminal peptide is a full length CTP. According to another embodiment of the present invention, the carboxy-terminal peptide is a truncated CTP. Each possibility represents a separate embodiment of the present invention.

[0068] According to another implementation variant of the present invention in this application, the terms "signal sequence" and "signal peptide" are used interchangeably. According to another embodiment of the present invention, the term "sequence", when used in relation to a polynucleotide, may refer to a coding portion. Each possibility represents a separate embodiment of the present invention.

[0069] According to another implementation variant of the present invention in this application, the terms "peptide of interest" and "polypeptide sequence of interest" are used interchangeably. According to another embodiment of the present invention, the peptide of interest is a full-length protein. According to another embodiment of the present invention, the peptide of interest is a protein fragment. Each possibility represents a separate embodiment of the present invention.

[0070] According to another embodiment, the present invention provides a pharmaceutical composition comprising a polypeptide that consists of a growth hormone, one carboxy-terminal chorionic gonadotropin peptide attached to the amino terminus of said growth hormone, and two carboxy-terminal chorionic gonadotropin peptides attached towards the carboxy terminus of said growth hormone. According to another implementation variant, the present invention provides a pharmaceutical composition containing a polypeptide that consists of a growth hormone, one carboxy-terminal chorionic gonadotropin peptide attached to the amino terminus of said growth hormone, two carboxy-terminal chorionic gonadotropin peptides attached to the carboxy-terminus said growth hormone, and a signal peptide fused to the amino terminus of one carboxy-terminal chorionic gonadotropin peptide. According to another embodiment of the present invention, the pharmaceutical composition further comprises a buffer and a tonicity agent. According to another embodiment of the present invention, the buffer is 10 mM citrate and the tonicity agent is 147 mM NaCl. According to one implementation variant of the present invention, the pH of the specified composition is approximately 6.0. According to another embodiment of the present invention, the pH of said composition is about 6.2. According to another embodiment of the present invention, the pH of said composition is about 6.4. According to another implementation variant of the present invention, the pH of the specified composition is in the range from approximately 6.0 to 6.4. In one embodiment of the present invention, the buffer is 10 mM citrate, the tonicity agent is 147 mM NaCl, and the pH is 6.0. According to another embodiment of the present invention, the formulation is a liquid formulation.

[0071] According to another embodiment, the present invention provides a dosage form for administration once a week, containing the pharmaceutical composition proposed in this application.

[0072] In another embodiment, the present invention provides a pharmaceutical composition comprising a polypeptide consisting of a growth hormone, one carboxy-terminal chorionic gonadotropin peptide attached to the amino terminus of said growth hormone, and two carboxy-terminal chorionic gonadotropin peptides attached to the carboxy-terminus of said growth hormone. According to another embodiment, the present invention provides a pharmaceutical composition comprising a polypeptide consisting of a growth hormone, one carboxy-terminal chorionic gonadotropin peptide attached to the amino terminus of said growth hormone, two carboxy-terminal chorionic gonadotropin peptides attached to the carboxy-terminus of said growth hormone. growth hormone, and a signal peptide attached to the amino end of one of the specified carboxy-terminal chorionic gonadotropin peptide.

[0073] According to another embodiment of the present invention, a growth hormone containing CTP blocks described in this application has an increased biological activity in vivo compared to a similar growth hormone that does not contain CTP. According to another embodiment of the present invention, a growth hormone containing at least one CTP attached to its amino-terminus and at least two CTPs attached to its carboxy-terminus has increased in vivo biological activity compared to the same hormone. growth that does not contain CTP. According to another embodiment of the present invention, a growth hormone containing one CTP attached to its amino-terminus and two CTPs attached to its carboxy-terminus has increased in vivo biological activity compared to a similar growth hormone that does not contain CTP.

[0074] In another embodiment, the present invention provides a polypeptide comprising sequences of at least two human chorionic gonadotropin carboxy-terminal peptides (CTPs) fused to a polypeptide sequence of interest, wherein a first CTP sequence of at least two CTP sequences is fused to the amino terminus of the polypeptide sequence of interest, and a second CTP sequence of at least two CTP sequences is fused to the carboxy terminus of the polypeptide sequence of interest. In another embodiment of the present invention, the carboxy-terminal peptide (CTP) sequence is a human chorionic gonadotropin sequence.

[0075] According to another embodiment of the present invention, the subject is a human subject. According to one embodiment of the present invention, the human subject is deficient in growth hormone. According to one embodiment of the present invention, the subject is deficient in growth hormone. According to another embodiment of the present invention, the subject is a pet. According to another embodiment of the present invention, the subject is a mammal. According to another embodiment of the present invention, the subject is a farm animal. According to another embodiment of the present invention, the subject is a dog. According to another embodiment of the present invention, the subject is a cat. According to another embodiment of the present invention, the subject is a monkey. According to another embodiment of the present invention, the subject is a horse. According to another embodiment of the present invention, the subject is a cow. According to another embodiment of the present invention, the subject is a mouse. According to another embodiment of the present invention, the subject is a rat. According to one embodiment of the present invention, the subject is male. According to another embodiment of the present invention, the subject is female. According to another embodiment of the present invention, the subject is an adult subject having growth hormone (GH) deficiency. According to another embodiment of the present invention, the subject is a prepubescent child with growth hormone deficiency (GHD). As described in this application, various doses of MOD-4023 (CTP-Human GH-CTP-CTP) provided good catch-up growth in prepubertal children (see example 10).

[0076] The expression "polypeptide sequence of interest" refers, in another embodiment of the present invention, to any polypeptide sequence, such as a sequence having biological activity. According to another embodiment of the present invention, the peptide is glycosylated. According to another embodiment of the present invention, the peptide is non-glycosylated. Examples of polypeptides that benefit from increased half-life include, but are not limited to, erythropoietin (EPO), interferons, human growth hormone (HGH), and glucagon-like peptide-1 (GLP-1). According to one implementation variant of the present invention, the polypeptide is a growth hormone (GHR). In another embodiment, the polypeptide is human growth hormone (HGH).

[0077] According to one embodiment of the present invention, the CTP-GH-CTP-CTP configuration described in this application contains growth hormone or its active fragment connected via a linker to at least one CTP block. According to one embodiment of the present invention, the linker is a peptide bond. According to another embodiment of the present invention, the CTP-GH-CTP-CTP configuration described in this application contains growth hormone or its active fragment connected via a peptide bond to at least one CTP unit. According to another embodiment of the present invention, the CTP-GH-CTP-CTP configuration described in this application contains growth hormone or its active fragment connected via a peptide bond to at least one CTP unit, which is connected to an additional CTP unit via a peptide bond. According to another embodiment of the present invention, the polypeptide described in this application contains growth hormone fragments and CTP blocks and/or fragments thereof, which are interconnected by a peptide bond. According to another embodiment of the present invention, one nucleic acid molecule encodes a polypeptide described in this application, containing growth hormone and/or fragments thereof and CTP blocks and/or fragments thereof.

[0078] According to another embodiment of the present invention, a carboxy-terminal peptide (CTP) is attached to the sequence of a polypeptide of interest via a linker. According to another embodiment of the present invention, at least one CTP is optionally attached to said polypeptide sequence of interest via a linker. According to another embodiment of the present invention, the linker that connects the CTP sequence and the polypeptide sequence of interest is a covalent bond. According to another embodiment of the present invention, the linker that connects the CTP sequence and the polypeptide sequence of interest is a peptide bond. According to another embodiment of the present invention, the linker that connects the CTP sequence and the polypeptide sequence of interest is a substituted peptide bond. According to another embodiment of the present invention, the carboxy-terminal peptide (CTP) sequence comprises an amino acid sequence selected from the sequences shown in SEQ ID NO: 48.

[0079] According to another embodiment of the present invention, the sequence of SEQ ID NO: 48 contains the following amino acid sequence (AA): DPRFQDSSSSKAPPPSLPSPSRLPGPSDTPILQ (SEQ ID NO: 48).

[0080] According to another embodiment of the present invention, the carboxy-terminal peptide (CTP) of human chorionic gonadotropin (hCG) is hybridized to a protein. According to another embodiment of the present invention, the carboxy-terminal peptide (CTP) of human chorionic gonadotropin (hCG) is hybridized to a peptide of said protein. In one embodiment of the present invention, the protein or peptide thereof is a growth hormone. In one embodiment of the present invention, the protein or peptide thereof is a human growth hormone. According to another embodiment of the present invention, the carboxy-terminal peptide (CTP) of human chorionic gonadotropin is hybridized to a glycoprotein. According to another embodiment of the present invention, the carboxy-terminal peptide (CTP) of hCG is hybridized to a hormone-glycoprotein. According to another embodiment of the present invention, hCG CTP is hybridized to a peptide derived from a hormone glycoprotein. In some embodiments, the glycoprotein hormones include erythropoietin (EPO), FSH, or TSH, as well as peptides derived therefrom.

[0081] According to some variants of implementation of the present invention, the CTP sequence at the amino-terminus and carboxy-terminus of the polypeptide provides increased protection of the protein from degradation. In some embodiments of the present invention, the amino-terminal and carboxy-terminal CTP sequences of the polypeptide provide for a longer half-life of the attached protein.

[0082] According to some embodiments of the present invention, a CTP sequence at the amino terminus of the polypeptide, a CTP sequence at the carboxy terminus of the polypeptide, and at least one additional CTP sequence attached in tandem to the CTP sequence at the carboxy terminus provide increased protection of the protein from degradation . In some embodiments of the present invention, a CTP sequence at the amino terminus of a polypeptide, a CTP sequence at the carboxy terminus of a polypeptide, and at least one additional CTP sequence fused in tandem to a CTP sequence at the carboxy terminus provide for a longer half-life of the attached protein. In some embodiments of the present invention, a CTP sequence at the amino terminus of the polypeptide, a CTP sequence at the carboxy terminus of the polypeptide, and at least one additional CTP sequence fused in tandem to the CTP sequence at the carboxy terminus provide increased activity of the attached protein.

[0083] According to some embodiments of the present invention, a CTP sequence at the amino terminus of the polypeptide, a CTP sequence at the carboxy terminus of the polypeptide, and at least one additional CTP sequence attached in tandem to the CTP sequence at the amino terminus provide increased protection of the attached protein from degradation. In some embodiments of the present invention, a CTP sequence at the amino terminus of the polypeptide, a CTP sequence at the carboxy terminus of the polypeptide, and at least one additional CTP sequence fused in tandem to the CTP sequence at the amino terminus provide a longer half-life of the attached protein. In some embodiments of the present invention, a CTP sequence at the amino terminus of a polypeptide, a CTP sequence at the carboxy terminus of a polypeptide, and at least one additional CTP sequence fused in tandem to a CTP sequence at the amino terminus provide increased activity of the attached protein.

[0084] In some embodiments of the present invention, the CTP sequences at the amino terminus of growth hormone and the carboxy terminus of growth hormone provide increased protection of growth hormone from degradation. According to another embodiment of the present invention, at least one CTP sequence at the amino terminus of growth hormone and two CTP blocks at the carboxy terminus of growth hormone provide increased clearance protection. According to another embodiment of the present invention, at least one CTP sequence at the amino terminus of the growth hormone and two CTP blocks at the carboxy terminus of the growth hormone provide a longer clearance period. According to another embodiment of the present invention, at least one CTP sequence at the amino terminus of growth hormone and two CTP blocks at the carboxy terminus of growth hormone increase _Cmax of growth hormone. According to another embodiment of the present invention, at least one CTP sequence at the amino terminus of the growth hormone and two CTP blocks at the carboxy terminus of the growth hormone increase the T _max of the growth hormone. According to another embodiment of the present invention, at least one CTP sequence at the amino terminus of growth hormone and two CTP blocks at the carboxy terminus of growth hormone increase T _1/2 .

[0085] In some embodiments of the present invention, the CTP sequences at the amino terminus of the growth hormone and the carboxy terminus of the growth hormone increase the half-life of the modified growth hormone. According to another embodiment of the present invention, at least one CTP sequence at the amino terminus of the growth hormone and at least two CTP sequences at the carboxy terminus of the growth hormone provide a longer half-life of the modified growth hormone. According to another embodiment of the present invention, one CTP sequence at the amino terminus of the growth hormone and two CTP sequences at the carboxy terminus of the growth hormone provide for a longer half-life of the attached growth hormone. According to another embodiment of the present invention, one CTP sequence at the amino terminus of the growth hormone and two CTP sequences fused in tandem to the carboxy terminus of the growth hormone provide a longer half-life of the modified growth hormone.

[0086] According to some embodiments of the present invention, a CTP sequence at the amino-terminus of a polypeptide, a CTP sequence at the carboxy-terminus of growth hormone, and at least one additional CTP sequence attached in tandem to the CTP sequence at the carboxy-terminus provide increased protection of growth hormone from degradation. In some embodiments of the present invention, a growth hormone amino-terminal CTP sequence, a growth hormone carboxy-terminal CTP sequence, and at least one additional CTP sequence fused in tandem to the carboxy-terminal CTP sequence increase the half-life of the growth hormone. According to some embodiments of the present invention, a CTP sequence at the amino terminus of growth hormone, a CTP sequence at the carboxy terminus of the growth hormone, at least one additional CTP sequence attached in tandem to the CTP sequence at the carboxy terminus enhances the biological activity of the growth hormone.

[0087] According to one embodiment of the present invention, at least one CTP sequence consists of an amino acid sequence selected from the group consisting of: SEQ ID NO: 17 and SEQ ID NO: 18. According to another embodiment of the present invention, a carboxy-terminal peptide ( CTP) according to the present invention contains the amino acid sequence from the amino acid at position 112 to the amino acid at position 145 of human chorionic gonadotropin, shown in SEQ ID NO: 17. According to another embodiment of the present invention, the sequence of the CTP according to the present invention contains the amino acid sequence from the amino acid at position 118 to amino acid at position 145 of human chorionic gonadotropin as set forth in SEQ ID NO: 18. According to another embodiment of the present invention, the CTP sequence also starts at any position between positions 112-118 and ends at position 145 of human chorionic gonadotropin. In some embodiments of the present invention, the CTP peptide sequence is 28, 29, 30, 31, 32, 33, or 34 amino acids long and begins at position 112, 113, 114, 115, 116, 117, or 118 amino acids of the CTP sequence.

[0088] According to another embodiment of the present invention, the CTP peptide is a variant of human chorionic gonadotropin CTP that differs from native CTP by having 1-5 conservative amino acid substitutions as described in US Pat. No. 5,712,122. According to another embodiment of the present invention, the CTP peptide is a variant of human chorionic gonadotropin CTP that differs from native CTP by having 1 conservative amino acid substitution. According to another embodiment of the present invention, the CTP peptide is a variant of human chorionic gonadotropin CTP that differs from native CTP by having 2 conservative amino acid substitutions. According to another embodiment of the present invention, the CTP peptide is a variant of human chorionic gonadotropin CTP that differs from native CTP by having 3 conservative amino acid substitutions. According to another embodiment of the present invention, the CTP peptide is a variant of human chorionic gonadotropin CTP that differs from native CTP by having 4 conservative amino acid substitutions. According to another embodiment of the present invention, the CTP peptide is a variant of human chorionic gonadotropin CTP that differs from native CTP by having 5 conservative amino acid substitutions. According to another embodiment of the present invention, the amino acid sequence of the CTP peptide of the present invention has at least 70% homology with the amino acid sequence of native CTP or a peptide thereof. According to another embodiment of the present invention, the amino acid sequence of the CTP peptide of the present invention has at least 80% homology with the amino acid sequence of native CTP or a peptide thereof. According to another embodiment of the present invention, the amino acid sequence of the CTP peptide of the present invention has at least 90% homology with the amino acid sequence of native CTP or a peptide thereof. According to another embodiment of the present invention, the amino acid sequence of the CTP peptide of the present invention has at least 95% homology with the amino acid sequence of native CTP or a peptide thereof.

[0089] According to another embodiment of the present invention, the DNA sequence of the CTP peptide of the present invention has at least 70% homology with the DNA sequence of native CTP or its peptide. According to another embodiment of the present invention, the DNA sequence of the CTP peptide according to the present invention has at least 80% homology with the DNA sequence of native CTP or its peptide. According to another embodiment of the present invention, the DNA sequence of the CTP peptide according to the present invention has at least 90% homology with the DNA sequence of native CTP or its peptide. According to another embodiment of the present invention, the DNA sequence of the CTP peptide according to the present invention has at least 95% homology with the DNA sequence of native CTP or its peptide.

[0090] In one embodiment of the present invention, at least one of the human chorionic gonadotropin CTP amino acid sequences is truncated. In one embodiment of the present invention, both human chorionic gonadotropin CTP amino acid sequences are truncated. According to another embodiment of the present invention, 2 of the human chorionic gonadotropin CTP amino acid sequences are truncated. According to another embodiment of the present invention, 2 or more amino acid sequences of CTP human chorionic gonadotropin are truncated. According to another embodiment of the present invention, all human chorionic gonadotropin CTP amino acid sequences are truncated. In one embodiment, the truncated CTP comprises the first 10 amino acids of the sequence shown in SEQ ID NO: 43. In one embodiment, the truncated CTP contains the first 11 amino acids of the sequence shown in SEQ ID NO: 43. In one embodiment, of the present invention, the truncated CTP contains the first 12 amino acids of the sequence shown in SEQ ID NO: 43. In one embodiment of the present invention, the truncated CTP contains the first 13 amino acids of the sequence shown in SEQ ID NO: 43. In one embodiment, the truncated CTP contains the first 14 amino acids from the sequence shown in SEQ ID NO: 43. In one embodiment, the truncated CTP contains the first 15 amino acids from the sequence shown in SEQ ID NO: 43. In one embodiment, the truncated CTP contains the first 16 amino acids from the sequence shown in SEQ ID NO: 43. In one embodiment of the present invention, the truncated CTP contains the last 14 amino acids from the sequence shown in SEQ ID NO: 43.

[0091] In one embodiment of the present invention, at least one of the human chorionic gonadotropin CTP amino acid sequences is glycosylated. According to another embodiment of the present invention, both human chorionic gonadotropin CTP amino acid sequences are glycosylated. According to another embodiment of the present invention, 2 of the human chorionic gonadotropin CTP amino acid sequences are glycosylated. According to another embodiment of the present invention, 2 or more of the human chorionic gonadotropin CTP amino acid sequences are glycosylated. According to another embodiment of the present invention, all human chorionic gonadotropin CTP amino acid sequences are glycosylated. According to one embodiment of the present invention, the CTP sequence of the present invention contains at least one glycosylation site. According to one embodiment of the present invention, the CTP sequence of the present invention contains 2 glycosylation sites. According to one embodiment of the present invention, the CTP sequence of the present invention contains 3 glycosylation sites. According to one embodiment of the present invention, the CTP sequence of the present invention contains 4 glycosylation sites. Each possibility represents a separate embodiment of the present invention.

[0092] According to some embodiments of the present invention, the term "homology" in accordance with the present invention also includes deletions, insertions, or substitution variants, including amino acid substitutions therein and their biologically active polypeptide fragments.

[0093] According to some embodiments of the present invention, human growth hormone (HGH) is used in accordance with the principles of the present invention. In some embodiments of the present invention, the addition of a CTP sequence to the amino-terminus and carboxy-terminus of a human GH protein results in increased efficiency (FIGS. 2, 3, 5, 7 and 9). In some embodiments of the present invention, the addition of a CTP sequence to the amino-terminus and carboxy-terminus of a human GH protein results in longer in vivo activity. In one embodiment of the present invention, the human CTP-GH polypeptides of the present invention are shown in SEQ ID NOs: 39-41.

[0094] The present application discloses that in pituitary-ablated rats (which lack growth hormone secretion), growth enhancement was observed after injection of human CTP-GH. The present application further discloses that growth enhancement, which correlated well with the catch-up growth of patients, was found in prepubertal children with growth hormone deficiency (see example 10 in this application).

[0095] In one embodiment, the present invention provides a method for achieving normal growth restoration in a prepubertal growth hormone deficient child, the method comprising administering a pharmaceutical composition comprising a CTP-modified growth hormone of the present invention. In another embodiment, the present invention provides a method for achieving regrowth in a growth hormone deficient prepubertal child, said method comprising administering a pharmaceutical composition comprising a CTP-modified growth hormone of the present invention.

[0096] According to one implementation variant of the present invention, the expression "human growth hormone" (HGH) refers to a polypeptide, for example, registered in GenBank under the accession number P01241 (SEQ ID NO: 47), which exhibits the activity of human growth hormone (i.e. growth stimulation).

[0097] According to another embodiment of the present invention, the term "human growth hormone" (human GH) refers to a polypeptide, for example, registered in GenBank under accession number P01241, which exhibits human GH activity (i.e., growth promotion). In another embodiment of the present invention, the term "GH" according to the present invention also refers to homologues. In another embodiment of the present invention, the amino acid sequence of GH in accordance with the methods and compositions according to the present invention is at least 50% homologous to the GH sequence given in this application, as determined using the BLASTP software of the National Center for Biotechnology Information (NCBI) using default settings. According to another embodiment of the present invention, the percentage of homology is 60%. According to another embodiment of the present invention, the percentage of homology is 70%. According to another embodiment of the present invention, the percentage of homology is 80%. According to another embodiment of the present invention, the percentage of homology is 90%. According to another embodiment of the present invention, the percentage of homology is at least 95%. According to another embodiment of the present invention, the percentage of homology is greater than 95%. Each possibility represents a separate embodiment of the present invention.

[0098] Representative CTP-GH polypeptides and human CTP-GH polypeptides of the present invention are shown in the sequences shown in SEQ ID NO: 39, SEQ ID NO: 40, and SEQ ID NO: 41.

[0099] According to another embodiment of the present invention, the methods of the present invention provide a growth hormone peptide (GH) further comprising at least one CTP peptide at the N-terminus and at least one CTP peptide at the C-terminus for promoting muscle growth. . According to another embodiment of the present invention, the methods of the present invention provide a GH peptide, further comprising one CTP peptide at the N-terminus and two CTP peptides at the C-terminus, intended to stimulate muscle growth. According to another embodiment of the present invention, the methods of the present invention provide the GH peptide shown in the sequence of SEQ ID NO: 23, further comprising at least one CTP peptide at the N-terminus and at least one CTP peptide at the C-terminus, intended for stimulation muscle growth. According to another embodiment of the present invention, the methods of the present invention provide the GH peptide shown in the sequence of SEQ ID NO: 36, further comprising at least one CTP peptide at the N-terminus and at least one CTP peptide at the C-terminus, intended for stimulation muscle growth. According to another embodiment of the present invention, the methods of the present invention provide the GH peptide shown in the sequence of SEQ ID NO: 37, further comprising at least one CTP peptide at the N-terminus, intended to stimulate muscle growth. According to another embodiment of the present invention, the methods of the present invention provide the GH peptide shown in the sequence of SEQ ID NO: 38, further comprising at least one CTP peptide at the N-terminus, intended to stimulate muscle growth. According to another embodiment of the present invention, the methods of the present invention provide the GH peptide shown in SEQ ID NO: 39 for promoting muscle growth. According to another embodiment of the present invention, the methods of the present invention provide the GH peptide shown in SEQ ID NO: 40 for promoting muscle growth. According to another embodiment of the present invention, the methods of the present invention provide the GH peptide shown in SEQ ID NO: 41 for promoting muscle growth. According to another embodiment of the present invention, the methods of the present invention provide the GH peptide shown in the sequence of SEQ ID NO: 42, further comprising at least one CTP peptide at the N-terminus, intended to stimulate muscle growth. According to another embodiment of the present invention, the methods of the present invention provide a CTP-modified GH peptide of the present application, which is intended to stimulate muscle growth.

[00100] According to another embodiment of the present invention, the methods of the present invention provide a nucleic acid sequence encoding a GH peptide, further comprising at least one CTP peptide at the N-terminus and at least one CTP peptide at the C-terminus, which is intended to stimulate muscle growth. According to another embodiment of the present invention, the methods of the present invention provide a nucleic acid sequence encoding a GH peptide, further comprising one CTP peptide at the N-terminus and two CTP peptides at the C-terminus, which is intended to stimulate muscle growth. According to one embodiment of the present invention, the methods of the present invention provide a nucleic acid shown in SEQ ID NO: 44 encoding a GH peptide containing one CTP peptide at the N-terminus and one CTP peptide at the C-terminus, which is intended to stimulate muscle growth. According to another embodiment of the present invention, the methods of the present invention provide a nucleic acid shown in SEQ ID NO: 45 encoding a GH peptide containing one CTP peptide at the N-terminus and two CTP peptides at the C-terminus, which is intended to stimulate muscle growth. According to another embodiment of the present invention, the methods of the present invention provide a nucleic acid shown in SEQ ID NO: 46 encoding a GH peptide, one CTP peptide at the N-terminus and two CTP peptides at the C-terminus to stimulate muscle growth.

[00101] According to one implementation variant, the present invention provides a method of reducing the frequency of dosing of growth hormone in a subject, comprising administering to said subject a therapeutically effective amount of a polypeptide consisting of growth hormone, one carboxy-terminal peptide (CTP) of human chorionic gonadotropin attached to the amino terminus said growth hormone, and two chorionic gonadotropin CTPs attached to the carboxy terminus of said growth hormone, said polypeptide optionally having a signal peptide attached to the amino terminus of said one CTP, thereby reducing the frequency of growth hormone dosing in a subject.

[00102] According to another embodiment, the present invention provides a method of improving the area under the concentration-time curve (AUC) for growth hormone in a subject, comprising administering to said subject a therapeutically effective amount of a polypeptide consisting of growth hormone, one carboxy-terminal peptide ( CTP) human chorionic gonadotropin attached to the amino terminus of said growth hormone, and two human chorionic gonadotropin CTPs attached to the carboxy terminus of said growth hormone, wherein said polypeptide optionally contains a signal peptide attached to the amino terminus of said one CTP, thereby reducing the frequency dosing growth hormone in a subject.

[00103] In another embodiment, the present invention provides a composition comprising a polypeptide that consists of a growth hormone, one human chorionic gonadotropin carboxy-terminal peptide (CTP) attached to the amino terminus of said growth hormone, and two human chorionic gonadotropin CTPs attached to the carboxy-terminus of said growth hormone, wherein said polypeptide optionally contains a signal peptide attached to the amino-terminus of said one CTP, wherein said composition has increased stability. According to one embodiment of the present invention, the formulation is stable for at least one year. According to another embodiment of the present invention, the composition is stable for at least two years.

[00104] According to one embodiment, the present invention provides a method of treating a subject in need of GH therapy, comprising administering to said subject a therapeutically effective amount of a polypeptide consisting of growth hormone, one carboxy-terminal peptide (CTP) of human chorionic gonadotropin attached to the amino terminus said growth hormone, and two chorionic gonadotropin CTPs attached to the carboxy terminus of said growth hormone, said polypeptide optionally having a signal peptide attached to the amino terminus of said one CTP, thereby reducing the frequency of growth hormone dosing in a subject.

[00105] According to another embodiment, the present invention provides a method of increasing insulin-like growth factor (IGF-1) levels in a subject, comprising administering to said subject a therapeutically effective amount of a polypeptide consisting of growth hormone, one carboxy-terminal peptide (CTP) of human chorionic gonadotropin, attached to the amino terminus of said growth hormone, and two human chorionic gonadotropin CTPs attached to the carboxy terminus of said growth hormone, wherein said polypeptide optionally contains a signal peptide attached to the amino terminus of said one CTP, thereby providing an increase in the level of insulin-like growth factor ( IGF-1) in a subject.

[00106] According to another embodiment, the present invention provides a method of maintaining insulin-like growth factor (IGF-1) levels in a subject, comprising administering to said subject a therapeutically effective amount of a polypeptide consisting of growth hormone, one carboxy-terminal peptide (CTP) of human chorionic gonadotropin, attached to the amino terminus of said growth hormone, and two human chorionic gonadotropin CTPs attached to the carboxy terminus of said growth hormone, wherein said polypeptide optionally contains a signal peptide attached to the amino terminus of said one CTP, thereby maintaining insulin-like growth factor levels ( IGF-1) in a subject. In another embodiment, IGF-1 levels are maintained within a certain range, as further provided herein.

[00107] According to another implementation variant, the present invention provides a method of increasing and maintaining levels of insulin-like growth factor (IGF-1) within a certain range in a subject, comprising administering to said subject a therapeutically effective amount of a polypeptide consisting of growth hormone, one carboxy-terminal peptide chorionic gonadotropin (CTP) attached to the amino terminus of said growth hormone, and two chorionic gonadotropin CTPs attached to the carboxy terminus of said growth hormone, said polypeptide optionally containing a signal peptide attached to the amino terminus of said one CTP, thereby increasing and maintaining the subject's levels of insulin-like growth factor (IGF-1) within a certain range.

[00108] According to another embodiment of the present invention, the defined range is the therapeutic dosage range achieved by administering the CTP-modified growth hormone provided herein. According to another embodiment of the present invention, the defined range is that in which the C _max and C _trough values of the IGF-1 sinusoidal profile are maintained after successive administrations of CTP-modified growth hormone, as further provided herein (see Example 15). According to another embodiment of the present invention, the defined range is a therapeutic dose range for sequential administration of the CTP-modified growth hormone provided herein that elicits an appropriate response in the subject and requires minimal dose modification. According to another embodiment of the present invention, the defined range is comparable to the range of IGF-1 levels in individuals who are considered normal. According to another embodiment of the present invention, the defined range is the normal range of IGF-1 levels/values in normal individuals. In another embodiment, the determined range is within the normal range when the IGF-1 Standard Deviation Index (SDS) values are within ±2 SDS.

[00109] According to another embodiment of the present invention, the methods of the present invention provide any of the CTP-modified GH peptides described in this application to stimulate bone growth.

[00110] According to another embodiment of the present invention, the methods of the present invention provide a nucleic acid sequence encoding any of the CTP-modified GH peptides described herein to promote bone growth.

[00111] According to another embodiment of the present invention, the conjugated growth hormones of the present invention are used in a manner similar to that of unmodified growth hormones. According to another embodiment of the present invention, the conjugated growth hormones of the present invention have a longer plasma half-life and retention time, reduced clearance, and increased in vivo clinical activity. According to another embodiment of the present invention, due to the improved properties of the conjugated growth hormones described in this application, these conjugates are administered at a lower frequency than unmodified growth hormones. According to another embodiment of the present invention, the conjugated growth hormones described in this application are administered at a frequency of from once a week to once every two weeks. According to another embodiment of the present invention, the conjugated growth hormones described herein are administered at a frequency of once every two weeks to once every three weeks. According to another embodiment of the present invention, the conjugated growth hormones described in this application are administered at a frequency of from once a day to three times a week. According to another embodiment of the present invention, the reduced frequency of administration will result in improved patient adherence to the treatment regimen, resulting in improved treatment outcomes as well as improved patient quality of life. According to another embodiment of the present invention, it has been found that, compared to conventional polyethylene glycol-linked growth hormone conjugates, growth hormone-CTP conjugates having a molecular weight and linker structure that is similar to that of the conjugates of the present invention have improved efficacy, improved stability, elevated AUC levels and longer half-life. According to another embodiment of the present invention, it has been found that, compared with conventional polyethylene glycol-linked growth hormone conjugates, growth hormones having a molecular weight and linker structure that is similar to those of the conjugates of the present invention have improved potency, improved stability, increased AUC levels and longer half-life. According to another embodiment of the present invention, a therapeutically effective amount of conjugated growth hormone is the amount of conjugate necessary to obtain the expected biological activity, which is measurable in vivo . According to another embodiment of the present invention, the growth hormone used in accordance with the principles of the present invention exhibits improved efficacy. In some embodiments of the present invention, the addition of a CTP sequence to the amino-terminus and carboxy-terminus of a growth hormone provides long-term activity in vivo .

[00112] According to another embodiment of the present invention, a therapeutically effective amount of conjugated growth hormone is determined in accordance with factors such as the exact type of condition being treated, the condition of the patient being treated, and the presence of other ingredients in the composition. According to another embodiment of the present invention, a therapeutically effective amount of conjugated growth hormone is from 0.01 to 10 μg per kg of body weight when administered once a week. According to another implementation variant of the present invention, a therapeutically effective amount of conjugated growth hormone is from 0.1 to 1 μg per kg of body weight when administered once a week. According to another embodiment of the present invention, a pharmaceutical composition containing a conjugated growth hormone is formulated to contain an active ingredient that will be effective for administration to a human patient by various means.

[00113] According to another embodiment of the present invention, the growth hormone is any growth hormone known to those skilled in the art. According to another embodiment of the present invention, the growth hormone is a human growth hormone. According to another embodiment of the present invention, the growth hormone is a non-human growth hormone. According to another embodiment of the present invention, the nucleotide sequence and/or amino acid sequence of the growth hormone is available in a gene bank database. According to another embodiment of the present invention, the growth hormone is a homologue. According to another embodiment of the present invention, a homologue also refers to a deletion, insertion, or substitution variant, including an amino acid substitution therein, and its biologically active polypeptide fragments.

[00114] According to another embodiment of the present invention, the growth hormone is a human GH variant that does not contain exons 2, 3, 4, or any combination thereof. According to another embodiment of the present invention, the growth hormone contains a signal peptide. According to another embodiment of the present invention, the growth hormone contains a signal sequence cleavage site. According to another embodiment of the present invention, the CTP-modified growth hormone-containing polypeptides of the present invention comprise recombinant growth hormone.

[00115] In another embodiment of the present invention, the methods of the present invention provide the GH peptide of the present invention to maintain muscle quality.

[00116] In another embodiment of the present invention, the methods of the present invention provide the growth hormone of the present invention to maintain bone quality.

[00117] In another embodiment of the present invention, the methods of the present invention provide the GH-CTP nucleic acid sequence of the present invention to maintain bone quality.

[00118] In another embodiment of the present invention, the methods of the present invention provide any of the CTP-modified growth hormone peptides described herein for the treatment of wasting syndrome.

[00119] In another embodiment of the present invention, the methods of the present invention provide a nucleic acid sequence encoding any of the CTP-modified GH peptides described herein for the treatment of wasting syndrome.

[00120] In another embodiment of the present invention, the methods of the present invention provide any of the CTP-modified GH peptides described herein to increase cardiac function.

[00121] In another embodiment of the present invention, the methods of the present invention provide a nucleic acid sequence encoding any of the CTP-modified GH peptides described herein to increase cardiac function.

[00122] In another embodiment of the present invention, the methods of the present invention provide the GH peptides described herein to increase lipolysis.

[00123] In another embodiment of the present invention, the methods of the present invention provide a nucleic acid sequence encoding any of the CTP-modified GH peptides described herein to increase lipolysis.

[00124] In another embodiment, the methods of the present invention provide any of the CTP-modified GH peptides described herein to improve fluid balance.

[00125] In another embodiment of the present invention, the growth hormone of the present invention comprises the amino acid sequence registered in GeneBank under accession number AAA72260. According to another implementation variant of the present invention, the growth hormone according to the present invention contains the amino acid sequence registered in GeneBank under accession number AAK69708. According to another embodiment of the present invention, the growth hormone of the present invention comprises the amino acid sequence registered in GeneBank under accession number CAA01435. According to another embodiment of the present invention, the growth hormone of the present invention comprises the amino acid sequence registered in GeneBank under accession number CAA01329. According to another embodiment of the present invention, the growth hormone of the present invention comprises the amino acid sequence registered in GeneBank under accession number CAA00380. According to another embodiment of the present invention, the growth hormone of the present invention comprises the amino acid sequence registered in GeneBank under accession number AAA72555. According to another implementation variant of the present invention, the growth hormone according to the present invention contains the amino acid sequence registered in GeneBank under accession number NP_000506.2. According to another embodiment of the present invention, the growth hormone of the present invention comprises the amino acid sequence registered in GeneBank under accession number NP_072053.1. According to another embodiment of the present invention, the growth hormone of the present invention comprises the amino acid sequence registered in GeneBank under accession number NP_072054.1. According to another embodiment of the present invention, the growth hormone of the present invention comprises the amino acid sequence registered in GeneBank under accession number NP_072055.1. According to another embodiment of the present invention, the growth hormone of the present invention comprises the amino acid sequence registered in GeneBank under accession number NP_072056.1.

[00126] According to another embodiment of the present invention, the methods of the present invention provide a nucleic acid sequence encoding a GH peptide, further comprising at least one CTP peptide at the N-terminus and at least one CTP peptide at the C-terminus, designed to improve balance liquids. According to another embodiment of the present invention, the methods of the present invention provide a nucleic acid sequence encoding a GH peptide, further comprising one CTP peptide at the N-terminus and two CTP peptides at the C-terminus, designed to improve fluid balance. According to one embodiment of the present invention, the methods of the present invention provide a nucleic acid shown in the sequence of SEQ ID NO: 44 encoding a GH peptide containing one CTP peptide at the N-terminus and one CTP peptide at the C-terminus, designed to improve fluid balance. According to another embodiment of the present invention, the methods of the present invention provide a nucleic acid shown in the sequence of SEQ ID NO: 45 encoding a GH peptide containing one CTP peptide at the N-terminus and two CTP peptides at the C-terminus, designed to improve fluid balance. According to another embodiment of the present invention, the methods of the present invention provide a nucleic acid shown in SEQ ID NO: 46 encoding a GH peptide, one CTP peptide at the N-terminus, and two CTP peptides at the C-terminus to improve fluid balance.

[00127] According to another embodiment of the present invention, the methods of the present invention provide a GH peptide further comprising at least one CTP peptide at the N-terminus and at least one CTP peptide at the C-terminus for the treatment of osteoporosis. According to another embodiment of the present invention, the methods of the present invention provide a GH peptide, further comprising one CTP peptide at the N-terminus and two CTP peptides at the C-terminus, for the treatment of osteoporosis. According to another embodiment of the present invention, the methods of the present invention provide the GH peptide shown in the sequence of SEQ ID NO: 23, further comprising at least one CTP peptide at the N-terminus and at least one CTP peptide at the C-terminus, intended for treatment osteoporosis. According to another embodiment of the present invention, the methods of the present invention provide the GH peptide shown in the sequence of SEQ ID NO: 36, further comprising at least one CTP peptide at the N-terminus and at least one CTP peptide at the C-terminus, intended for treatment osteoporosis. According to another embodiment of the present invention, the methods of the present invention provide the GH peptide shown in the sequence of SEQ ID NO: 37, further comprising at least one CTP peptide at the N-terminus, intended for the treatment of osteoporosis. According to another embodiment of the present invention, the methods of the present invention provide the GH peptide shown in the sequence of SEQ ID NO: 38, further comprising at least one CTP peptide at the N-terminus, intended for the treatment of osteoporosis. According to another embodiment of the present invention, the methods of the present invention provide the GH peptide shown in SEQ ID NO: 39 for the treatment of osteoporosis. According to another embodiment of the present invention, the methods of the present invention provide the GH peptide shown in SEQ ID NO: 40 for the treatment of osteoporosis. According to another embodiment of the present invention, the methods of the present invention provide the GH peptide shown in SEQ ID NO: 41 for the treatment of osteoporosis. According to another embodiment of the present invention, the methods of the present invention provide the GH peptide shown in the sequence of SEQ ID NO: 42, further comprising at least one CTP peptide at the N-terminus, intended for the treatment of osteoporosis. According to another embodiment of the present invention, the methods of the present invention provide a CTP-modified GH peptide as provided herein for the treatment of osteoporosis.

[00128] According to another embodiment of the present invention, the methods of the present invention provide a nucleic acid sequence encoding a GH peptide, further comprising at least one CTP peptide at the N-terminus and at least one CTP peptide at the C-terminus, intended for the treatment of osteoporosis . According to another embodiment of the present invention, the methods of the present invention provide a nucleic acid sequence encoding a GH peptide, further comprising one CTP peptide at the N-terminus and two CTP peptides at the C-terminus, for the treatment of osteoporosis. According to one embodiment of the present invention, the methods of the present invention provide a nucleic acid shown in the sequence of SEQ ID NO: 44 encoding a growth hormone peptide containing one CTP peptide at the N-terminus and one CTP peptide at the C-terminus, intended for the treatment of osteoporosis. According to another embodiment of the present invention, the methods of the present invention provide a nucleic acid shown in the sequence of SEQ ID NO: 45 encoding a growth hormone peptide containing one CTP peptide at the N-terminus and two CTP peptides at the C-terminus, intended for the treatment of osteoporosis. According to another embodiment of the present invention, the methods of the present invention provide a nucleic acid shown in SEQ ID NO: 46 encoding a GH peptide, one CTP peptide at the N-terminus and two CTP peptides at the C-terminus for the treatment of osteoporosis.

[00129] According to another embodiment of the present invention, the methods of the present invention provide a GH peptide further comprising at least one CTP peptide at the N-terminus and at least one CTP peptide at the C-terminus for inhibiting osteoporosis. According to another embodiment of the present invention, the methods of the present invention provide a GH peptide, further comprising one CTP peptide at the N-terminus and two CTP peptides at the C-terminus, designed to inhibit osteoporosis. According to another embodiment of the present invention, the methods of the present invention provide the GH peptide shown in the sequence of SEQ ID NO: 23, further comprising at least one CTP peptide at the N-terminus and at least one CTP peptide at the C-terminus, designed to inhibit osteoporosis. According to another embodiment of the present invention, the methods of the present invention provide the GH peptide shown in the sequence of SEQ ID NO: 36, further comprising at least one CTP peptide at the N-terminus and at least one CTP peptide at the C-terminus, designed to inhibit osteoporosis. According to another embodiment of the present invention, the methods of the present invention provide the GH peptide shown in the sequence of SEQ ID NO: 37, further comprising at least one CTP peptide at the N-terminus, designed to inhibit osteoporosis. According to another embodiment of the present invention, the methods of the present invention provide the growth hormone shown in the sequence of SEQ ID NO: 38, additionally containing at least one CTP peptide at the N-terminus, designed to inhibit osteoporosis. According to another embodiment of the present invention, the methods of the present invention provide the GH peptide shown in SEQ ID NO: 39 for inhibiting osteoporosis. According to another embodiment of the present invention, the methods of the present invention provide the GH peptide shown in SEQ ID NO: 40 for inhibiting osteoporosis. According to another embodiment of the present invention, the methods of the present invention provide the GH peptide shown in SEQ ID NO: 41 for inhibiting osteoporosis. According to another embodiment of the present invention, the methods of the present invention provide the GH peptide shown in the sequence of SEQ ID NO: 42, further comprising at least one CTP peptide at the N-terminus, designed to inhibit osteoporosis. According to another embodiment of the present invention, the methods of the present invention provide a CTP-modified GH peptide, as provided herein, to inhibit osteoporosis.

[00130] According to another embodiment of the present invention, the methods of the present invention provide a nucleic acid sequence encoding a GH peptide, further comprising at least one CTP peptide at the N-terminus and at least one CTP peptide at the C-terminus, designed to inhibit osteoporosis. According to another embodiment of the present invention, the methods of the present invention provide a nucleic acid sequence encoding a GH peptide, further comprising one CTP peptide at the N-terminus and two CTP peptides at the C-terminus, designed to inhibit osteoporosis. According to one embodiment of the present invention, the methods of the present invention provide a nucleic acid shown in the sequence of SEQ ID NO: 44 encoding a growth hormone peptide containing one CTP peptide at the N-terminus and one CTP peptide at the C-terminus, designed to inhibit osteoporosis. According to another embodiment of the present invention, the methods of the present invention provide a nucleic acid shown in the sequence of SEQ ID NO: 45 encoding a growth hormone peptide containing one CTP peptide at the N-terminus and two CTP peptides at the C-terminus, designed to inhibit osteoporosis. According to another embodiment of the present invention, the methods of the present invention provide a nucleic acid shown in SEQ ID NO: 46 encoding a GH peptide, one CTP peptide at the N-terminus, and two CTP peptides at the C-terminus for inhibition of osteoporosis.

[00131] In another embodiment of the present invention, the methods of the present invention provide the GH peptide of the present invention to improve exercise capacity.

[00132] According to another embodiment of the present invention, the methods of the present invention provide a GH peptide further comprising at least one N-terminal CTP peptide and at least one C-terminal CTP peptide designed to improve lung function. According to another embodiment of the present invention, the methods of the present invention provide a GH peptide, further comprising one CTP peptide at the N-terminus and two CTP peptides at the C-terminus, intended to improve lung function. According to another embodiment of the present invention, the methods of the present invention provide the growth hormone shown in the sequence of SEQ ID NO: 23, additionally containing at least one CTP peptide at the N-terminus and at least one CTP peptide at the C-terminus, designed to improve function lungs. According to another embodiment of the present invention, the methods of the present invention provide the growth hormone shown in the sequence of SEQ ID NO: 36, additionally containing at least one CTP peptide at the N-terminus and at least one CTP peptide at the C-terminus, designed to improve function lungs. According to another embodiment of the present invention, the methods of the present invention provide the growth hormone shown in the sequence of SEQ ID NO: 37, additionally containing at least one CTP peptide at the N-terminus, designed to improve lung function. According to another embodiment of the present invention, the methods of the present invention provide the growth hormone shown in the sequence of SEQ ID NO: 38, additionally containing at least one CTP peptide at the N-terminus, designed to improve lung function. According to another implementation variant of the present invention, the methods according to the present invention provide growth hormone, shown in the sequence of SEQ ID NO: 39, designed to improve lung function. According to another implementation variant of the present invention, the methods according to the present invention provide growth hormone, shown in the sequence of SEQ ID NO: 40, designed to improve lung function. According to another implementation variant of the present invention, the methods according to the present invention provide growth hormone, shown in the sequence of SEQ ID NO: 41, designed to improve lung function. According to another embodiment of the present invention, the methods of the present invention provide the growth hormone shown in the sequence of SEQ ID NO: 42, additionally containing at least one CTP peptide at the N-terminus, intended to improve lung function. According to another embodiment of the present invention, the methods of the present invention provide a CTP-modified GH peptide as provided herein to improve lung function.

[00133] According to another embodiment of the present invention, the methods of the present invention provide a nucleic acid sequence encoding a GH peptide, further comprising at least one CTP peptide at the N-terminus and at least one CTP peptide at the C-terminus, designed to improve function lungs. According to another embodiment of the present invention, the methods of the present invention provide a nucleic acid sequence encoding a GH peptide, further comprising one CTP peptide at the N-terminus and two CTP peptides at the C-terminus, intended to improve lung function. According to one embodiment of the present invention, the methods of the present invention provide a nucleic acid shown in the sequence of SEQ ID NO: 44 encoding a growth hormone peptide containing one CTP peptide at the N-terminus and one CTP peptide at the C-terminus, intended to improve lung function. According to another embodiment of the present invention, the methods of the present invention provide a nucleic acid shown in the sequence of SEQ ID NO: 45 encoding a growth hormone peptide containing one CTP peptide at the N-terminus and two CTP peptides at the C-terminus, intended to improve lung function. According to another embodiment of the present invention, the methods of the present invention provide a nucleic acid shown in SEQ ID NO: 46 encoding a GH peptide, one CTP peptide at the N-terminus and two CTP peptides at the C-terminus to improve lung function.

[00134] According to another embodiment of the present invention, the methods of the present invention provide a GH peptide, further comprising at least one CTP peptide at the N-terminus and at least one CTP peptide at the C-terminus, designed to improve immunity. According to another embodiment of the present invention, the methods of the present invention provide a GH peptide additionally containing one CTP peptide at the N-terminus and two CTP peptides at the C-terminus, designed to improve immunity. According to another embodiment of the present invention, the methods of the present invention provide the growth hormone shown in the sequence of SEQ ID NO: 23, additionally containing at least one CTP peptide at the N-terminus and at least one CTP peptide at the C-terminus, designed to improve immunity . According to another embodiment of the present invention, the methods of the present invention provide the growth hormone shown in the sequence of SEQ ID NO: 36, additionally containing at least one CTP peptide at the N-terminus and at least one CTP peptide at the C-terminus, designed to improve immunity . According to another embodiment of the present invention, the methods of the present invention provide the growth hormone shown in the sequence of SEQ ID NO: 37, additionally containing at least one CTP peptide at the N-terminus, designed to improve immunity. According to another embodiment of the present invention, the methods of the present invention provide the growth hormone shown in the sequence of SEQ ID NO: 38, additionally containing at least one CTP peptide at the N-terminus, designed to improve immunity. According to another implementation variant of the present invention, the methods according to the present invention provide growth hormone, shown in the sequence of SEQ ID NO: 39, designed to improve immunity. According to another implementation variant of the present invention, the methods according to the present invention provide growth hormone, shown in the sequence of SEQ ID NO: 40, designed to improve immunity. According to another implementation variant of the present invention, the methods according to the present invention provide growth hormone, shown in the sequence of SEQ ID NO: 41, designed to improve immunity. According to another embodiment of the present invention, the methods of the present invention provide the growth hormone shown in the sequence of SEQ ID NO: 42, additionally containing at least one CTP peptide at the N-terminus, designed to improve immunity. According to another embodiment of the present invention, the methods of the present invention provide a CTP-modified GH peptide as provided herein to improve immunity.

[00135] According to another embodiment of the present invention, the methods of the present invention provide a nucleic acid sequence encoding a GH peptide, further comprising at least one CTP peptide at the N-terminus and at least one CTP peptide at the C-terminus, designed to improve immunity . According to another embodiment of the present invention, the methods of the present invention provide a nucleic acid sequence encoding a GH peptide, further comprising one CTP peptide at the N-terminus and two CTP peptides at the C-terminus, intended to improve immunity. According to one embodiment of the present invention, the methods of the present invention provide a nucleic acid shown in the sequence of SEQ ID NO: 44 encoding a GH peptide containing one CTP peptide at the N-terminus and one CTP peptide at the C-terminus, intended to improve immunity. According to another embodiment of the present invention, the methods of the present invention provide a nucleic acid shown in the sequence of SEQ ID NO: 45 encoding a GH peptide containing one CTP peptide at the N-terminus and two CTP peptides at the C-terminus, intended to improve immunity. According to another embodiment of the present invention, the methods of the present invention provide a nucleic acid shown in the sequence of SEQ ID NO: 46 encoding a GH peptide, one CTP peptide at the N-terminus and two CTP peptides at the C-terminus to improve immunity.

[00136] According to another embodiment of the present invention, the methods of the present invention provide a GH peptide further comprising at least one CTP peptide at the N-terminus and at least one CTP peptide at the C-terminus for vital organ regrowth. According to another embodiment of the present invention, the methods of the present invention provide a GH peptide, further comprising one CTP peptide at the N-terminus and two CTP peptides at the C-terminus, for vital organ regrowth. According to another embodiment of the present invention, the methods of the present invention provide the growth hormone shown in the sequence of SEQ ID NO: 23, additionally containing at least one CTP peptide at the N-terminus and at least one CTP peptide at the C-terminus, intended for growth regrowth vital organs. According to another embodiment of the present invention, the methods of the present invention provide the growth hormone shown in the sequence of SEQ ID NO: 36, additionally containing at least one CTP peptide at the N-terminus and at least one CTP peptide at the C-terminus, intended for growth regrowth vital organs. According to another embodiment of the present invention, the methods of the present invention provide the growth hormone shown in the sequence of SEQ ID NO: 37, additionally containing at least one CTP peptide at the N-terminus, intended for regrowth of vital organs. According to another embodiment of the present invention, the methods of the present invention provide the growth hormone shown in the sequence of SEQ ID NO: 38, additionally containing at least one CTP peptide at the N-terminus, intended for regrowth of vital organs. According to another implementation variant of the present invention, the methods according to the present invention provide growth hormone, shown in the sequence of SEQ ID NO: 39, intended for regrowth of vital organs. According to another implementation variant of the present invention, the methods according to the present invention provide growth hormone, shown in the sequence of SEQ ID NO: 40, intended for regrowth of vital organs. According to another implementation variant of the present invention, the methods according to the present invention provide growth hormone, shown in the sequence of SEQ ID NO: 41, intended for regrowth of vital organs. According to another embodiment of the present invention, the methods of the present invention provide the growth hormone shown in the sequence of SEQ ID NO: 42, additionally containing at least one CTP peptide at the N-terminus, intended for regrowth of vital organs. According to another embodiment of the present invention, the methods of the present invention provide a CTP-modified GH peptide as provided herein for regrowth of vital organs.

[00137] According to another embodiment of the present invention, the methods of the present invention provide a nucleic acid sequence encoding a GH peptide, further comprising at least one CTP peptide at the N-terminus and at least one CTP peptide at the C-terminus, intended for regrowth vital organs. According to another embodiment of the present invention, the methods of the present invention provide a nucleic acid sequence encoding a GH peptide, additionally containing one CTP peptide at the N-terminus and two CTP peptides at the C-terminus, intended for regrowth of vital organs. According to one embodiment of the present invention, the methods of the present invention provide a nucleic acid, shown in the sequence of SEQ ID NO: 44, encoding a GH peptide containing one CTP peptide at the N-terminus and one CTP peptide at the C-terminus, designed to restore the growth of vital organs. According to another embodiment of the present invention, the methods of the present invention provide a nucleic acid, shown in the sequence of SEQ ID NO: 45, encoding a growth hormone peptide containing one CTP peptide at the N-terminus and two CTP peptides at the C-terminus, designed to restore the growth of vital organs. According to another embodiment of the present invention, the methods of the present invention provide a nucleic acid, shown in the sequence of SEQ ID NO: 46, encoding a GH peptide containing one CTP peptide at the N-terminus and two CTP peptides at the C-terminus, for regrowth of vital organs .

[00138] In another embodiment of the present invention, the methods of the present invention provide the GH peptide of the present invention to improve feelings of well-being.

[00139] According to another embodiment of the present invention, the methods of the present invention provide a GH peptide further comprising at least one N-terminal CTP peptide and at least one C-terminal CTP peptide designed to restore REM sleep. According to another embodiment of the present invention, the methods of the present invention provide a GH peptide, further comprising one CTP peptide at the N-terminus and two CTP peptides at the C-terminus, for restoring REM sleep. According to another embodiment of the present invention, the methods of the present invention provide the growth hormone shown in the sequence of SEQ ID NO: 23, additionally containing at least one CTP peptide at the N-terminus and at least one CTP peptide at the C-terminus, designed to restore the phase fast sleep. According to another embodiment of the present invention, the methods of the present invention provide the growth hormone shown in the sequence of SEQ ID NO: 36, additionally containing at least one CTP peptide at the N-terminus and at least one CTP peptide at the C-terminus, designed to restore the phase fast sleep. According to another embodiment of the present invention, the methods of the present invention provide a growth hormone shown in the sequence of SEQ ID NO: 37, additionally containing at least one CTP peptide at the N-terminus, designed to restore REM sleep. According to another embodiment of the present invention, the methods of the present invention provide the growth hormone shown in the sequence of SEQ ID NO: 38, additionally containing at least one CTP peptide at the N-terminus, designed to restore REM sleep. According to another implementation variant of the present invention, the methods according to the present invention provide STG, shown in the sequence of SEQ ID NO: 39, designed to restore REM sleep. According to another implementation variant of the present invention, the methods according to the present invention provide STG, shown in the sequence of SEQ ID NO: 40, designed to restore REM sleep. According to another implementation variant of the present invention, the methods according to the present invention provide STG, shown in the sequence of SEQ ID NO: 41, designed to restore REM sleep. According to another embodiment of the present invention, the methods of the present invention provide a growth hormone shown in the sequence of SEQ ID NO: 42, additionally containing at least one CTP peptide at the N-terminus, designed to restore REM sleep. According to another embodiment of the present invention, the methods of the present invention provide a CTP-modified GH peptide as provided herein for restoring REM sleep.

[00140] According to another embodiment of the present invention, the methods of the present invention provide a nucleic acid sequence encoding a GH peptide, further comprising at least one CTP peptide at the N-terminus and at least one CTP peptide at the C-terminus, designed to restore phase fast sleep. According to another embodiment of the present invention, the methods of the present invention provide a nucleic acid sequence encoding a GH peptide, additionally containing one CTP peptide at the N-terminus and two CTP peptides at the C-terminus, designed to restore REM sleep. According to one embodiment of the present invention, the methods of the present invention provide a nucleic acid shown in the sequence of SEQ ID NO: 44 encoding a GH peptide containing one CTP peptide at the N-terminus and one CTP peptide at the C-terminus, designed to restore REM sleep. . According to another embodiment of the present invention, the methods of the present invention provide a nucleic acid shown in the sequence of SEQ ID NO: 45 encoding a GH peptide containing one CTP peptide at the N-terminus and two CTP peptides at the C-terminus, designed to restore REM sleep. . According to another embodiment of the present invention, the methods of the present invention provide a nucleic acid shown in the sequence of SEQ ID NO: 46 encoding a GH peptide containing one CTP peptide at the N-terminus and two CTP peptides at the C-terminus to restore REM sleep.

[00141] According to another embodiment of the present invention, the methods of the present invention provide a nucleic acid sequence encoding the GH protein described in this application. According to another embodiment of the present invention, the methods of the present invention provide a nucleic acid sequence encoding a CTP-modified GH-containing polypeptide for promoting muscle growth, increasing heart function, promoting bone growth, maintaining muscle integrity, maintaining muscle metabolic balance, inducing muscle gain , induction of de novo muscle building, increase in bone loading, treatment of symptoms associated with osteoporosis, treatment of wasting syndrome, increase in lipolysis, improvement of fluid balance, treatment of osteoporosis, improvement of lung function, improvement of immunity, regrowth of a vital organ, increased sense of well-being , restoring REM sleep, or any combination thereof.

[00142] According to some embodiments of the present invention, the term "homology" in accordance with the present invention also includes deletions, insertions or substitution variants, including amino acid substitutions therein, and their biologically active polypeptide fragments. According to one embodiment of the present invention, the substitution variant is one in which the glutamine at position 65 of the growth hormone is replaced by valine [Gellerfors et al., J Pharm Biomed Anal 1989, 7:173-83].

[00143] According to another embodiment, the nucleic acid molecule encoding the growth hormone described in this application encodes any amino acid sequence of growth hormone known to a person skilled in the art. In another embodiment, the nucleic acid molecule encoding the growth hormone described in this application encodes human growth hormone. According to another embodiment, the nucleic acid molecule encoding growth hormone contains a nucleic acid sequence registered in GeneBank under accession number NM_000515.3. According to another embodiment, the nucleic acid molecule encoding growth hormone contains a nucleic acid sequence registered in GeneBank under accession number NM_022559.2. According to another embodiment, the nucleic acid molecule encoding growth hormone contains a nucleic acid sequence registered in GeneBank under accession number NM_022560.2. According to another embodiment, the nucleic acid molecule encoding growth hormone contains a nucleic acid sequence registered in GeneBank under accession number NM_022561.2. According to another embodiment, the nucleic acid molecule encoding growth hormone contains a nucleic acid sequence registered in GeneBank under accession number NM_022562.2.

[00144] According to one embodiment of the present invention, a homologue also refers to a deletion, insertion, or substitution variant, including an amino acid substitution therein, and its biologically active polypeptide fragments.

[00145] According to another embodiment of the present invention, the polypeptide sequence of interest is human GH. According to another embodiment of the present invention, the polypeptide sequence of interest is a peptide or protein, including any modified form.

[00146] According to another embodiment of the present invention, the methods of the present invention provide human GH, further comprising at least one CTP peptide at the N-terminus and at least one CTP peptide at the C-terminus, for the treatment of wasting syndrome, AIDS, cachexia or human GH deficiency.

[00147] In one embodiment, the present invention is used in veterinary medicine. In one embodiment, the present invention provides a method of treating domesticated mammals kept as human companion animals (e.g., dogs, cats, horses) that are of significant commercial value (e.g., dairy cows, cattle, sport animals), which are of significant scientific importance (for example, captive or free-living specimens of endangered species), or which have other significance.

[00148] According to some embodiments of the present invention, modification of the CTP sequences is preferred because it allows lower dosages to be used.

[00149] According to some embodiments of the present invention, in this application, the term "polypeptide" includes native polypeptides (degradants, synthetically synthesized polypeptides, or recombinant polypeptides) and peptidomimetics (generally synthetic polypeptides), as well as peptoids and semipeptoids, which are polypeptide analogs containing, in some embodiments, modifications that give the polypeptides increased stability in the body or a higher ability to penetrate cells.

[00150] According to some embodiments of the present invention, modifications include, but are not limited to, N-terminal modifications, C-terminal modifications, polypeptide bond modifications, including, but not limited to, CH ₂ -NH, CH ₂ -S, CH ₂ -S=O, O=C-NH, CH ₂ -O, CH ₂ -CH ₂ , S=C-NH, CH=CH or CF=CH, backbone modifications and residue modifications. Methods for preparing peptidomimetic compounds are well known in the art and are described, for example, in Quantitative Drug Design, CA Ramsden Gd., Chapter 17.2, F. Choplin Pergamon Press (1992), which is incorporated herein by reference in its entirety. More information regarding this aspect is given below.

[00151] In some embodiments of the present invention, the (-CO-NH-) polypeptide bonds in the polypeptide are substituted. In some embodiments of the present invention, the polypeptide bonds are substituted with N-methylated bonds (-N(CH ₃ )-CO-). In some embodiments of the present invention, the polypeptide bonds are substituted with ester bonds (-C(R)HCOOC(R)-N-). In some embodiments of the present invention, the polypeptide bonds are substituted with ketomethylene bonds (-CO-CH ₂ -). In some embodiments of the present invention, the polypeptide bonds are replaced by α-aza bonds (-NH-N(R)-CO-) in which R is any alkyl, eg methyl, carbo bonds (-CH ₂ -NH-). In some embodiments of the present invention, the polypeptide bonds are substituted with hydroxyethylene bonds (-CH(OH)-CH ₂ -). In some embodiments of the present invention, the polypeptide bonds are substituted with thioamide bonds (-CS-NH-). In some embodiments of the present invention, the polypeptide bonds are substituted with olefinic double bonds (-CH=CH-). In some embodiments of the present invention, the polypeptide bonds are replaced by retro-amide bonds (-NH-CO-). In some embodiments of the present invention, the polypeptide bonds are substituted with polypeptide derivatives (-N(R)-CH ₂ -CO-) in which R is a "straight" side chain naturally occurring on a carbon atom. According to some variants of implementation of the present invention, these modifications occur in any of the bonds along the polypeptide chain and even in several bonds (2-3 bonds) at the same time.

[00152] According to some embodiments of the present invention, the natural aromatic amino acids of the polypeptide, such as Trp, Tyr, and Phe, are substituted with a synthetic non-natural acid, such as phenylglycine, TIC, naphthylalanine (Nol), Phe derivatives with a methylated ring, halogenated Phe derivatives, or o- methyltyrosine. In some embodiments, the polypeptides of the invention comprise one or more modified amino acids or one or more non-amino acid monomers (eg, fatty acids, complex carbohydrates, etc.).

[00153] According to one implementation variant of the present invention, the terms "amino acid" or "amino acids" include 20 naturally occurring amino acids; those amino acids that frequently undergo post-translational modifications under in vivo conditions, including, for example, hydroxyproline, phosphoserine, and phosphothreonine; and other atypical amino acids including, but not limited to, 2-aminoadipic acid, hydroxylysine, isodesmosine, norvaline, norleucine, and ornithine. According to one implementation variant of the present invention, the term "amino acid" includes both D-amino acids and L-amino acids.

[00154] According to some embodiments of the present invention, the polypeptides of the present invention are used in therapy, for which the polypeptides must be in soluble form. In some embodiments of the present invention, the polypeptides of the present invention comprise one or more non-natural or natural polar amino acids, including, but not limited to, serine and threonine, which are capable of increasing the solubility of the polypeptide due to their hydroxyl-containing side chain.

[00155] According to some embodiments of the present invention, the polypeptides of the present invention are used in a linear form, although one skilled in the art will understand that in cases where cyclization does not significantly affect the characteristics of the polypeptides, cyclic forms of the polypeptide can also be used.

[00156] According to some embodiments of the present invention, the polypeptides of the present invention are synthesized biochemically, for example, using standard solid phase synthesis techniques. According to some embodiments of the present invention, these biochemical methods include total solid phase synthesis, partial solid phase synthesis, fragment condensation, or classical solution synthesis. According to some embodiments of the present invention, these methods are used when the polypeptide is relatively short (about 5-15 kDa) and/or when it cannot be obtained using recombinant techniques (i.e., not encoded by a nucleic acid sequence) and, therefore, contains various chemical fragments.

[00157] According to some embodiments of the present invention, solid phase polypeptide synthesis techniques are well known to those skilled in the art and are further described by John Morrow Stewart and Janis Dillaha Young, Solid Phase Polypeptide Syntheses (2nd Ed., Pierce Chemical Company, 1984). According to some embodiments of the present invention, synthetic polypeptides are purified using preparative high performance liquid chromatography [Creighton T. (1983) Proteins, structures and molecular principles. W. H. Freeman and Co. N.Y.], and their composition can be confirmed by amino acid sequencing using methods known to the person skilled in the art.

[00158] In some embodiments of the present invention, recombinant protein production techniques are used to synthesize the polypeptides of the present invention. In some embodiments of the present invention, recombinant protein production techniques are used to synthesize relatively long polypeptides (eg, greater than 18-25 amino acids). In some embodiments of the present invention, recombinant protein production techniques are used to synthesize a large amount of a polypeptide of the present invention. In some embodiments of the present invention, recombinant techniques are described in Bitter et al., (1987) Methods in Enzymol. 153:516-544, Studier et al. (1990) Methods in Enzymol. 185:60-89, Brisson et al. (1984) Nature 310:511-514, Takamatsu et al. (1987) EMBO J. 6:307-311, Coruzzi et al. (1984) EMBO J. 3:1671-1680 and Brogli et al, (1984) Science 224:838-843, Gurley et al. (1986) Mol. cell. Biol. 6:559-565 and Weissbach & Weissbach, 1988, Methods for Plant Molecular Biology, Academic Press, NY, Section VIII, pp 421-463.

[00159] According to one embodiment of the present invention, a polypeptide of the present invention is synthesized using a polynucleotide encoding a polypeptide of the present invention. In some embodiments of the invention, a polynucleotide encoding a polypeptide of the invention is ligated into an expression vector in which transcription is controlled by a cis regulatory sequence (eg, a promoter sequence). In some embodiments of the present invention, the cis regulatory sequence is suitable for directing constitutive expression of a polypeptide of the present invention. In some embodiments of the present invention, a cis regulatory sequence is useful for directing tissue-specific expression of a polypeptide of the present invention. In some embodiments of the present invention, a cis regulatory sequence is useful for directing inducible expression of a polypeptide of the present invention.

[00160] According to some embodiments of the present invention, polynucleotides that express the polypeptides of the present invention are shown in the sequences of SEQ ID NOS: 44, 45 and 46.

[00161] According to some embodiments of the present invention, tissue-specific promoters suitable for use in accordance with the present invention include sequences that are functional in a particular population of cells, for example, include, but are not limited to, promoters such as the albumin promoter, which is specific for liver [Pinkert et al., (1987) Genes Dev. 1:268-277], lymphoid tissue-specific promoters [Calame et al., (1988) Adv. Immunol. 43:235-275]; in particular promoters of T-cell receptors [Winoto et al., (1989) EMBO J. 8:729-733] and immunoglobulins; [Banerji et al. (1983) Cell 33729-740], neuron-specific promoters such as the neurofilament promoter [Byrne et al. (1989) Proc. Natl. Acad. sci. USA 86:5473-5477], pancreas-specific promoters [Edlunch et al. (1985) Science 230:912-916], or breast-specific promoters such as the whey promoter (US Pat. No. 4,873,316 and European Patent Application Publication No. 264,166). Inducible promoters suitable for use in accordance with the present invention include, for example, the tetracycline inducible promoter (Srour, M.A., et al., 2003. Thromb. Haemost. 90: 398-405).

[00162] According to one embodiment of the present invention, the expression "polynucleotide" refers to a single- or double-stranded nucleic acid sequence that will be isolated and provided as an RNA sequence, a complementary polynucleotide sequence (cDNA), a genomic polynucleotide sequence, and/or composite polynucleotide sequences ( for example, combinations of the sequences above).

[00163] According to one implementation variant of the present invention, the term "complementary polynucleotide sequence" refers to a sequence that is synthesized by reverse transcription of RNA using reverse transcriptase or any other RNA-dependent DNA polymerase. According to one implementation variant of the present invention, the sequence can then be amplified in vivo or in vitro using DNA polymerase.

[00164] According to one implementation variant of the present invention, the term "genomic polynucleotide sequence" refers to a sequence obtained (isolated) from a chromosome and, therefore, it is a contiguous part of the chromosome.

[00165] According to one implementation variant of the present invention, the term "composite polynucleotide sequence" refers to a sequence that is at least partially complementary and at least partially genomic. According to one implementation variant of the present invention, the composite sequence may contain some of the exon sequences necessary to encode the polypeptide according to the present invention, as well as some of the intron sequences inserted between them. In one embodiment of the present invention, the intron sequences may be derived from any source, including other genes, and will typically include conserved splicing signal sequences. In one embodiment of the present invention, the intron sequences contain cis-acting expression control elements.

[00166] According to one embodiment of the present invention, the polynucleotides of the present invention further comprise a signal sequence encoding a signal peptide for secretion of the polypeptides of the present invention. In some embodiments of the present invention, the signal sequences comprise, but are not limited to, an endogenous EPO signal sequence set forth in SEQ ID NO: 19 or an endogenous IFN-β1 signal sequence set forth in SEQ ID NO: 26. In another an embodiment of the present invention, the signal sequence is located at the N-terminus with respect to the CTP sequence, which in turn is located at the N-terminus with respect to the polypeptide sequence of interest; for example, the sequence is (a) a signal sequence, (b) a CTP-, (c) a sequence of interest, (d) optionally 1 or more additional CTP sequences. According to another embodiment of the present invention, 1 or more CTP sequences are inserted between the signal sequence of the polypeptide sequence of interest and the polypeptide sequence of interest itself, thereby interrupting the wild-type sequence of interest. Each possibility represents a separate embodiment of the present invention.

[00167] According to another embodiment of the present invention, the growth hormone further comprises a signal peptide. In some embodiments of the present invention, the signal sequences comprise, but are not limited to, an endogenous signal sequence. In some embodiments of the present invention, the signal sequences comprise, but are not limited to, an endogenous signal sequence of any known growth hormone or growth hormones. According to another embodiment of the present invention, the polypeptides and methods of the present invention provide a growth hormone further comprising a signal peptide comprising the following amino acid sequence: MATGSRTSLLLAFGLLCLPWLQEGSA (SEQ ID NO: 49).

[00168] According to one embodiment of the present invention, once expressed and secreted, signal peptides are cleaved from precursor proteins, resulting in mature proteins.

[00169] According to some embodiments of the present invention, the polynucleotides of the present invention are obtained using PCR techniques using procedures and methods known to a person skilled in the art. In some embodiments of the present invention, the procedure involves ligating two different DNA sequences (see, for example, "Current Protocols in Molecular Biology", eds. Ausubel et al., John Wiley & Sons, 1992).

[00170] According to one embodiment of the present invention, the polynucleotides of the present invention are inserted into expression vectors (ie, a nucleic acid construct) to allow expression of the recombinant polypeptide. According to one embodiment of the present invention, the expression vector of the present invention contains additional sequences that make the vector suitable for replication and integration in prokaryotes. According to one embodiment of the present invention, the expression vector of the present invention contains additional sequences that make the vector suitable for replication and integration in eukaryotes. According to one embodiment of the present invention, the expression vector of the present invention contains a shuttle vector which makes the vector suitable for replication and integration in both prokaryotes and eukaryotes. In some embodiments of the present invention, cloning vectors include transcription and translation initiation sequences (eg, promoters, enhancer) and transcription and translation terminators (eg, polyadenylation signals).

[00171] According to one implementation variant of the present invention, a number of prokaryotic or eukaryotic cells can be used as a host system for the expression of polypeptides according to the present invention. According to some variants of implementation of the present invention, these cells include, but are not limited to, microorganisms, such as bacteria, transformed with an expression vector containing recombinant bacteriophage DNA, plasmid DNA or cosmid DNA, which contains a sequence encoding a polypeptide; yeast transformed with recombinant yeast expression vectors containing a sequence encoding a polypeptide; plant cell systems infected with recombinant viral expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant expression plasmid vectors, such as the Ti plasmid, containing the sequence encoding the polypeptide.

[00172] According to some embodiments of the present invention, non-bacterial expression systems (eg, mammalian expression systems such as CHO cells) are used to express the polypeptide of the present invention. In one embodiment of the present invention, the expression vector used to express the polynucleotides of the present invention in mammalian cells is a pCI-DHFR vector containing a cytomegalovirus (CMV) promoter and a neomycin resistance gene. The construction of the pCI-DHFR vector is described, in accordance with one embodiment, in Example 1 and in FIG. 3.

[00173] In some embodiments of the present invention in bacterial systems according to the present invention, a number of vectors for expression may be preferably selected depending on the method of application intended for the expressed polypeptide. In one embodiment of the present invention, a large amount of a polypeptide is desirable. According to one embodiment of the present invention, vectors are desirable that direct the expression of high levels of the protein product, possibly as a fusion protein with a hydrophobic signal sequence that directs the expressed product to the bacterial periplasm or culture medium from which the protein product can be easily purified. In one embodiment of the present invention, a specific fusion protein is designed to include a specific cleavage site to facilitate isolation of the polypeptide. According to one implementation variant of the present invention, vectors suitable for such manipulation include, but are not limited to, a series of vectors for the expression of pET in E. coli [Studier et al ., Methods in Enzymol. 185:60-89 (1990)].

[00174] According to one embodiment of the present invention, yeast expression systems are used. In one embodiment of the present invention, a number of vectors containing constitutive or inducible promoters can be used in yeast as described in US Pat. No. 5,932,447. According to another embodiment of the present invention, vectors are used that stimulate the integration of foreign DNA sequences into the yeast chromosome.

[00175] According to one embodiment of the present invention, the expression vector of the present invention may further comprise additional polynucleotide sequences that allow, for example, translation of multiple proteins from a single mRNA, such as an internal ribosome entry site (IRES) and sequences for genomic integration of a promoter- chimeric polypeptide.

[00176] In some embodiments, mammalian expression vectors include, but are not limited to, pcDNA3, pcDNA3.1(+/-), pGL3, pZeoSV2(+/-), pSecTag2, pDisplay, pEF/myc/cyto, pCMV /myc/cyto, pCR3.1, pSinRep5, DH26S, DHBB, pNMT1, pNMT41, pNMT81 available from Invitrogen, pCI available from Promega, pMbac, pPbac, pBK-RSV and pBK-CMV available from Strategene, pTRES available from Clontech and derivatives thereof.

[00177] According to some embodiments of the present invention, expression vectors containing regulatory elements from eukaryotic viruses, such as retroviruses, are used in accordance with the present invention. SV40 vectors include pSVT7 and pMT2. In some embodiments of the present invention, bovine papilloma virus-derived vectors include pBV-1MTHA and Epstein-Barr virus-derived vectors include pHEBO and p2O5. Examples of other vectors include pMSG, pAV009/A ⁺ , pMTO10/A ⁺ , pMAMneo-5, pDSVE baculovirus, and any other vector that expresses proteins under the control of the SV-40 early gene promoter, SV-40 late gene promoter, metallothionein promoter, promoter murine mammary tumor virus, Rous sarcoma virus promoter, polyhedrin promoter, or other promoters that have been shown to be effective for expression in eukaryotic cells.

[00178] According to some embodiments of the present invention, recombinant viral vectors can be used for in vivo expression of the polypeptides of the present invention as they provide a number of advantages such as lateral infection and targeting specificity. According to one embodiment of the present invention, lateral infection is inherent in the life cycle of, for example, retroviruses, and is the process by which one infected cell produces many progeny virions that bud off and infect neighboring cells. According to one implementation variant of the present invention, the result is that a large area quickly becomes infected, most of which was not originally infected with the original viral particles. According to one implementation variant of the present invention receive viral vectors that are not able to spread laterally. According to one implementation variant of the present invention, this feature may be beneficial if the desired goal is the introduction of a particular gene only in a localized number of targeted cells.

[00179] According to one embodiment of the present invention, various methods can be used to introduce the expression vector of the present invention into cells. Such methods are generally described in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Springs Harbor Laboratory, New York (1989, 1992), in Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Md. (1989), Chang et al., Somatic Gene Therapy, CRC Press, Ann Arbor, Mich. (1995), Vega et al., Gene Targeting, CRC Press, Ann Arbor Mich. (1995), Vectors: A Survey of Molecular Cloning Vectors and Their Uses, Butterworths, Boston Mass. (1988) and Gilboa et al. [Biotechniques 4 (6): 504-512, 1986] and include, for example, stable or transient transfection, lipofection, electroporation, and infection with recombinant viral vectors. See also US Pat. Nos. 5,464,764 and 5,487,992 which describe positive-negative selection methods.

[00180] According to some embodiments of the present invention, the introduction of a nucleic acid by viral infection has a number of advantages over other methods such as lipofection and electroporation, since higher transfection efficiency can be obtained due to the infectious nature of viruses.

[00181] According to one embodiment of the present invention, it is to be understood that the polypeptides of the present invention can also be expressed from a nucleic acid construct introduced into an individual using any suitable mode of administration described above (i.e., in vivo gene therapy). According to one embodiment of the present invention, the nucleic acid construct is introduced into a suitable cell using an appropriate gene delivery vehicle/method (transfection, transduction, homologous recombination, etc.) and expression system, if necessary, and then the modified cells are expanded in culture and transplanted into an individual (ie, ex vivo gene therapy).

[00182] In one embodiment of the present invention, plant expression vectors are used. According to one implementation variant of the present invention, the expression of the sequence encoding the polypeptide is controlled by a number of promoters. In some embodiments of the present invention, viral promoters are used, such as the CaMV 35S RNA and 19S RNA promoters [Brisson et al ., Nature 310:511-514 (1984)], or the TMV envelope protein promoter [Takamatsu et al ., EMBO J. 6:307-311 (1987)]. According to another embodiment of the present invention, plant promoters are used, such as, for example, the RUBISCO small subunit promoter [Coruzzi et al ., EMBO J. 3:1671-1680 (1984); and Brogli et al ., Science 224:838-843 (1984)], or promoters of heat shock proteins such as soybean hsp17.5-E or hsp17.3-B [Gurley et al ., Mol. cell. Biol. 6:559-565 (1986)]. According to one embodiment of the present invention, constructs are introduced into plant cells using Ti-plasmid, Ri-plasmid, plant viral vectors, direct DNA transformation, microinjection, electroporation, and other methods well known to the person skilled in the art. See, for example, Weissbach & Weissbach [Methods for Plant Molecular Biology, Academic Press, NY, Section VIII, pp 421-463 (1988)]. Other expression systems, such as those based on insect and mammalian host cells, which are well known in the art, can also be used in accordance with the present invention.

[00183] It should be understood that in addition to the necessary elements for transcription and translation of the inserted coding sequence (encoding the polypeptide), the expression construct of the present invention may also contain sequences designed to optimize the stability, production, purification, yield, or activity of the expressed polypeptide.

[00184] In some embodiments, various methods can be used to introduce an expression vector of the present invention into a host cell system. In some embodiments of the present invention, such methods are generally described in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Springs Harbor Laboratory, New York (1989, 1992), in Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Md. (1989), Chang et al., Somatic Gene Therapy, CRC Press, Ann Arbor, Mich. (1995), Vega et al., Gene Targeting, CRC Press, Ann Arbor Mich. (1995), Vectors: A Survey of Molecular Cloning Vectors and Their Uses, Butterworths, Boston Mass. (1988) and Gilboa et al. [Biotechniques 4 (6): 504-512, 1986] and include, for example, stable or transient transfection, lipofection, electroporation, and infection with recombinant viral vectors. See also US Pat. Nos. 5,464,764 and 5,487,992 which describe positive-negative selection methods.

[00185] According to some embodiments of the present invention, the transformed cells are cultured under efficient conditions that allow for the expression of large amounts of the recombinant polypeptide. In some embodiments of the present invention, effective culture conditions include, but are not limited to, effective media, bioreactor, temperature, pH, and oxygen conditions that promote protein production. In one embodiment of the present invention, an effective medium refers to any medium in which a cell is cultured to produce a recombinant polypeptide of the present invention. In some embodiments of the present invention, the medium typically includes an aqueous solution containing sources of assimilable carbon, nitrogen, and phosphate, as well as appropriate salts, minerals, metals, and other nutrients such as vitamins. In some embodiments of the present invention, the cells of the present invention may be cultured in standard fermentation bioreactors, shake flasks, test tubes, microtiter plates, and Petri dishes. According to some variants of implementation of the present invention, the cultivation is carried out at a temperature, pH and oxygen content suitable for the recombinant cell. According to some embodiments of the present invention, the selection of culture conditions is within the skill of the art.

[00186] According to some embodiments of the present invention, depending on the vector and host system used for production, the resulting polypeptides of the present invention remain in the recombinant cell, are secreted into the fermentation medium, are secreted into the space between two cell membranes, such as the periplasmic space in E. coli ; or linger on the outer surface of the cell or viral membrane.

[00187] According to one embodiment of the present invention, after a predetermined culture period, the recombinant polypeptide is isolated.

[00188] According to one implementation variant of the present invention, in this application, the expression "isolation of the recombinant polypeptide" refers to the collection of the entire fermentation medium containing the polypeptide, and does not necessarily imply additional separation or purification steps.

[00189] According to one embodiment of the present invention, the polypeptides of the present invention are purified using various standard protein purification techniques, including, but not limited to, affinity chromatography, ion exchange chromatography, filtration, electrophoresis, hydrophobic chromatography, size exclusion chromatography, reverse phase chromatography, chromatography using concanavalin A, chromatographic focusing and differential solubilization.

[00190] According to one embodiment of the present invention, to facilitate isolation, the expressed coding sequence may be designed to encode the polypeptide of the present invention and the hybridized cleavage fragment. According to one implementation variant of the present invention, the hybrid protein can be designed so that the polypeptide can be easily isolated using affinity chromatography; for example, by immobilization on a column specific for the fragment to be cleaved off. According to one embodiment of the present invention, a cleavage site is constructed between the polypeptide and the cleavage fragment, and the polypeptide can be released from the chromatographic column by treatment with an appropriate enzyme or agent that specifically cleaves the fusion protein at that site [e.g., see Booth et al ., Immunol . Lett. 19:65-70 (1988); and Gardella et al ., J. Biol. Chem. 265:15854-15859 (1990)].

[00191] In one embodiment, the polypeptide of the present invention is isolated in a "substantially pure" form.

[00192] According to one implementation variant of the present invention, the expression "substantially pure" refers to the purity, which allows the effective use of the protein in accordance with the methods of use described in this application.

[00193] In one embodiment, the polypeptide of the present invention can also be synthesized using in vitro expression systems. In one embodiment of the present invention , in vitro synthesis methods are well known in the art and the components of the system are commercially available.

[00194] According to some embodiments of the present invention, recombinant polypeptides are synthesized and purified; their therapeutic efficacy can be evaluated in vivo or in vitro . According to one embodiment of the present invention, the production of CTP-modified GH is carried out using recombinant DNA technology.

[00195] In some embodiments of the present invention, recombinant polypeptides are synthesized and purified; their therapeutic efficacy can be evaluated in vivo or in vitro . According to one embodiment of the present invention, the binding activity of recombinant CTP-modified GH according to the present invention can be determined using various quantitative assay methods.

[00196] In one embodiment, the present invention provides CTP-GH-CTP polypeptides. According to one embodiment of the present invention, recombinant DNA techniques are used to produce CTP-GH-CTP polypeptides. According to one embodiment of the present invention, CTP-GH-CTP-CTP polypeptides are provided. According to one implementation variant of the present invention, the recombinant DNA techniques described in example 1 and in Fig. 1 are used to obtain CTP-GH-CTP-CTP polypeptides. The CTP-CTP of the present invention is quantified under in vivo conditions. In one embodiment of the present invention, the therapeutic efficacy of the CTP-GH-CTP or CTP-GH-CTP-CTP polypeptides of the present invention is quantified in vitro . According to one embodiment of the present invention, the binding activity of the recombinant GH polypeptides of the present invention is measured using Nb2 (prolactin-dependent rat lymphoma cell line (ECACC cell bank)) or mouse FCD-P1 cell line previously transfected with human growth hormone receptor. According to one embodiment of the present invention, GH binding to these receptors induces cell proliferation, which, in one embodiment of the present invention, is measured based on MTT cell staining levels as a function of GH activity. In one embodiment of the present invention, in vivo activity is assessed by measuring weight gain over time in growth hormone deficient treated animals.

[00197] According to one embodiment of the present invention, there is provided a method of inducing growth or weight gain in a subject, comprising administering to the subject a therapeutically effective amount of a polypeptide containing growth hormone, one carboxy-terminal peptide (CTP) of human chorionic gonadotropin attached to the amino terminus of said growth hormone , and two human chorionic gonadotropin CTPs attached to the carboxy terminus of the growth hormone, thereby inducing height or weight gain in the subject.

[00198] According to another embodiment of the present invention, there is provided a method for inducing height or weight gain in a non-human subject, comprising the step of administering to said non-human subject a therapeutically effective amount of an expression vector comprising a polynucleotide consisting of a nucleic acid encoding a polypeptide wherein said polypeptide consists of a non-human growth hormone, one carboxy-terminal peptide (CTP) of human chorionic gonadotropin attached to the amino terminus of said non-human growth hormone, and two CTPs of chorionic gonadotropin attached to the carboxy-terminus of said non-human growth hormone, wherein said polypeptide optionally contains a signal peptide attached to the amino terminus of said one CTP, thereby inducing growth or weight gain in a non-human subject.

[00199] According to another embodiment of the present invention, there is provided a method for inducing weight loss or reducing body fat in a subject, comprising administering to said subject a therapeutically effective amount of a polypeptide containing growth hormone, one carboxy-terminal peptide (CTP) of human chorionic gonadotropin attached to an amino -terminus of said growth hormone, and two human chorionic gonadotropin CTPs attached to the carboxy terminus of said growth hormone, thereby inducing weight loss or reduction in body fat in said subject. According to one implementation variant of the present invention, the specified subject is obese. According to another embodiment of the present invention, said subject is overweight.

[00200] In another embodiment, the present invention provides a method for reducing body fat in a non-human subject, comprising administering to said subject a therapeutically effective amount of an expression vector comprising a polynucleotide, said polynucleotide consisting of a non-human growth hormone, one carboxy- end peptide (CTP) of human chorionic gonadotropin attached to the amino end of the specified non-human growth hormone, and two CTPs of chorionic gonadotropin attached to the carboxy-terminus of the specified non-human growth hormone, while the specified polypeptide additionally contains a signal peptide attached to the amino end of the specified one CTP, thereby inducing height or weight gain in a non-human subject.

[00201] In another embodiment, the present invention provides a method for reducing body fat in a subject. In another embodiment, the present invention provides a method for increasing muscle mass in a subject. In another embodiment, the present invention provides a method for promoting muscle growth in a subject. According to another implementation variant, the present invention provides a method for increasing the ratio of muscle mass to fat. In another embodiment, the present invention provides a method for reducing body mass index (BMI) or Quetelet index.

[00202] According to another embodiment of the present invention, height is measured by weight gain. According to another embodiment of the present invention, height is measured by height increase. According to another embodiment of the present invention, height is measured by weight gain. According to another embodiment of the present invention, growth is measured by the increase in muscle mass. According to another embodiment of the present invention, height is measured by weight gain. According to another embodiment of the present invention, growth is measured as an increase in bone mass. According to another embodiment of the present invention, height is measured by weight gain. According to another embodiment of the present invention, growth is measured by the increase in muscle mass. According to another embodiment of the present invention, weight gain is due to increased bone and/or muscle weight. According to another embodiment of the present invention, growth is measured using any known estimate known to a person skilled in the art.

[00203] According to some embodiments of the present invention, the human growth hormone polypeptides of the present invention can be used to treat a subject having diseases associated with impaired growth and weight, such as growth deficiency, AIDS-associated wasting, aging, impaired immune system function HIV -infected subjects, catabolic disease, recovery from surgery, congestive cardiomyopathy, liver transplant, liver regeneration after liver resection, chronic renal failure, renal osteodystrophy, osteoporosis, achondroplasia/hypochondroplasia, skeletal dysplasia, chronic inflammatory disease or metabolic disorder such as Crohn's disease, short bowel syndrome, juvenile chronic arthritis, cystic fibrosis, male infertility, X-linked hypophosphatemic rickets, Down syndrome, spina bifida, Noonan syndrome, obesity, impaired muscle strength, and fibromyalgia. According to some embodiments of the present invention, the interferon polypeptides of the present invention are used to treat a subject having various diseases such as hairy cell leukemia (HCL), Kaposi's sarcoma (KS), chronic myeloid leukemia (CML), chronic hepatitis C (CHC), genital warts (CA), chronic hepatitis B, malignant melanoma, follicular non-Hodgkin's lymphoma, multiple sclerosis, chronic granulomatous disease, Mycobacterium avium complex (MAC), pulmonary fibrosis and osteoporosis.

[00204] According to one embodiment of the present invention, the polypeptides of the present invention may be offered to an individual individually. According to one implementation variant of the present invention, the polypeptides according to the present invention can be offered to the individual as part of a pharmaceutical composition in which they are mixed with a pharmaceutically acceptable carrier.

[00205] According to one implementation variant of the present invention, the term "pharmaceutical composition" refers to the preparation of one or more active ingredients described in this application, in combination with other chemical components, such as physiologically acceptable carriers and excipients. The pharmaceutical composition is intended to facilitate the introduction of the compounds into the body.

[00206] According to one implementation variant of the present invention, the term "active ingredient" refers to a polypeptide sequence of interest, which provides a biological effect.

[00207] According to some embodiments of the present invention, any of the compositions of the present invention will contain at least two CTP sequences linked to a protein of interest, in any form. In one embodiment, the present invention provides combination formulations. According to one embodiment of the present invention, the term "combination preparation" means "set of components" in the sense that the components of the combination, as defined above, can be dosed independently of each other or by using different fixed combinations with different amounts of components of the composition, i. simultaneously, jointly, separately or sequentially. According to some embodiments of the present invention, parts of the set of components can then be administered, for example, simultaneously or in chronological order intermittently, that is, at different points in time and at the same or different intervals of time for any part of the set of components. Full amounts of the components of the composition in a certain ratio, in some embodiments of the invention, can be entered in a combined preparation. According to one embodiment of the present invention, one skilled in the art can readily modify the combination formulation, for example, to meet the needs of a subpopulation of patients to be treated, or the needs of a particular patient whose varying needs may be due to a particular disease, disease severity, age, sex. or body weight.

[00208] According to one embodiment of the present invention, the terms "physiologically acceptable carrier" and "pharmaceutically acceptable carrier", which can be used interchangeably, refer to a carrier or diluent that does not cause significant irritation in the body and does not eliminate the biological activity and properties of the administered compound. . Said expressions include an adjuvant. According to one embodiment of the present invention, one of the ingredients included in a pharmaceutically acceptable carrier may be, for example, polyethylene glycol (PEG), a biocompatible polymer with a wide range of solubility in organic and aqueous media (Mutter et al. (1979).

[00209] According to one embodiment of the present invention, the term "adjuvant" refers to an inert substance added to a pharmaceutical composition to further facilitate the administration of the active ingredient. According to one embodiment of the present invention, excipients include calcium carbonate, calcium phosphate, various sugars and various types of starch, cellulose derivatives, gelatin, vegetable oils, and polyethylene glycols.

[00210] Methods for making and administering drugs can be found in "Remington's Pharmaceutical Sciences," Mack Publishing Co., Easton, PA, latest edition, which is incorporated herein by reference.

[00211] According to one embodiment of the present invention, suitable routes of administration include, for example, oral, rectal, transmucosal, transnasal, intestinal, or parenteral delivery, including intramuscular, subcutaneous, and intramedullary injections, as well as intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal or intraocular injections.

[00212] According to one implementation variant of the present invention, the drug is administered, as a rule, locally, and not systemically, for example, by injecting the drug directly into a specific area of the patient's body.

[00213] According to one implementation variant of the present invention, in which the pharmaceutical composition or pharmaceutical composition is administered to the patient by injection, the introduction is carried out using a syringe or pen.

[00214] According to another embodiment of the present invention, polypeptides containing growth hormone modified with CTP according to the present invention are administered at a dose of 1-90 micrograms in 0.1-5 ml of solution. According to another embodiment of the present invention, CTP-modified growth hormone-containing polypeptides are administered at a dose of 1-50 micrograms in 0.1-5 ml of solution. According to another embodiment of the present invention, CTP-modified growth hormone-containing polypeptides are administered at a dose of 1-25 micrograms in 0.1-5 ml of solution. According to another embodiment of the present invention, CTP-modified growth hormone-containing polypeptides are administered at a dose of 50-90 micrograms in 0.1-5 ml of solution. According to another embodiment of the present invention, CTP-modified growth hormone-containing polypeptides are administered at a dose of 10-50 micrograms in a 0.1-5 ml solution. According to another embodiment of the present invention, CTP-modified growth hormone-containing polypeptides are administered at a dose of 5 milligrams (mg) in 1 ml of solution, or 10 mg in 1 ml of solution, or 20 mg in 1 ml of solution, or 40 mg in 1 ml of solution .

[00215] According to another embodiment of the present invention, CTP-modified growth hormone-containing polypeptides are administered at a dose of 1-90 micrograms in 0.1-5 ml of solution by intramuscular (IM) injection, subcutaneous (SC) injection, or intravenous (IV) injection once a week. According to another embodiment of the present invention, CTP-modified growth hormone-containing polypeptides are administered at a dose of 1-90 micrograms in 0.1-5 ml of solution by intramuscular (IM) injection, subcutaneous (SC) injection, or intravenous (in /c) injections twice a week. According to another embodiment of the present invention, CTP-modified growth hormone-containing polypeptides are administered at a dose of 1-90 micrograms in 0.1-5 ml of solution by intramuscular (IM) injection, subcutaneous (SC) injection, or intravenous (in /c) injections three times a week. According to another embodiment of the present invention, CTP-modified growth hormone-containing polypeptides are administered at a dose of 1-90 micrograms in 0.1-5 ml of solution by intramuscular (IM) injection, subcutaneous (SC) injection, or intravenous (in /c) injections once every two weeks. According to another embodiment of the present invention, CTP-modified growth hormone-containing polypeptides are administered at a dose of 1-90 micrograms in 0.1-5 ml of solution by intramuscular (IM) injection, subcutaneous (SC) injection, or intravenous (in /c) injections once every 17 days. According to another embodiment of the present invention, CTP-modified growth hormone-containing polypeptides are administered at a dose of 1-90 micrograms in 0.1-5 ml of solution by intramuscular (IM) injection, subcutaneous (SC) injection, or intravenous (in /c) injections once every 19 days. According to one implementation variant of the present invention, the introduction is carried out by intramuscular (IM) injection. According to one implementation variant of the present invention, the introduction is carried out by subcutaneous (SC) injection. According to one implementation variant of the present invention, the introduction is carried out by intravenous (in/in) injection.

[00216] Various dosage ranges are included within the scope of the present invention. The dosage of the polypeptide according to the present invention, in one embodiment of the present invention, is in the range of 0.05-80 mg/day. According to another embodiment of the present invention, the dosage is in the range of 0.05-50 mg/day. According to another embodiment of the present invention, the dosage is in the range of 0.1-20 mg/day. According to another embodiment of the present invention, the dosage is in the range of 0.1-10 mg/day. According to another embodiment of the present invention, the dosage is in the range of 0.1-5 mg/day. According to another embodiment of the present invention, the dosage is in the range of 0.5-5 mg/day. According to another embodiment of the present invention, the dosage is in the range of 0.5-50 mg/day. According to another embodiment of the present invention, the dosage is in the range of 5-80 mg/day. According to another embodiment of the present invention, the dosage is in the range of 35-65 mg/day. According to another embodiment of the present invention, the dosage is in the range of 35-65 mg/day. According to another embodiment of the present invention, the dosage is in the range of 20-60 mg/day. According to another embodiment of the present invention, the dosage is in the range of 40-60 mg/day. According to another embodiment of the present invention, the dosage is in the range of 45-60 mg/day. According to another embodiment of the present invention, the dosage is in the range of 40-60 mg/day. According to another embodiment of the present invention, the dosage is in the range of 60-120 mg/day. According to another embodiment of the present invention, the dosage is in the range of 120-240 mg/day. According to another embodiment of the present invention, the dosage is in the range of 40-60 mg/day. According to another embodiment of the present invention, the dosage is in the range of 240-400 mg/day. According to another embodiment of the present invention, the dosage is in the range of 45-60 mg/day. According to another embodiment of the present invention, the dosage is in the range of 15-25 mg/day. According to another embodiment of the present invention, the dosage is in the range of 5-10 mg/day. According to another embodiment of the present invention, the dosage is in the range of 55-65 mg/day.

[00217] According to one embodiment of the present invention, the dosage is 20 mg/day. According to another embodiment of the present invention, the dosage is 30 mg/day. According to another embodiment of the present invention, the dosage is 40 mg/day. According to another embodiment of the present invention, the dosage is 50 mg/day. According to another embodiment of the present invention, the dosage is 60 mg/day. According to another embodiment of the present invention, the dosage is 70 mg/day. According to another embodiment of the present invention, the dosage is 80 mg/day. According to another embodiment of the present invention, the dosage is 90 mg/day. According to another embodiment of the present invention, the dosage is 100 mg/day.

[00218] The dosage of CTP-modified GH according to the present invention, in one embodiment of the present invention, is in the range of 0.005-100 mg/week. According to another embodiment of the present invention, the dosage is in the range of 0.005-5 mg/week. According to another embodiment of the present invention, the dosage is in the range of 0.01-50 mg/week. According to another embodiment of the present invention, the dosage is in the range of 0.1-20 mg/week. According to another embodiment of the present invention, the dosage is in the range of 0.1-10 mg/week. According to another embodiment of the present invention, the dosage is in the range of 0.01-5 mg/week. According to another embodiment of the present invention, the dosage is in the range of 0.001-0.01 mg/week. According to another embodiment of the present invention, the dosage is in the range of 0.001-0.1 mg/week. According to another embodiment of the present invention, the dosage is in the range of 0.1-5 mg/week. According to another embodiment of the present invention, the dosage is in the range of 0.5-50 mg/week. According to another embodiment of the present invention, the dosage is in the range of 0.2-15 mg/week. According to another embodiment of the present invention, the dosage is in the range of 0.8-65 mg/week. According to another embodiment of the present invention, the dosage is in the range of 1-50 mg/week. According to another embodiment of the present invention, the dosage is in the range of 5-10 mg/week. According to another embodiment of the present invention, the dosage is in the range of 8-15 mg/week. According to another embodiment of the present invention, the dosage is in the range of 10-20 mg/week. According to another embodiment of the present invention, the dosage is in the range of 20-40 mg/week. According to another embodiment of the present invention, the dosage is in the range of 60-120 mg/week. According to another embodiment of the present invention, the dosage is in the range of 12-40 mg/week. According to another embodiment of the present invention, the dosage is in the range of 40-60 mg/week. According to another embodiment of the present invention, the dosage is in the range of 50-100 mg/week. According to another embodiment of the present invention, the dosage is in the range of 1-60 mg/week. According to another embodiment of the present invention, the dosage is in the range of 15-25 mg/week. According to another embodiment of the present invention, the dosage is in the range of 5-10 mg/week. According to another embodiment of the present invention, the dosage is in the range of 55-65 mg/week. According to another embodiment of the present invention, the dosage is in the range of 1-5 mg/week.

[002019] In another embodiment, the dosage of GH administered to a subject is 50% of the standard dosage administered to a control subject from the same population of subjects (e.g., children, elderly patients, men, women, GH-deficient subjects, subjects of a particular nationality, etc.). .d.). According to another implementation variant of the present invention, the dosage is 30% of the dose administered to a subject from a particular population of subjects. According to another embodiment of the present invention, the dosage is 45% of the dose administered to a subject from a particular population of subjects. According to another implementation variant of the present invention, the dosage is 100% of the dose administered to a subject from a particular population of subjects.

[002020] According to another embodiment of the present invention, the dosage is 1-5 mg/week. According to another embodiment of the present invention, the dosage is 2 mg/week. According to another embodiment of the present invention, the dosage is 4 mg/week. According to another embodiment of the present invention, the dosage is 1.2 mg/week. According to another embodiment of the present invention, the dosage is 1.8 mg/week. According to another implementation variant of the present invention, the dosage approximately corresponds to the dosages described in this application.

[00221] According to another embodiment of the present invention, the dosage is 1-5 mg/administration. According to another embodiment of the present invention, the dosage is 2 mg/administration. According to another embodiment of the present invention, the dosage is 4 mg/administration. According to another embodiment of the present invention, the dosage is 1.2 mg/administration. According to another embodiment of the present invention, the dosage is 1.8 mg/administration. According to one embodiment of the present invention, the composition is administered once a week. According to another embodiment of the present invention, the composition is administered once every two weeks. According to another embodiment of the present invention, the composition is administered monthly. According to another embodiment of the present invention, the composition is administered daily.

[00222] According to one embodiment of the present invention, CTP-modified GH is made in the form of a liquid composition.

[00223] According to another embodiment of the present invention, CTP-modified GH is formulated into a dosage form for intranasal administration. According to another embodiment of the present invention, CTP-modified GH is formulated into an injectable dosage form. According to another embodiment of the present invention, CTP-modified GH is administered to a subject at a dose that is in the range of 0.0001 mg to 0.6 mg. According to another embodiment of the present invention, CTP-modified GH is administered to a subject at a dose that is in the range of 0.001 mg to 0.005 mg. According to another embodiment of the present invention, CTP-modified GH is administered to a subject at a dose that is in the range of 0.005 mg to 0.01 mg. According to another embodiment of the present invention, CTP-modified GH is administered to a subject at a dose that is in the range of 0.01 mg to 0.3 mg. According to another embodiment of the present invention, CTP-modified GH is administered to a subject at a dose that is in the range of 0.2 mg to 0.6 mg.

[00224] According to another embodiment of the present invention, CTP-modified GH is administered to a subject at a dose that is in the range of 1 to 100 μg. According to another embodiment of the present invention, CTP-modified GH is administered to a subject at a dose that is in the range of 10 to 80 micrograms. According to another embodiment of the present invention, CTP-modified GH is administered to a subject at a dose that is in the range of 20 to 60 micrograms. According to another embodiment of the present invention, CTP-modified GH is administered to a subject at a dose that is in the range of 10 to 50 micrograms. According to another embodiment of the present invention, CTP-modified GH is administered to a subject at a dose that is in the range of 40 to 80 micrograms. According to another embodiment of the present invention, CTP-modified GH is administered to a subject at a dose that is in the range of 10 to 30 micrograms. According to another embodiment of the present invention, CTP-modified GH is administered to a subject at a dose that is in the range of 30 to 60 micrograms.

[00225] According to another embodiment of the present invention, CTP-modified GH is administered to a subject at a dose that is in the range of 0.2 mg to 2 mg. According to another embodiment of the present invention, CTP-modified GH is administered to a subject at a dose that is in the range of 2 mg to 6 mg. According to another embodiment of the present invention, CTP-modified GH is administered to a subject at a dose that is in the range of 4 to 10 mg. According to another embodiment of the present invention, CTP-modified GH is administered to a subject at a dose that is between 5 mg and 15 mg.

[00226] According to another embodiment of the present invention, CTP-modified GH is administered by injection into a muscle (intramuscular injection). According to another embodiment of the present invention, CTP-modified GH is administered by injection under the skin (subcutaneous injection). According to another embodiment of the present invention, CTP-modified GH is administered by injection into a muscle. According to another embodiment of the present invention, CTP-modified GH is administered by injection under the skin.

[00227] According to another embodiment of the present invention, the methods of the present invention include increasing patient adherence to a GH treatment regimen, including providing a subject in need thereof with CTP-modified GH, thereby increasing patient adherence to a growth hormone treatment regimen.

[00228] According to another embodiment of the present invention, protein drugs with a molecular weight of less than 50,000 daltons, such as CTP-modified GH, according to the present invention, are generally short-lived molecules in vivo with a short half-life of a few hours. According to another implementation variant of the present invention, the subcutaneous route of administration generally provides a slower release into the bloodstream. According to another implementation variant of the present invention, the CTP-modified polypeptide according to the present invention increases the half-life of protein drugs with a molecular weight of less than 50,000 daltons, such as growth hormone. According to another implementation variant of the present invention, the CTP-modified polypeptide according to the present invention allows interferons to have a beneficial effect over a longer period of time.

[00229] According to another embodiment of the present invention, the immunogenicity of a CTP-modified polypeptide containing CTP-modified growth hormone corresponds to that of isolated growth hormone. According to another embodiment of the present invention, the immunogenicity of a CTP-modified polypeptide containing CTP-modified growth hormone is comparable to that of isolated growth hormone. According to another embodiment of the present invention, the modification of GH described in this application using CTP peptides reduces the immunogenicity of GH. According to another embodiment of the present invention, the activity of the CTP-modified polypeptide containing growth hormone corresponds to the activity of the isolated GH protein. According to another embodiment of the present invention, the CTP-modified growth hormone-containing polypeptide is more active than isolated growth hormone. According to another embodiment of the present invention, the CTP-modified polypeptide containing growth hormone maximizes the protective ability of growth hormone against degradation and minimally reduces its biological activity.

[00230] According to another embodiment of the present invention, the methods of the present invention include increasing adherence to the requirements of a treatment regimen in subjects suffering from chronic diseases who are in need of GH therapy. According to another implementation variant of the present invention, the methods according to the present invention reduce the frequency of GH dosing by modifying GH using CTP, as described above. According to another implementation variant of the present invention, the term "adherence" includes compliance with the prescribed regimen. According to another embodiment of the present invention, the methods of the present invention include increasing the adherence of patients in need of GH therapy by reducing the frequency of GH administration. According to another embodiment of the present invention, the reduction in the frequency of GH administration is achieved by modifications of CTP, which increase the stability of the CTP-modified GH. According to another embodiment of the present invention, a decrease in the frequency of GH administration is achieved as a result of an increase in T _1/2 of growth hormone. According to another embodiment of the present invention, a decrease in the frequency of GH administration is achieved by increasing the GH clearance time. According to another implementation variant of the present invention, the reduction in the frequency of administration of growth hormone is achieved by increasing the AUC value of growth hormone.

[00231] In another embodiment, the present invention provides a method for reducing body fat in a non-human subject, comprising administering to said subject a therapeutically effective amount of an expression vector comprising a polynucleotide, said polynucleotide consisting of a non-human growth hormone, one carboxy- end peptide (CTP) of chorionic gonadotropin attached to the amino end of the specified non-human growth hormone, as well as two CTPs of chorionic gonadotropin attached to the carboxy-terminus of the specified non-human growth hormone, while the specified polypeptide additionally contains a signal peptide attached to the amino end of the specified one CTP, thereby inducing height or weight gain in a non-human subject.

[00232] According to another embodiment, the present invention provides a method of increasing insulin-like growth factor (IGF-1) levels in a human subject, comprising administering to said subject a therapeutically effective amount of a polypeptide containing growth hormone, one carboxy-terminal peptide (CTP) of human chorionic gonadotropin , attached to the amino terminus of said growth hormone, and two human chorionic gonadotropin CTPs attached to the carboxy terminus of said growth hormone, thereby increasing IGF-1 levels in said subject.

[00233] In another embodiment, the present invention provides a method for increasing insulin-like growth factor (IGF-1) levels in a non-human subject, comprising administering to said subject a therapeutically effective amount of an expression vector comprising a polynucleotide, said polynucleotide consisting of a non-human hormone growth, one carboxy-terminal peptide (CTP) of the chorionic gonadotropin attached to the amino-terminus of the specified non-human growth hormone, and two CTPs of the chorionic gonadotropin attached to the carboxy-terminus of the specified non-human growth hormone, while the specified polypeptide optionally contains a signal peptide attached to the amino terminus of said one CTP, thereby inducing height or weight gain in a non-human subject.

[00234] According to one embodiment of the present invention, an increase in IGF-1 levels in a human subject may be effective in the treatment, prevention or suppression of type 1 diabetes mellitus, type 2 diabetes mellitus, amyotrophic lateral sclerosis (ALS, also known as "Lou Gehrig's disease ”), severe burn injury and myotonic muscular dystrophy (MMD).

[00235] According to another embodiment of the present invention, CTP-modified GH is administered to the subject once a day. According to another embodiment of the present invention, a CTP-modified GH-containing polypeptide is administered to a subject once every two days. According to another embodiment of the present invention, CTP-modified GH is administered to the subject once every three days. According to another embodiment of the present invention, CTP-modified GH is administered to the subject once every four days. According to another embodiment of the present invention, CTP-modified GH is administered to the subject once every five days. According to another embodiment of the present invention, CTP-modified GH is administered to the subject once every six days. According to another embodiment of the present invention, CTP-modified GH is administered to the subject once a week. According to another embodiment of the present invention, CTP-modified GH is administered to the subject once every 7-14 days. According to another embodiment of the present invention, CTP-modified GH is administered to the subject once every 10-20 days. According to another embodiment of the present invention, CTP-modified GH is administered to the subject once every 5-15 days. According to another embodiment of the present invention, CTP-modified GH is administered to the subject once every 15-30 days.

[00236] According to another embodiment of the present invention, the dosage is in the range of 50-500 mg/day. According to another embodiment of the present invention, the dosage is in the range of 50-150 mg/day. According to another embodiment of the present invention, the dosage is in the range of 100-200 mg/day. According to another embodiment of the present invention, the dosage is in the range of 150-250 mg/day. According to another embodiment of the present invention, the dosage is in the range of 200-300 mg/day. According to another embodiment of the present invention, the dosage is in the range of 250-400 mg/day. According to another embodiment of the present invention, the dosage is in the range of 300-500 mg/day. According to another embodiment of the present invention, the dosage is in the range of 350-500 mg/day.

[00237] According to one embodiment of the present invention, the dosage is 20 mg/day. According to one embodiment of the present invention, the dosage is 30 mg/day. According to one embodiment of the present invention, the dosage is 40 mg/day. According to one embodiment of the present invention, the dosage is 50 mg/day. According to one embodiment of the present invention, the dosage is 0.01 mg/day. According to another embodiment of the present invention, the dosage is 0.1 mg/day. According to another embodiment of the present invention, the dosage is 1 mg/day. According to another embodiment of the present invention, the dosage is 0.530 mg/day. According to another embodiment of the present invention, the dosage is 0.05 mg/day. According to another embodiment of the present invention, the dosage is 50 mg/day. According to another embodiment of the present invention, the dosage is 10 mg/day. According to another embodiment of the present invention, the dosage is 20-70 mg/day. According to another embodiment of the present invention, the dosage is 5 mg/day.

[00238] According to another embodiment of the present invention, the dosage is 1-90 mg/day. According to another embodiment of the present invention, the dosage is 1-90 mg/2 days. According to another embodiment of the present invention, the dosage is 1-90 mg/3 days. According to another embodiment of the present invention, the dosage is 1-90 mg/4 days. According to another embodiment of the present invention, the dosage is 1-90 mg/5 days. According to another embodiment of the present invention, the dosage is 1-90 mg/6 days. According to another embodiment of the present invention, the dosage is 1-90 mg/week. According to another embodiment of the present invention, the dosage is 1-90 mg/9 days. According to another embodiment of the present invention, the dosage is 1-90 mg/11 days. According to another embodiment of the present invention, the dosage is 1-90 mg/14 days.

[00239] According to another embodiment of the present invention, the dosage of growth hormone is 10-50 mg/day. According to another embodiment of the present invention, the dosage is 10-50 mg/2 days. According to another embodiment of the present invention, the dosage is 10-50 mg/3 days. According to another embodiment of the present invention, the dosage is 10-50 mg/4 days. According to another embodiment of the present invention, the dosage is 10-50 mg/5 days. According to another embodiment of the present invention, the dosage is 10-50 mg/6 days. According to another embodiment of the present invention, the dosage is 10-50 mg/week. According to another embodiment of the present invention, the dosage is 10-50 mg/9 days. According to another embodiment of the present invention, the dosage is 10-50 mg/11 days. According to another embodiment of the present invention, the dosage is 10-50 mg/14 days.

[00240] Oral administration, according to one embodiment of the present invention, includes a unit dosage form including tablets, capsules, lozenges, chewable tablets, suspensions, emulsions, and the like. The listed unit dosage forms contain a safe and effective amount of the desired compound or compounds, each of which, according to one embodiment of the present invention, is from about 0.7 or 3.5 mg to about 280 mg/70 kg, or, in another embodiment, from about 0.5 or 10 mg up to approximately 210 mg/70 kg. Pharmaceutically acceptable carriers suitable for oral unit dosage forms are well known in the art. According to some variants of implementation of the present invention, tablets, as a rule, contain standard pharmaceutically compatible adjuvants as inert diluents, such as calcium carbonate, sodium carbonate, mannitol, lactose and cellulose; binders such as starch, gelatin and sucrose; disintegrants such as starch, alginic acid and croscarmellose; lubricants such as magnesium stearate, stearic acid and talc. In one embodiment of the present invention, slip agents such as silica can be used to improve the flow characteristics of the powder mixture. According to one embodiment of the present invention, dyes such as FD&C dyes may be added to improve appearance. Sweeteners and flavors such as aspartame, saccharin, menthol, peppermint and fruit flavors can be used as adjuvants for chewable tablets. Capsules typically contain one or more of the solid diluents disclosed above. According to some embodiments of the present invention, the choice of carriers depends on minor factors such as taste, cost, and storage stability, which are not critical for the purposes of the present invention, and can be easily made by a person skilled in the art.

[00241] According to one embodiment of the present invention, the oral dosage form has a predetermined release profile. According to one implementation variant of the present invention dosage form for oral administration according to the present invention includes tablets, capsules, lozenges or chewable tablets with prolonged release. According to one implementation variant of the present invention, the dosage form for oral administration according to the present invention includes tablets, capsules, lozenges or chewable tablets with slow release. According to one embodiment of the present invention, the oral dosage form of the present invention comprises immediate release tablets, capsules, lozenges or chewable tablets. According to one implementation variant of the present invention, the dosage form for oral administration is made in accordance with the desired release profile of the pharmaceutical active ingredient known to the person skilled in the art.

[00242] Compositions for oral administration, in some embodiments, include liquid solutions, emulsions, suspensions, and the like. According to some variants of implementation of the present invention, pharmaceutically acceptable carriers suitable for obtaining these compositions are well known in the art. According to some embodiments of the present invention, liquid compositions for oral administration contain from about 0.012% to about 0.933% of the desired compound or compounds, or, in another embodiment of the present invention, from about 0.033% to about 0.7%.

[00243] According to some embodiments of the present invention, compositions for use in the methods of the present invention include solutions or emulsions, which, in some embodiments, are aqueous solutions or emulsions, comprising a safe and effective amount of the compounds of the present invention and optionally other compounds, intended for local intranasal administration. According to some embodiments of the present invention, the compositions contain from about 0.01% to about 10.0%, wt./about. compounds according to the present invention, more preferably from about 0.1% to about 2.0%, which is used for systemic delivery of compounds by the intranasal route.

[00244] According to another embodiment of the present invention, the pharmaceutical compositions are administered by intravenous, intra-arterial, or intramuscular injection of a liquid preparation. In some embodiments of the present invention, liquid formulations include solutions, suspensions, dispersions, emulsions, oils, and the like. According to one embodiment of the present invention, the pharmaceutical compositions are administered intravenously, and for this purpose they are made in a form suitable for intravenous administration. According to another embodiment of the present invention, the pharmaceutical compositions are administered intra-arterially, and for this purpose they are prepared in a form suitable for intra-arterial administration. According to another embodiment of the present invention, the pharmaceutical compositions are administered intramuscularly, and for this purpose they are made in a form suitable for intramuscular administration.

[00245] According to another embodiment of the present invention, pharmaceutical compositions are applied topically to the surface of the body, and for this purpose they are made in a form suitable for topical administration. Suitable topical formulations include gels, ointments, creams, lotions, drops, and the like. For topical administration, the compounds of the present invention are combined with an additional appropriate therapeutic agent or agents, prepared and applied as solutions, suspensions or emulsions in a physiologically acceptable diluent with or without the addition of a pharmaceutically acceptable carrier.

[00246] According to one embodiment of the present invention, the pharmaceutical compositions of the present invention are made using methods well known in the art, for example, using standard methods of mixing, dissolving, granulating, making pills, triturating, emulsifying, encapsulating, embedding or lyophilization.

[00247] According to one embodiment of the present invention, pharmaceutical compositions for use in accordance with the present invention are manufactured in the usual way using one or more physiologically acceptable carriers containing excipients and excipients that facilitate processing of the active ingredients into preparations that can be used for pharmaceutical purposes. goals. According to one implementation variant of the present invention, the composition depends on the chosen route of administration.

[00248] According to one embodiment of the present invention, the injectable compositions of the present invention are formulated as aqueous solutions. According to one embodiment of the present invention, the injectable compositions of the present invention are formulated in physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological saline buffer. In some embodiments of the present invention, transmucosal formulations contain penetrants appropriate to the type of barrier to be overcome. Such penetrants are generally known in the art.

[00249] According to one implementation variant of the present invention, the preparations described in this application are made for parenteral administration, for example, by bolus injection or continuous infusion. According to some embodiments of the present invention, injectable formulations are provided in unit dosage form, eg, in ampoules or in multi-dose containers, optionally with an added preservative. In some embodiments of the present invention, the compositions are suspensions, solutions or emulsions in oily or aqueous vehicles and contain adjuvants such as suspending, stabilizing and/or dispersing agents.

[00250] The compositions also contain, in some embodiments, preservatives such as benzalkonium chloride, thimerosal, and the like; chelating agents such as sodium edetate and others; buffers, for example based on phosphate, citrate and acetate; tonicity agents such as sodium chloride, potassium chloride, glycerol, mannitol and others; antioxidants such as ascorbic acid, acetylcysteine, sodium metabisulphite and others; aromatic agents; viscosity regulators such as polymers, including cellulose and its derivatives; and polyvinyl alcohol and acids and bases to adjust the pH of these aqueous compositions, if necessary. The compositions also contain, in some embodiments, local anesthetics or other active agents. The compositions can be used as sprays, aerosols, drops and the like.

[00251] According to some variants of implementation of the present invention, pharmaceutical compositions for parenteral administration include aqueous solutions of the active drug in water-soluble form. In addition, suspensions of the active ingredients, in some embodiments, are prepared as suitable oily or aqueous injectable suspensions. Suitable lipophilic solvents or carriers include, in some embodiments, fatty oils such as sesame oil, or synthetic fatty acid esters such as ethyl oleate, triglycerides, or liposomes. Aqueous injection suspensions contain, in some embodiments, substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. According to another embodiment of the present invention, the suspension also contains suitable stabilizers or agents that increase the solubility of the active ingredients in order to provide highly concentrated solutions.

[00252] According to another embodiment of the present invention, the active compound can be delivered in a vesicle, in particular in a liposome (see Langer, Science 249:1527-1533 (1990); Treat et al., in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353-365 (1989); Lopez-Berestein, ibid., pp. 317-327; see mostly ibid.).

[00253] According to another embodiment of the present invention, a pharmaceutical composition delivered using a controlled release system is formulated for administration by intravenous infusion, implantable osmotic pump, transdermal patch, syringe, liposomes, or other modes of administration. According to one embodiment of the present invention, a pump is used (see Langer, supra; Sefton, CRC Crit. Ref. Biomed. Eng. 14:201 (1987); Buchwald et al., Surgery 88:507 (1980); Saudek et al. , N. Engl. J. Med. 321:574 (1989) According to another embodiment of the present invention, polymeric materials can be used.In another embodiment, the controlled release system can be placed in close proximity to the therapeutic target, i. into the brain, allowing only a fraction of the systemic dose to be administered (see, for example, Goodson, in Medical Applications of Controlled Release , supra, vol. 2, pp. 115-138 (1984). Other controlled release systems are discussed in the Langer review ( Science 249: 1527-1533 (1990).

[00254] According to some embodiments of the present invention, the active ingredient is in the form of a powder for reconstitution with a suitable vehicle, for example, a sterile pyrogen-free aqueous solution immediately prior to use. Compositions are made, in some embodiments, for administration by nebulization and inhalation. According to another embodiment of the present invention, the compositions are contained in a container with spray means attached.

[00255] According to one embodiment of the present invention, the formulation of the present invention is formulated into rectal compositions such as suppositories or retention enemas using, for example, conventional suppository bases such as cocoa butter or other glycerides.

[00256] According to one implementation variant of the present invention, the preparation according to the present invention is made in the form of liquid formulations for injection using a syringe or pen. According to some variants of implementation of the present invention, pharmaceutical compositions suitable for use in accordance with the methods disclosed in the present invention include compositions in which the active ingredients are contained in an amount effective to achieve the intended purpose. According to some embodiments of the present invention, a therapeutically effective amount means an amount of active ingredients that is effective to prevent, alleviate, or ameliorate the symptoms of a disease or prolong the life of the subject being treated.

[00257] According to one embodiment of the present invention, determining a therapeutically effective amount is within the skill of the art.

[00258] According to one implementation variant of the present invention, the compositions proposed in this application also contain preservatives such as benzalkonium chloride, thimerosal and the like; chelating agents such as sodium edetate and others; buffers, for example based on phosphate, citrate and acetate; tonicity agents such as sodium chloride, potassium chloride, glycerol, mannitol and others; antioxidants such as ascorbic acid, acetylcysteine, sodium metabisulphite and others; aromatic agents; viscosity regulators such as polymers, including cellulose and its derivatives; and polyvinyl alcohol, as well as acids and bases to adjust the pH of these aqueous compositions, if necessary. The compositions also contain local anesthetics and other active substances. The compositions can be used as sprays, aerosols, drops and the like.

[00259] Some examples of substances that can serve as pharmaceutically acceptable carriers or components thereof include sugars such as lactose, glucose and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethylcellulose, ethylcellulose and methylcellulose; powdered tragacanth; malt; gelatin; talc; solid lubricants such as stearic acid and magnesium stearate; calcium sulfate; vegetable oils such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil and cocoa butter; polyols such as propylene glycol, glycerin, sorbitol, mannitol and polyethylene glycol; alginic acid; emulsifiers such as Tween™ emulsifiers; wetting agents such as sodium lauryl sulfate; dyes; flavors; tableting agents, stabilizers; antioxidants; preservatives; pyrogen-free water; isotonic saline; and phosphate buffer solutions. The choice of a pharmaceutically acceptable carrier for use in combination with a compound is largely determined by the route by which the compound is to be administered. When the compound of the present invention is to be administered by injection, in one embodiment, the pharmaceutically acceptable carrier is sterile physiological saline containing a blood-compatible suspending agent that has been adjusted to a pH of about 7.4.

[00260] In addition, the compositions additionally contain binders (for example, gum arabic, corn starch, gelatin, carbomer, ethyl cellulose, guar gum, hydroxypropyl cellulose, hydroxypropyl methylcellulose, povidone), disintegrating agents (for example, corn starch, potato starch, alginic acid, silicon dioxide , croscarmellose sodium, crospovidone, guar gum, sodium starch glycolate), buffers (e.g. Tris-HCl, acetate, phosphate) having different pH and ionic strengths, additives such as albumin or gelatin to prevent absorption on surfaces, detergents (e.g. , tween-20, tween-80, pluronic acid F68, bile salts), protease inhibitors, surfactants (eg, sodium lauryl sulfate), penetration enhancers, solubilizing agents (eg, glycerol, polyethylene glycol glycerol), antioxidants (eg, ascorbic acid, sodium metabisulfite, butylated hydroxyanisole), stabilizers (e.g. hydroxypropyl cellulose, hydroxypropyl methylcellulose), viscosity increasing agents (e.g. carbomer, colloidal silicon dioxide, ethyl cellulose, guar gum), sweeteners (e.g. aspartame, citric acid), preservatives (e.g. , thimerosal, benzyl alcohol, parabens), lubricants (e.g. stearic acid, magnesium stearate, polyethylene glycol, sodium lauryl sulfate), slip agents (e.g. colloidal silicon dioxide), plasticizers (e.g. diethyl phthalate, triethyl citrate), emulsifiers (e.g. carbomer, hydroxypropylcellulose, sodium lauryl sulfate), polymeric coatings (eg poloxamers or poloxamines), coatings and film forming agents (eg ethylcellulose, acrylates, polymethacrylates) and/or adjuvants.

[00261] Typical carrier components for syrups, elixirs, emulsions, and suspensions include ethanol, glycerin, propylene glycol, polyethylene glycol, liquid sucrose, sorbitol, and water. Typical suspending agents to form a suspension include methylcellulose, sodium carboxymethylcellulose, cellulose (eg Avicel™, RC-591), tragacanth, and sodium alginate; typical wetting agents include lecithin and polyethylene oxide sorbitol (eg polysorbate 80). Typical preservatives include methyl paraben and sodium benzoate. According to another implementation variant of the present invention, liquid compositions for oral administration also contain one or more components, such as sweeteners, flavors and colors, described above.

[00262] the Formulations proposed in this application also include the incorporation of the active material into or on the surface of particulate preparations of polymeric compounds, such as polylactic acid, polyglycolic acid, hydrogels, etc., or on the surface of liposomes, microemulsions, micelles, single-layer or multilayered vesicles, shadows of erythrocytes or spheroplasts. Such compositions will affect the physical state, solubility, stability, in vivo release rate and in vivo clearance rate.

[00263] Also included within the scope of the present invention are particulate compositions coated with polymers (eg, poloxamers or poloxamines) and a compound associated with antibodies directed against tissue-specific receptors, ligands, or antigens, or associated with tissue-specific receptor ligands.

[00264] In some embodiments, the present invention provides compounds modified by covalent attachment of water-soluble polymers such as polyethylene glycol, polyethylene glycol-polypropylene glycol copolymers, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, or polyproline. According to another embodiment of the present invention, the modified compounds have a significantly longer half-life after intravenous injection than the corresponding unmodified compounds. According to one embodiment of the present invention, the modifications also increase the solubility of the compound in aqueous solution, eliminate aggregation, enhance the physical and chemical stability of the compound, and significantly reduce the immunogenicity and reactivity of the compound. According to another embodiment of the present invention, the desired in vivo biological activity is achieved by administering such polymer-compound adducts at a lower frequency or in lower doses than the unmodified compound.

[00265] According to some embodiments of the present invention, obtaining an effective amount or dose can be initially estimated based on the results of quantitative in vitro studies. According to one implementation variant of the present invention, the dose can be determined in animal models, and such information can be used to more accurately determine the appropriate doses in humans.

[00266] According to one implementation variant of the present invention, the toxicity and therapeutic efficacy of the active ingredients described in this application can be determined using standard pharmaceutical techniques in vitro , in cell cultures or in experimental animals. According to one embodiment of the present invention, data obtained from quantitative studies in vitro and using cell cultures, as well as studies on animals, can be used in determining the range of doses for use in humans. According to one implementation variant of the present invention, the doses vary depending on the dosage form used and the route of administration. According to one implementation variant of the present invention, the exact composition, route of administration and dosage can be chosen by the attending physician, taking into account the condition of the patient. [See, for example, Fingl, et al., (1975) "The Pharmacological Basis of Therapeutics", Ch. 1 p.1].

[00267] According to one embodiment of the present invention, depending on the severity and susceptibility of the condition being treated, the dose can be administered with one or more injections, while the course of treatment can last from several days to several weeks or until recovery, or until reduction the severity of the pathological condition.

[00268] According to one embodiment of the present invention, the amount of composition or composition to be administered will obviously depend on the subject being treated, the severity of the disease, the route of administration, the judgment of the attending physician, and so on.

[00269] According to one implementation variant of the present invention, the compositions, including the preparation according to the present invention, made in a compatible pharmaceutical carrier, must also be obtained, placed in an appropriate container and labeled for the treatment of the indicated condition.

[00270] According to another embodiment of the present invention, CTP-modified GH is administered systemically. According to another embodiment of the present invention, the growth hormone described in this application is administered by intravenous, intramuscular or subcutaneous injection. According to another embodiment of the present invention, CTP-modified GH is a lyophilized (i.e., freeze-dried) preparation in combination with complex organic excipients and stabilizers such as non-ionic surfactants (i.e., surfactants) , various sugars, organic polyols and/or human serum albumin. According to another implementation variant of the present invention, the pharmaceutical composition contains lyophilized growth hormone modified with CTP, described in this application, in sterile water for injection. According to another embodiment of the present invention, the pharmaceutical composition comprises the lyophilized growth hormone described herein in sterile phosphate-buffered saline (PBS) for injection. According to another implementation variant of the present invention, the pharmaceutical composition contains lyophilized growth hormone described in this application, in a sterile 0.9% NaCl solution for injection.

[00271] According to another embodiment of the present invention, a pharmaceutical composition containing CTP-modified GH described in this application is also made with the addition of complex carriers, such as human serum albumin, polyols, sugars, and anionic surface-active stabilizing agents. See, for example, WO 89/10756 (Hara et al., containing polyol and p-hydroxybenzoate). According to another embodiment of the present invention, the pharmaceutical composition contains the growth hormone described in this application, and is also made with the addition of lactobionic acid and an acetate/glycine buffer. According to another embodiment of the present invention, a pharmaceutical composition containing CTP-modified growth hormone described in this application is also made with the addition of amino acids, such as arginine or glutamate, which increase the solubility of interferon compositions in water. According to another embodiment of the present invention, the pharmaceutical composition contains lyophilized growth hormone modified with CTP described in this application, and is also made with the addition of glycine or human serum albumin (HSA), a buffer (for example, acetate) and an isotonic agent (for example, NaCl). According to another embodiment of the present invention, the pharmaceutical composition contains lyophilized growth hormone modified with CTP described in this application, and is also made with the addition of phosphate buffer, glycine and HSA.

[00272] According to another implementation variant of the present invention, a pharmaceutical composition containing growth hormone modified with CTP, described in this application, is stable when introduced into buffered solutions having a pH in the range from about 4 to 7.2. According to another implementation variant of the present invention, a pharmaceutical composition containing CTP-modified GH described in this application is stable when introduced into buffered solutions having a pH in the range from about 6 to 6.4. According to another embodiment of the present invention, a pharmaceutical composition containing CTP-modified GH described in this application is stable when added to buffered solutions having a pH of 6.0. According to another embodiment of the present invention, a pharmaceutical composition containing CTP-modified GH described in this application is stable when added to buffered solutions having a pH of 6.2. According to another embodiment of the present invention, a pharmaceutical composition containing CTP-modified GH described in this application is stable when added to buffered solutions having a pH of 6.4. According to another embodiment of the present invention, a pharmaceutical composition containing CTP-modified GH described in this application is stabilized with an amino acid as a stabilizing agent and, in some cases, with a salt (if the amino acid does not contain a charged side chain). According to one embodiment of the present invention, the pharmaceutical composition is stable at room temperature. According to another embodiment of the present invention, the pharmaceutical composition is stable at 4°C. According to another embodiment of the present invention, the pharmaceutical composition is stable at 5°C. According to another embodiment of the present invention, the pharmaceutical composition is stable at -20°C. According to one embodiment of the present invention, the pharmaceutical composition is stable for at least three months. According to one embodiment of the present invention, the pharmaceutical composition is stable for at least six months. According to one embodiment of the present invention, the pharmaceutical composition is stable for at least one year. According to one embodiment of the present invention, the pharmaceutical composition is stable for at least two years.

[00273] According to another embodiment of the present invention, a pharmaceutical composition containing CTP-modified growth hormone described in this application is made in the form of a liquid composition containing a stabilizing agent in an amount of from about 0.3% to 5% by weight, which is an amino acid .

[00274] According to another embodiment of the present invention, a pharmaceutical composition containing CTP-modified GH described in this application provides dosing accuracy and product safety. According to another implementation variant of the present invention, a pharmaceutical composition containing CTP-modified GH described in this application provides a biologically active, stable liquid composition for use by injection. According to another embodiment of the present invention, the pharmaceutical composition contains non-lyophilized GH modified with CTP described in this application.

[00275] According to another embodiment of the present invention, a pharmaceutical composition containing CTP-modified GH described in this application provides a liquid formulation that can be stored for an extended period of time in a liquid state, thereby facilitating storage and transportation prior to administration.

[00276] According to another embodiment of the present invention, a pharmaceutical composition containing CTP-modified GH described in this application contains solid lipids as a matrix material. According to another embodiment of the present invention, the injectable pharmaceutical composition containing CTP-modified growth hormone described in this application contains solid lipids as a matrix material. According to another embodiment of the present invention, the production of lipid microparticles by spray freezing has been described by Speiser (Speiser et al., Pharm. Res. 8 (1991) 47-54), followed by the production of lipid nanoprecipitates for oral administration (Speiser EP 0167825 (1990 )). According to another embodiment of the present invention, the lipids used are well tolerated by the body (for example, glycerides consisting of fatty acids that are present in emulsions for parenteral nutrition).

[00277] According to another embodiment of the present invention, a pharmaceutical composition containing CTP-modified GH described in this application is provided in the form of liposomes (J. E. Diederichs and al., Pharm./nd. 56 (1994) 267-275).

[00278] According to another embodiment of the present invention, a pharmaceutical composition containing CTP-modified GH described in this application contains polymeric microparticles. According to another implementation variant of the present invention, the pharmaceutical composition for injection containing growth hormone modified CTP described in this application contains polymeric microparticles. According to another implementation variant of the present invention, a pharmaceutical composition containing growth hormone modified with CTP, described in this application, contains nanoparticles. According to another embodiment of the present invention, a pharmaceutical composition containing CTP-modified GH described in this application contains liposomes. According to another embodiment of the present invention, a pharmaceutical composition containing CTP-modified growth hormone described in this application contains a lipid emulsion. According to another implementation variant of the present invention, a pharmaceutical composition containing growth hormone modified with CTP, described in this application, contains microspheres. According to another embodiment of the present invention, a pharmaceutical composition containing CTP-modified GH described in this application contains lipid nanoparticles. According to another embodiment of the present invention, a pharmaceutical composition containing CTP-modified GH described in this application contains lipid nanoparticles containing amphiphilic lipids. According to another embodiment of the present invention, a pharmaceutical composition containing CTP-modified GH described in this application contains lipid nanoparticles containing a drug, a lipid matrix and a surfactant. According to another implementation variant of the present invention, the content of monoglycerides in the lipid matrix is at least 50% wt./mass.

[00279] According to one embodiment of the present invention, the compositions of the present invention are provided in a package or dispenser device, such as a FDA-approved kit, that contains one or more unit dosage forms containing the active ingredient. According to one implementation variant of the present invention, the package, for example, may contain a metal or plastic foil, such as a blister pack. According to one embodiment of the present invention, the package or dispenser is accompanied by instructions for administration. According to one embodiment of the present invention, the package or dispenser is accompanied by a notice associated with the container in the form prescribed by a government agency regulating the manufacture, use, or sale of pharmaceuticals, the notice reflecting that agency's approval of the form of said compositions for human or animal administration. Such notice, in one embodiment, is a US Food and Drug Administration-approved label for prescription drugs or an approved package leaflet.

[00280] According to one embodiment of the present invention, it should be understood that CTP-modified GH according to the present invention can be offered to an individual with additional active agents to achieve an improved therapeutic effect compared to treatment with each agent alone. According to another implementation variant of the present invention, in order to minimize the number of undesirable side effects that are associated with combination therapy, certain measures are taken (for example, dosing and selection of an additional agent).

[00281] Additional objects, advantages and novel features of the present invention will become apparent to those skilled in the art upon examination of the following non-limiting examples. In addition, each of the various embodiments and aspects of the present invention described above and claimed in the claims, finds experimental confirmation in the following examples.

EXAMPLES

[00282] In general, the nomenclature used in this application and the laboratory techniques used in the present invention include molecular biology, biochemistry and microbiology techniques, as well as recombinant DNA techniques. Such techniques are described in detail in the literature. See, for example, "Molecular Cloning: A laboratory Manual" Sambrook et al., (1989); "Current Protocols in Molecular Biology" Volumes I-III Ausubel, R. M., ed. (1994); Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Maryland (1989); Perbal, "A Practical Guide to Molecular Cloning", John Wiley & Sons, New York (1988); Watson et al., "Recombinant DNA", Scientific American Books, New York; Birren et al. (eds) "Genome Analysis: A Laboratory Manual Series", Vols. 1-4, Cold Spring Harbor Laboratory Press, New York (1998); the techniques described in US Pat. Nos. 4,666,828; 4683202; 4801531; 5192659 and 5272057; "Cell Biology: A Laboratory Handbook," Volumes I-III Cellis, J. E., ed. (1994); "Culture of Animal Cells - A Manual of Basic Technique" by Freshney, Wiley-Liss, N. Y. (1994), Third Edition; "Current Protocols in Immunology" Volumes I-III Coligan J. E., ed. (1994); Stites et al. (eds), "Basic and Clinical Immunology" (8th Edition), Appleton & Lange, Norwalk, CT (1994); Mishell and Shiigi (eds), "Selected Methods in Cellular Immunology", W. H. Freeman and Co., New York (1980); available immunological quantitative research methods are described in detail in patents and specialized literature, see, for example, US patent No. 3791932; 3839153; 3850752; 3850578; 3853987; 3867517; 3879262; 3901654; 3935074; 3984533; 3996345; 4034074; 4098876; 4879219; 5011771 and 5281521; "Oligonucleotide Synthesis" Gait, M. J., ed. (1984); "Nucleic Acid Hybridization" Hames, B. D., and Higgins S. J., eds. (1985); "Transcription and Translation" Hames, B. D., and Higgins S. J., eds. (1984); "Animal Cell Culture" Freshney, R.I., ed. (1986); "Immobilized Cells and Enzymes" IRL Press, (1986); "A Practical Guide to Molecular Cloning" Perbal, B., (1984) and "Methods in Enzymology" Vol. 1-317, Academic Press; "PCR Protocols: A Guide To Methods And Applications", Academic Press, San Diego, CA (1990); Marshak et al., "Strategies for Protein Purification and Characterization - A Laboratory Course Manual" CSHL Press (1996); all of which are incorporated herein by reference. Other general references are given in the text of this document.

EXAMPLE 1

Obtaining human growth hormone constructs

MATERIALS AND METHODS

[00283] Four human GH clones (20 kD protein variants) were synthesized. XbaI-NotI fragments containing human GH sequences from four variants were ligated into the pCI-dhfr eukaryotic expression vector, previously cleaved with XbaI and NotI restriction enzymes. Received DNA from 4 clones (401-0, 1, 2, 3 and 4). Another partial human GH clone (bp 1-242) was also synthesized from a 22 kDa protein (0606114). Primers were ordered from Sigma-Genosys. The primer sequences used to generate the human GH-CTP polypeptides of the present invention are shown in Table 1 below.

Table 1 Primer number SEQID NO Subsequence Restriction site (underlined in sequence) 25 27 5' CTCTAGAGGACATGGCCAC 3' XbaI 32 ^R 28 5' ACAGGGAGGTCTGGGGGTTCTGCA 3' 33 29 5' TGCAGAACCCCCAGACCTCCCTGTGTC 3' 4 ^R thirty 5' CCAAACTCATCAATGTATCTA 3' 25 31 5' CTCTAGAGGACATGGCCAC 3' XbaI ^35R 32 5' CGAACTCCTGGTAGGTGTCAAAGGC 3' 34 33 5' GCCTTTGACACCTACCAGGAGTTCG 3' 37 ^R 34 5' ACCGCGGCCGCATCCAGACCTTCATCACTGAGGC 3' notI 39 ^R 35 5' GCGGCCGCGGACTCATCAGAAGCCGCAGCTGCCC 3'

[00284] Construct 402-0-p69-1 (Human GH) SEQ ID NO: 36: MOD-4020 is wild-type recombinant human growth hormone (without CTP) that was generated for use as a control in the experiments described below.

[00285] Three PCR reactions were performed. The first reaction was performed using primers 25 and 32 ^R and plasmid DNA 0606114 (partial human GH clone containing 1-242 bp) as a template; as a result of PCR amplification, a product containing 245 bp was obtained.

[00286] The second reaction was carried out using primers 33 and 4 ^R and plasmid DNA 401-0-p57-2 as a template; as a result of PCR amplification, a product containing 542 bp was obtained.

[00287] The last reaction was carried out using primers 25 and 4 ^R and a mixture of the products of the two previous reactions as a template; PCR amplification yielded a 705 bp product and ligated into a TA cloning vector (Invitrogen, cat. no. K2000-01). The XbaI-NotI fragment containing the human GH-0 sequence was ligated into the pCI-dhfr eukaryotic expression vector. CHO DG-44 cells were transfected with the indicated vector. Cells were grown in a protein-free medium.

[00288] Construct 402-1-p83-5 (human GH-CTP) - SEQ ID NO: 37 and 402-2-p72-3 (human GH-CTP×2) - SEQ ID NO: 38: MOD-4021 represents is a recombinant human growth hormone that has been hybridized to 1 copy of the C-terminal peptide of the human chorionic gonadotropin (CTP) beta chain. A CTP MOD-4021 cassette was attached to the C-terminus (one cassette). MOD-4022 is a recombinant human growth hormone that has been hybridized with 2 copies of the C-terminal peptide of the human chorionic gonadotropin (CTP) beta chain. Two CTP MOD-4022 cassettes were attached to the C-terminus (two cassettes).

[00289] The construction of human GH-CTP and human GH-CTP-CTP was carried out in the same manner as the construction of human GH-0. pCI-dhfr-401-1-p20-1 (hGH*-ctp) and pCI-dhfr-401-2-p21-2 (hGH*-ctp x2) were used as templates in the second PCR reaction.

[00290] MOD-4021 and MOD-4022 were expressed in CHO DG-44 cells. The cells were propagated in a protein-free medium. The molecular weight of MOD-4021 is ~30.5 kDa since human GH has a molecular weight of 22 kDa, while each "CTP cassette" adds 8.5 kDa to the total molecular weight (see figure 1). The molecular weight of MOD-4022 is ~39 kD (see figure 1).

[00291] Construction of 402-3-p81-4 (CTP-human GH-CTP-CTP) - SEQ ID NO: 39 and 402-4-p82-9 (CTP*human GH-CTP-CTP) - SEQ ID NO: 40: MOD-4023 is a recombinant human growth hormone that has been hybridized to 3 copies of the C-terminal peptide of the human chorionic gonadotropin (CTP) beta chain. Three CTP MOD-4023 cassettes were attached to the N-terminus (one cassette) and C-terminus (two cassettes). MOD-4024 is a recombinant human growth hormone that has been hybridized to 1 truncated and 2 full-length copies of the human chorionic gonadotropin (CTP) beta chain C-terminal peptide. A truncated CTP MOD-4024 cassette was attached to the N-terminus and two CTP cassettes were attached to the C-terminus (two cassettes).

[00292] Three PCR reactions were performed. The first reaction was carried out using primer 25 and primer 35 ^R and plasmid DNA p401-3-p12-5 or 401-4-p22-1 as a template; as a result of PCR amplification, a product containing 265 or 220 bp was obtained. The second reaction was carried out using primer 34 and primer 37 ^R and plasmid DNA TA-hGH-2-q65-1 as a template; as a result of PCR amplification, a product containing 695 bp was obtained. The last reaction was carried out using primers 25 and 37 ^R and a mixture of the products of the previous two reactions as a template; PCR amplification yielded a 938 or 891 bp product and ligated into a TA cloning vector (Invitrogen, cat. no. K2000-01). The XbaI-NotI fragment containing the human GH sequence was ligated into the pCI-dhfr eukaryotic expression vector. (figure 3)

[00293] MOD-4023 and MOD-4024 were expressed in CHO DG-44 cells. The cells were propagated in a protein-free medium. The molecular weight of MOD-4023 is ~47.5 kDa (see Figure 1) and the molecular weight of MOD-4024 is ~43.25 kDa (Figure 1).

[00294] Construct 402-6-p95a-8 (CTP-human GH-CTP) - SEQ ID NO: 41: Construction of human GH-6 was performed in the same manner as construction of human GH-3. pCI-dhfr-402-1-p83-5 (human GH-CTP) was used as a template in the second PCR reaction.

[00295] Construct 402-5-p96-4 (human CTP-GH) - SEQ ID NO: 42: PCR reaction was performed using primer 25 and primer 39 ^R and plasmid pCI-dhfr-ctp-EPO-ctp (402-6 -p95a-8) as a matrix; PCR amplification yielded a 763 bp product and ligated into a TA cloning vector (Invitrogen, cat. no. K2000-01). The XbaI-NotI fragment containing the human CTP-GH sequence was ligated into the pCI-dhfr eukaryotic expression vector to obtain clone 402-5-p96-4.

EXAMPLE 2

Studies of the biological activity of human GH-CTP polypeptides according to the present invention in vivo

[00296] The following experiment was performed to test the potential biological activity of human GH-CTP polypeptides over an extended period of time, compared to commercial recombinant human GH and MOD-4020.

MATERIALS AND METHODS

[00297] Pituitary-removed female rats (60-100 g) were weekly injected subcutaneously with 21.7 μg of human GH polypeptides-CTP or 5 μg of control commercial human rGH by SC injection once a day.

[00298] Weight was measured in all animals prior to treatment, 24 hours after the first injection, and then the day before the end of the study on day 21. Each point represents the percentage of mean weight gain in the group ((weight day 0 - weight last day)/weight 0th day). Mean weight gain was normalized to that of commercial human GH administered by injection once daily. The treatment regimen is presented in Table 2.

table 2 No. medicinal product N Route of administration Treatment regimen equimolar dose
(µg/rat) Cumulative dose (mcg/rat) Dose volume (ml) 1 Carrier 7 PC Days 1, 7 and 13; 1 week No data No data 0.25 2 Blank vector 7 PC Days 1, 7 and 13;1/week No data No data 0.25 3 MOD-4020
SEQ ID NO: 36 7 PC Days 1, 7 and 13; 1 week 21.7 65 0.25 4 MOD-4021
SEQ ID NO: 37 7 PC Days 1, 7 and 13; 1 week 21.7 65 0.25 5 MOD-4022
SEQ ID NO: 38 7 PC Days 1, 7 and 13; 1 week 21.7 65 0.25 6 MOD-4023
SEQ ID NO: 39 7 PC Days 1, 7 and 13; 1 week 21.7 65 0.25 7 MOD-4024
SEQ ID NO: 40 7 PC Days 1, 7 and 13; 1 week 21.7 65 0.25 8 Commercial human growth hormone v.1 7 PC Days 1, 7 and 13; 1 week 21.7 65 0.25 9 Commercial human growth hormone v.1 7 PC Days 1-13; 1 week 5 65 0.25

RESULTS

[00299] The results are summarized in Figure 2. The results indicate that MOD-4023 (SEQ ID NO: 39) and MOD-4024 (SEQ ID NO: 40) induced more than 120% weight gain compared to commercial human hGH that induced 100% weight gain.

CONCLUSION

[00300] Three weekly doses (injection days: 1, 7 and 13) of 21.7 μg of MOD-4023 (SEQ ID NO: 39) and MOD-4024 (SEQ ID NO: 40) induced weight gain in pituitary-removed rats, which was 30% greater than the weight gain for commercial human hGH, which was injected daily at a similar cumulative dose of 5 µg/day for 13 days.

EXAMPLE 3

Pharmacokinetic studies of CTP-modified growth hormone

[00301] Single dose pharmacokinetic studies were performed in Sprague-Dawley rats. All animal experiments were performed in accordance with the Animal Welfare Act, the requirements of the Manual for the Care and Use of Laboratory Animals, and under the supervision and approval of the Institutional Animal Care and Use Committees of Modigene, Biotechnology General Ltd. Rats were kept individually or two individuals in each cage in rooms with a 12-hour light/dark cycle. Access to water (public water supply) and non-certified rodent food was unrestricted.

[00302] To compare the pharmacokinetics of human CTP-GH-CTP-CTP and GH in rats, four groups of Sprague-Dawley rats (270-290 g) were formed, including three to six male rats per group. Rats were randomly assigned to four groups (Table 3). Rats were given a single SC or IV injection of GH (50 μg/kg iv or s) or human CTP-GH-CTP-CTP (108 μg/kg iv or s). With the exception of the pre-dose sample, which was obtained under isoflurane anesthesia, blood collection was performed from animals without anesthesia. Blood samples (approximately 0.25 ml) were collected in EDTA-coated Microtainer® tubes for plasma concentration studies of human CTP-GH-CTP-CTP by ELISA at the time points shown in Table 11. After each sampling, the blood volume was replenished with an equal volume of sterile 0.9% saline. Samples were kept on ice for no more than 1 hour before centrifugation and plasma collection. Plasma samples were stored at approximately -20°C until testing.

Table 3
Experimental design of a pharmacokinetic study in rats Treatment group Investigational drug Number of animals in the group /
time point Route of administration Floor Dose level
(µg/kg) Injected volume (µl) Conc. (µg/ml)/
Total volume (ml) Time points*
(hours after dosing) 1 Biotropin 6# PC males 50 500 20/5 0 (pre-dose), 0.5, 2, 4, 8, 24, 48 2 CTP-Human GH-CTP-CTP 6# PC males 108 500 43.2/5 0.5, 2, 4, 8, 24, 48, 72, 96 3 Biotropin 6# i/v males 50 300 20/3 0, 0.12, 2, 4, 8, 24 4 CTP-Human GH-CTP-CTP 6# i/v males 108 300 43.2/3 0.12, 2, 4, 8, 24, 48, 72 Blood sample volume/time point - 500 µl Latest blood samples #3 rats per time point.

[00303] A commercial human growth hormone specific sandwich ELISA kit (Roche Diagnostics) was used to evaluate rat plasma samples. This kit allows the detection of human growth hormone in plasma using an antibody in a sandwich ELISA assay. This kit was originally used to measure the concentration of human CTP-GH-CTP-CTP in rat blood plasma. For these plasma samples, a standard curve of human CTP-GH-CTP-CTP (1.2-100 ng/ml) was used, and rat plasma concentrations of CTP-human GH-CTP-CTP were interpolated from this curve.

[00304] Standard pharmacokinetic parameters, including clearance (CL or CL/F), volume of distribution (Vd or VD/F), half-life (T _1/2 ), area under the plasma concentration-time curve (AUC), maximum observed concentration in plasma (C _max ) and the time to reach the maximum observed plasma concentration (T _max ), were obtained from the curves of the concentration of albutropin or growth hormone in blood plasma from time to time using a non-compartmental analysis method using the WinNonlin modeling software (Pharsight, version 3.1). Plasma concentrations of human CTP-CTP-CTP or GH were evenly weighted for this analysis. AUC values were calculated using the log-linear trapezoidal method for intravenous data and the linear-log trapezoidal method for s.c. data. Plasma concentration profiles for each rat (except for data obtained with s.c. albutropin) or monkeys were studied separately, and mean values ± standard error of the mean (SEM) for pharmacokinetic parameters are shown in Table 4 and Figure 4.

[00305] Human CTP-GH-CTP-CTP is a single chain protein of 275 amino acids and no more than twelve O-linked carbohydrates. The structure consists of a modified human growth hormone (somatotropin) attached to three copies of the C-terminal peptide (CTP) of the human chorionic gonadotropin (hCG) beta chain; one copy is attached at the N-terminus and two copies (in tandem) at the C-terminus. Human growth hormone contains 191 amino acids. CTP contains 28 amino acids and four O-linked sugar chains.

EXAMPLE 4

Pharmacokinetics of CTP-modified growth hormone in Sprague-Dawley rats

[00306] The pharmacokinetics of human CTP-GH-CTP-CTP were evaluated and compared with the pharmacokinetics of commercial human GH (biotropin).

[00307]

Table 4
Mean values of pharmacokinetic parameters after i/v and s/c administration of a single dose of CTP-human GH-CTP-CTP and GH (biotropin) in Sprague-Dawley rats FC stats PC i/v Options Units Biotropin CTP-Human STG-
CTP-CTP Biotropin CTP-Human GH-CTP-CTP Dose mg/kg 50 50 50 50 AUClast h⋅ng/ml 41 680 162.7 1568.3 _Cmax ng/ml 13 36.8 275.8 926 _Tmax h 0.5 8 0 0 MRT h 2.5 12.9 0.5 9.9 T _1/2 α h 1.58 0.74 T _1/2 β h 1.73 9 0.5 6.9

Statistical data analysis

[00308] Serum samples were tested to determine specific concentrations for each sample. The concentration and data at each time point were processed using WinNonlin non-compartmental analysis.

[00309] The parameters that were determined included: AUC, MRT, T _1/2 , C _max and T _max . The figure 4 shows a more favorable pharmacokinetic profile of the concentration of CTP-Human GH-CTP-CTP in blood plasma compared with the concentration of GH (pg/ml) after a single IV or SC dose of CTP-Human GH-CTP-CTP or GH in rats (n=3-6 per dose/route of administration).

[00310] After a single subcutaneous dose of 50 μg/kg, the clearance of human CTP-GH-CTP-CTP from the blood of SD rats was significantly slower than the clearance of human CTP-GH-CTP and biotropin. The corresponding calculated half-life and AUC were:

Biotropin T _1/2 1.7 h AUC 41 h⋅ng/ml CTP-Human GH-CTP T _1/2 8.5 h AUC 424 h⋅ng/ml CTP-Human GH-CTP-CTP T _1/2 9.0 h AUC 680 h⋅ng/ml Conclusion: CTP-Human GH-CTP-CTP was selected as the final candidate from 6 other options. CTP-Human GH-CTP-CTP has demonstrated excellent functionality in terms of biological activity and pharmacokinetics.

EXAMPLE 5

Quantitative weight gain study (WGA) for CTP-modified GH when administered in a single dose/multiple doses

[00311] Male rats with pituitary gland removed (interaural method), 3-4 weeks of age, were obtained from CRL Laboratories. During a 3 week postoperative acclimatization period, rats were examined and weighed twice a week to exclude animals that were suspected to have incomplete pituitary resection as evidenced by body weight gain similar to that of sham-operated rats. Rats with incompletely resected pituitary glands were excluded from the study. During the experiment, the average body weight of rats with removed pituitary gland was 70-90 g. This is a standard quantitative biological study that meets the requirements of Pharm. US and Pharm. EU, to determine human growth hormone. Hypophysectomized rats (rats that have had their pituitary gland removed) lose their ability to gain weight. Injections of human GH (and CTP-human GH-CTP-CTP) to these rats resulted in weight gain. Based on the measured weight gain over a period of time and the amount of growth hormone administered, the specific activity of human GH (and CTP-human GH-CTP-CTP) is determined. Rats were administered either a single sc dose of 0.4, 0.8 and 4 mg/kg, or multiple sc doses of 0.6 and 1.8 mg/kg for 3 weeks with 4-day intervals between injections. Individual body weights of all animals were determined at randomization, before the first dose, then every two days or, in the case of deceased animals, at the time of death, and before sacrifice.

Quantitative Study of Weight Gain with Single and Multiple Doses

[00312] The results of a comparison of whole body growth response after various dosing regimens of human CTP-GH-CTP-CTP in pituitary-removed rats are shown in Figure 5. The results indicate that the effect of a single injection of 0.4 and 0.8 mg /kg/day human GH-CTP was equivalent to that of 4 daily injections of 0.1 mg/kg/day of biotropin. The peak effect of human GH-CTP was observed after two days.

[00313] Figure 6 again shows that the area under the curve after a single injection of human CTP-GH-CTP-CTP correlates with body weight gain in rats. Therefore, the pooled data indicate that weight gain is significantly correlated with cumulative AUC.

[00314] The human GH-CTP construct, when administered at 4-day intervals, provides weight gain that is similar to that following daily biotropin injections, as shown in Figure 7. Human GH is expected to have a 5-fold better half-life, than in rats, this indicates that the potential maximum effect in humans will be reached after 10 days for a single injection. These results support the feasibility of administering the human GH-CTP construct, CTP-human GH-CTP-CTP, once a week or every other week in humans.

EXAMPLE 6

Pharmacodynamic / pharmacokinetic studies of CTP-modified growth hormone

[00315] Male rats with removed pituitary gland (interaural method), 3-4 weeks of age, were obtained from CRL Laboratories. During a 3 week postoperative acclimatization period, rats were examined and weighed twice a week to exclude animals that were suspected to have incomplete pituitary resection as evidenced by body weight gain similar to that of sham-operated rats. Rats with incompletely resected pituitary glands were excluded from the study. During the experiment, the average body weight of rats with removed pituitary gland was 70-150 g.

[00316] Rats were injected s.c. CTP-human GH-CTP-CTP, vehicle, human growth hormone CTP-human GH-CTP-CTP, or human growth hormone (20 μg/rat) were administered by subcutaneous injection in a volume of 0. 2 ml/rat. The GH dose was 0.35 and 1.05 µg/kg, a growth hormone dose that was equimolar to the amount of GH found in the respective doses of human CTP-GH-CTP-CTP 0.6 and 1.8 µg/kg. A description of the study groups is shown in Table 5. After injection, plasma samples for IGF-1 concentration studies were obtained at the time points described in Table 5. Samples were analyzed for IGF-1 concentration using a commercial ELISA kit (R&D systems).

Table 5
Treatment regimen in a pituitary-removed rat study Treatment group Test compound Number of animals in the group / time point Route of administration Eq. dose
(mg/rat) Eq. dosage
(mg/kg) Conc. CTP-Human GH-CTP-CTP (mg/ml) Dose volume (ml) Time points*
(hours after dosing) M7 Biotropin 9 PC 0.032 0.35 0.16 0.2 0 (pre-dose), 0.5, 2, 4, 8, 24, 48, 72, 96 M8 Biotropin 9 PC 0.095 1.05 0.475 0.2 0 (pre-dose), 0.5, 2, 4, 8, 24, 48, 72, 96 M9 EN648-01-08-005 12 PC 0.032 (0.055) 0.35 (0.6) 0.275 0.2 1, 2, 4, 8, 24, 48, 72, 96 M10 EN648-01-08-005 12 PC 0.095 (0.165) 1.05 (1.8) 0.825 0.2 1, 2, 4, 8, 24, 48, 72, 96 Blood sample volume/time point - 500 µl Latest blood samples

[00317] A non-compartmental pharmacokinetic analysis was performed on mean plasma concentration versus time curves for each group. The C _max value of human CTP-GH-CTP-CTP was significantly higher than the C _max value of biotropin. The terminal half-life of CTP-human GH-CTP-CTP was 6 times longer than that of biotropin.

[00318]

Table 6
Evaluation of the pharmacokinetic parameters of human CTP-GH-CTP-CTP and biotropin after a single subcutaneous injection in pituitary-removed rats Group Dose Floor _{Cmax Tmax} AUC _0-∞ AUC _0-t CL/F T _1/2 mg/kg ng/ml h ng⋅h/ml ng⋅h/ml ml/h/kg h CTP-Human GH-CTP-CTP 1.8 M 2.150 8 37.713 37.695 0.928 6.86 0.6 M 681 8 11.505 11.489 3.042 6.8 human growth hormone 1.05 M 1.078 0.5 3.541 3,540 9.884 1 0.35 M 439 0.5 1.279 1.279 27.36 1

[00319] The AUC _0-t and AUC _0-∞ values were very similar, indicating that the duration of sampling was appropriate to characterize the pharmacokinetic profiles. The AUC value of human CTP-GH-CTP-CTP was 10 times higher than that of biotropin. In addition, the value of C _max was generally proportional to the dose, and for CTP-human GH-CTP-CTP it was twice as high as the C _max of biotropin. However, as shown in Figure 8, the T _max value of human CTP-GH-CTP-CTP was 8 hours compared to 0.5 hours for biotropin, and the terminal half-life did not appear to change with dose level. The T _1/2 CTP-STG-CTP-CTP was 6.8 times higher than that of biotropin.

[00320] The indirect effects of GH are primarily mediated by insulin-like growth factor I (IGF-1), a hormone that is secreted from the liver and other tissues in response to growth hormone. Much of the growth-enhancing effects of growth hormone are actually due to IGF-1 acting on its target cells. Accordingly, the effect of the human CTP-GH construct, human CTP-GH-CTP-CTP, on serum levels of IGF-1 in pituitary-removed rats was evaluated. The figure 9 shows the results of determining the levels of IGF-1 in the blood serum of rats with removed pituitary gland after SC injection of CTP-human growth hormone-CTP-CTP and commercial human growth hormone.

[00321] A single dose of human CTP-GH-CTP-CTP 0.6 or 1.8 mg/kg and a single dose of biotropin 0.35 or 1.05 mg/kg were administered by subcutaneous injection to pituitary-removed rats for PK profiling. FD. Serum IGF-1 levels after injection were measured using specific ELISA kits (Roche Diagnostics).

[00322] Aggregate serum levels of IGF-1 following injection of human CTP-GH-CTP-CTP were significantly higher than after injection of biotropin. The value of C _max was generally proportional to the dose, and C _max CTP-GH-CTP-CTP was 3-4 times higher than C _max biotropin. The T _max value of human CTP-GH-CTP-CTP was 36-48 hours compared to 20-24 hours for biotropin. In conclusion, higher levels of human GH and a longer presence in serum lead to a significant increase in IGF-1 levels.

EXAMPLE 7

Carbohydrate content and sialic acid content in CTP-modified GH

[00323] The study of O-glycans is based on the use of the Prozyme kit. O-glycans are chemically and enzymatically cleaved from the protein and separated from the peptides using paper chromatography. The sequencing of the O-glycan pool is carried out by sequential enzymatic digestions (using exoglycosidases) followed by HPLC analysis against standards.

Glycoprofiling with sequence analysis

[00324] Glycoprofiling was performed by Ludger Ltd. Two samples (EN648 and RS0708) were obtained by three replicate release, and each release was also examined by three replicate high performance liquid chromatography. Three 300 μg EN648 and RS0708 samples and one 100 μl sodium citrate/chloride buffer sample, as well as a positive control of fetuin (250 μg) and 100 μl of water as a negative control, were ultrafiltered by centrifugation using molecular weight membranes. 10,000 kDa cutoff to replace the buffer with water, and then subjected to hydrazinolysis under O-mode conditions (6 h at 60°C). The released glycans were re-N-acetylated and purified using LudgerClean CEX cartridges. An aliquot of the released glycans was then labeled with 2-aminobenzamide (2AB), purified using Ludger Clean S cartridges, and analyzed by HPLC based on hydrophilic interaction (HILIC-HPLC), LudgerSep-N2.

Content of monosaccharides

[00325] The study of neutral monosaccharides requires the hydrolysis of glycans from their constituent monosaccharide components. Hydrolysis was carried out by Ludger Ltd on intact glycoprotein samples. Specifically, 50 μg of intact glycoprotein was subjected to acid hydrolysis, labeled with 2-AB (2-aminobenzamide) and analyzed on a reverse phase HPLC column. This method allows the hydrolysis of all glycans present on the glycoprotein, including N- and O-linked types.

Obtaining sialic acid profiles

[00326] Two samples (EN648 and RS0708) and control buffer were tested. Sialic acid testing requires weak acid release of monosaccharides followed by DMB fluorophore labeling and HPLC testing on a LudgerSep-R1 column. For each study, 50 μg of intact glycoprotein was subjected to acid hydrolysis.

Human CTP-GHG-CTP-CTP glycan study

[00327]

Table 7
Research of glycans. Structural relationships and area percentages are based on HPLC and enzymatic digestion data.

[00328] The monosaccharide profiles indicate that the human CTP-CTP-CTP glycoprotein samples contain predominantly O-linked glycans. The main peak of O-glycans is sialylated backbone 1 (Neu5Acα2-3Galβ1-3GalNAc). The main sialic acid is Neu5Ac, there are also several minor peaks that indicate the existence of 3-4% diacetylated sialic acid, N-acetyl-9-O-acetylneuraminic acid (Neu5,9Ac2), and less than 1% N-glycolylneuraminic acid. Small amounts of Neu5Acα2-6(Galβ1-3)GalNAc are also present.

EXAMPLE 8

Pharmacokinetic/toxicokinetic studies of CTP-modified growth hormone in rhesus monkeys

[00329] Plasma concentration versus time curves were obtained for each animal. Non-compartmental analysis was performed using WinNonlin, professional version 5.2.1 (Pharsight Corporation, Mount View, CA, USA). The calculated pharmacokinetic parameters are shown in Table 8 below.

[00330]

Table 8
Estimates of pharmacokinetic parameters of human CTP-GH-CTP-CTP (mean ±SD) obtained by non-compartmental analysis after a single subcutaneous injection in rhesus monkeys Parameter 1.8 mg/kg 90 mg/kg _Cmax (µg/ml) 2.073±0.417 108.7±46.0 T _max (h) 4±0 11±7 AUC _0-t (µg⋅h/ml) 38.7±7.4 2.444±394 AUC _0-∞ (mcg⋅h/ml) 39.0±7.3 2.472±388 CL/F (ml/h/kg) 47.5±9.0 37.04±4.78 T _1/2 (h) 10.00±1.47 9.85±1.07 Vz/F (ml/kg) 701±236 529±104

[00331] The AUC _0-t and AUC _0-∞ values were comparable, indicating that the duration of sampling was appropriate to characterize the pharmacokinetic profiles. The value of C _max was proportional to the dose. T _max was reached later at a higher dose. The T _max was 4 hours in all animals in the low dose group and 8 or 24 hours in the high dose group. Terminal half-lives were similar for both dosing groups.

[00332] AUC was approximately proportional to dose, with AUC at higher dose slightly greater than the calculated proportional value resulting in lower CL/A and Vz/A than at lower dose. It is not possible to draw conclusions about whether the CL and Vz values are lower at a higher dose, or whether the F value is lower at a lower dose. There was overlap between groups, so it is unlikely that the observed difference in CL/F and Vz/F is significant.

[00333] The pharmacokinetic parameters calculated using the model were largely similar to those obtained using non-compartmental analysis. The absorption rate and half-life are shown in Table 9 below.

[00334]

Table 9
Estimates of absorption and half-life of CTP-Human GH-CTP-CTP (mean ±SD) after subcutaneous injection derived from pharmacokinetic modeling in rhesus monkeys Dose T _{1/2 abs} (h) T _{1/2 el} (h) 1.8 mg/kg 1.17±0.40 10.41±2.36 90 mg/kg 6.49±1.87 7.26±1.85

[00335] The data indicate that the clearance rates are largely similar between the groups, with a lower T _1/2el in the low dose group. However, absorption is more than 5 times slower after subcutaneous administration of 90 mg/kg compared to that after administration of 1.8 mg/kg. As in the case of non-compartmental analysis, the simulation results indicate a larger value of T _max at high dose.

[00336] Although GH supplementation is effective in the treatment of growth hormone deficiency in children and adults, the disadvantages of daily injections for long periods of time limit its use by physicians in some patient populations, as well as increase the risk of dosing errors, the number of individuals providing care, cost of treatment, and non-compliance with the treatment regimen. In certain populations, such as short children who cannot understand the consequences of non-compliance with the prescribed GH dosing regimen, the need to adhere to the requirements of the treatment regimen to achieve the optimal beneficial effect of GH treatment is especially important. The problem of finding a more suitable alternative to daily injections of growth hormone and subsequent adherence to the requirements of the treatment regimen takes on additional importance, as GH-deficient children are moving into a group of adult patients with an ongoing need for GH treatment. The need for daily therapy is largely driven by the short circulating half-life of recombinant GH and has led to the development of sustained release growth hormone (Reiter EO. Attire KM., Mashing TJ. Silverman BL. Kemp SF. Neolith RB. Ford KM. and Sanger P. A multimember study of the efficacy and safety of sustained release GH in the treatment of naive pediatric patients with GH deficiency J Clin Endocrinol Metab 86 (2001) pp 4700-4706.

[00337] In rats, the pharmacokinetic profile of GH-CTP, a hybrid protein of recombinant human growth hormone and CTP, described in this application, has a longer duration than that of GH. This unique pharmacokinetic profile allows intermittent administration of GH-CTP to achieve pharmacodynamic action in growth hormone deficient rats as evidenced by rising and increasing plasma levels of IGF-1, respectively.

[00338] GH-CTP provides a better pharmacokinetic profile compared to that of GH when administered sc in rats. There are significant differences in plasma clearance of GH-CTP compared to GH. In particular, after subcutaneous dosing, the plasma clearance of GH-CTP is more than 6 times slower than that of GH. The terminal half-life and mean retention time of GH-CTP were approximately six times longer than those of GH in rats after subcutaneous injection. In addition, Cl/F after subcutaneous dosing was 10 times lower for GH-CTP than for GH.

[00339] In order to study the possibility of transferring pharmacokinetic benefits in rats to pharmacodynamic benefits, the ability of GH-CTP to stimulate growth in GH-deficient rats with removed pituitary gland was investigated using a dosing regimen with less frequent administration, compared with daily administration, at equimolar levels doses of CTP-Human GH-CTP-CTP and GH. A single s.c. injection of GH-CTP produced incremental weight gain that was equivalent to that of 4 consecutive daily injections of growth hormone. In addition, the GH-CTP regimen for every fourth day provides a greater increase in body weight compared to GH.

[00340] Based on pharmacodynamics, the long circulation time of GH-CTP compared to GH in pituitary-removed rats resulted in a longer IGF-1 response as measured by plasma levels of IGF-1 after a single subcutaneous injection. In pituitary-removed rats, subcutaneous administration of a single dose of GH-CTP increased the concentration of circulating IGF-1 in a dose-dependent manner. At the highest dose of albutropin, IGF-1 concentrations increased above baseline values within 75 hours after a single dose. Therefore, the increased circulation time of a single dose of GH-CTP resulted in a significant improvement in pharmacodynamic parameters compared to a single dose of GH, increasing the likelihood that GH-CTP could provide a similar improvement in growth at a reduced dosing frequency compared to standard GH treatment regimens.

[00341] A single dose of CTP-modified human GH at 90 mg/kg in rhesus monkeys and 180 mg/kg in rats was well tolerated in both species. The allometric factor between rats and primates is approximately X2 based on the expected clearance of proteins in these animals. These data are in line with approved industry extrapolation models to increase the half-life of therapeutic proteins between species (FDA guidelines). In primates, the PK profile for a dose of 90 mg/kg is not significantly better than the PK profile for 180 mg/kg CTP-modified GH in rats. Therefore, allometric extrapolation of the data to humans supports a single weekly injection or biweekly injection regimen.

[00342] The concept of the use of the CTP-GH construct proposed in the present invention provided a reduction in the frequency of dosing compared to a commercial recombinant GH product. Nutropin Depot® is a sustained release GH formulation approved for use in pediatric patients. However, comparison with historical control revealed that the growth rate for 1 and 2 years is significantly (p<0.001) lower in children treated with Nutropin Depot® (growth rate for 1 year was 8.2±1.8 cm/year), than in children treated with GH (growth rate for 1 year was 10.1 ± 2.8 cm / year) (Silverman BL, et al. J. Pediatr. Endocrinol. Metab. 15 (2002), pp. 715-722 ). Local undesirable effects of Nutropin Depot® administered subcutaneously include nodules, erythema, pain at the injection site, headache and vomiting. Preclinical toxicology studies in rats and monkeys showed that subcutaneous administration of human CTP-GH-CTP-CTP did not cause local reactions compared to vehicle. Given the medical need for a less frequently administered GH formulation, the pharmacological properties of human CTP-GH-CTP-CTP in this rat study suggest that this product also provides benefits in terms of toxicology and patient compliance. Data on prolonged activity of human CTP-GH-CTP-CTP in rats support its potential usefulness as an agent requiring only intermittent administration to achieve the therapeutic benefit currently achieved using daily administration.

EXAMPLE 9

A long-acting CTP-modified variant of human growth hormone (HGH-CTP) was highly effective in adult patients with growth hormone deficiency - phase II clinical trial

[00343] A randomized, open-label, Phase II clinical trial was conducted to evaluate the safety, tolerability, pharmacokinetic, and pharmacodynamic properties of human GH-CTP administered by weekly or bimonthly injection in patients currently receiving daily injections. growth hormone. The study was conducted at several centers in six countries. The three main cohorts in the study received a single weekly dose of human GH-CTP containing 30%, 45%, or 100% of the equivalent cumulative dose of commercial human GH that adults with growth hormone deficiency receive for seven days as daily injections (referred to as cohorts "30%", "45%" and "100%", respectively). The data obtained reflect the results of 39 patients, 13 in each cohort. Each cohort included 2 women.

[00344] In addition to the three main cohorts, adult patients with growth hormone deficiency were enrolled in an experimental fourth cohort, the study of which was not part of a formal Phase II clinical trial. Patients in the experimental fourth cohort received a single injection of human GH-CTP once every two weeks, which contained 50% of the total commercial dose that adult patients with growth hormone deficiency received over a two-week period as daily injections.

[00345] Efficacy for the three main cohorts treated with a single weekly injection of human GH-CTP is determined by measuring daily levels of insulin-like growth factor 1 (IGF-1) within the desired therapeutic range for seven days (during the last week of treatment in the study) . The desired therapeutic range is defined as a range of +2 standard deviations to -2 standard deviations relative to the mean levels of IGF-1 expected in the normal population, disaggregated by age group and sex. In addition, the study measured IGF 1 levels within a narrower range of ±1.5 SD to observe patient variance within the normal range.

Results:

[00346] Table 10 shows the average percentage of days within the normal therapeutic range (±2 SD), the average percentage of days within the narrower normal therapeutic range (±1.5 SD), and the average C _max (highest concentration) of IGF- 1 in men measured during the last week of treatment, expressed as standard deviations from the mean IGF-1 level in the normal population.

Table 10
Results of a Phase II Human Clinical Trial Cohort % days within narrow normal range of IGF-1 levels (±1.5 SD) % days within normal range for IGF-1 levels (±2 SD) Mean C _max IGF-1 (preferably below +2 SD) thirty% 57% 100% -0.9 45% 100% 100% 0.1 100% 86% 100% 0.4

[00347] A dose of human GH-CTP of 2 mg/week containing 50% of the total weekly dose of human GH that would typically be given to an adult patient as the initial dose of treatment could be determined with high probability as the starting dose for men and women. women in a phase III adult clinical trial.

[00348] There were no safety and/or tolerability issues, and there was no indication that human GH-CTP, when used at high doses, induced excessive levels of IGF-1 in patients or even levels above the normal range.

[00349] Findings and Outlook from a Phase II Clinical Trial Evaluating IGF-1 Levels

Design and Objectives of Phase II Clinical Study MOD-4023:

[00350] A two-stage phase II clinical study was completed to confirm the feasibility of a weekly regimen of administration of human CTP-GH-CTP-CTP (MOD-4023) (see figure 10). This study was a transition from a study conducted in growth hormone deficient (GHD) patients currently receiving daily human GH, who were considered stable during their daily treatment period prior to MOD-4023 administration, as evidenced by SDS IGF levels -1 within normal range (±2SDS). Phase I of this study was a 4-week dose titration study (4 injections) supported by a full pharmacokinetic/pharmacodynamic (PK-PD) study within a week of the 4th dose of MOD-4023. The main goal of this part of the study was to determine the therapeutic dose range in which the level of IGF-1 is maintained within a certain range. Another goal was to evaluate the PK/PD profile of MOD-4023 at 3 different doses/multipliers and to confirm dose response. The second phase of this study (phase II) was a 16-week period of treatment and dose titration of MOD-4023. All patients who continued in Phase II of this study began receiving a similar dose level of MOD-4023 (61.7% of their personal, optimized, weekly cumulative dose of human GH), however, the dose of these patients could be modified based on controlled levels of IGF -1 for them.

[00351] In the first part of the study, doses were administered based on a percentage of the weekly cumulative human GH dose to assess initial response after a weekly MOD-4023 dosing regimen. For example: a patient treated with 1 mg/day human GH who was randomized to a 55% cohort received MOD-4023 at a dose of 1 mg * 7 days * 0.55 by injection on a weekly basis.

results

[00352] The primary efficacy endpoint of this study was the mean time interval for IGF-1 levels that were within the normal range after the last dose during Phase I, expressed in hours. According to the results of the final analysis, the levels of IGF-1 in most patients during this week were within the normal range throughout the week (table 11). Patients who were within the specified SDS range at the end time point were assigned a time interval value of 168 hours. None of the patients exceeded +2 SDS for C _max , indicating no excess levels of IGF-1. Eighty-five percent of the men (28/33 men) had an average IGF-1 SDS within the normal range (±2 SDS) (Figure 11). The median time interval for IGF-1 levels that are within ± normal range of all three cohorts showed no significant change as all median time intervals were within 1 standard deviation of each other.

[00353]

Table 11
Time interval for IGF-1 levels that are within ±2SDS after the 4th dose during Phase I Cohort 2
37% of the weekly dose of human GH Cohort 3
55% of the weekly dose of human GH Cohort 1a
123.4% of the weekly dose of human GH 156.37 h
120.9±66.24 (h) 168 h
146.49±50.62(h) 151.17 h
119.9±66.51(h)

[00354] As expected, administration of 37% of the human GH weekly dose resulted in IGF-1 SDS values in the lower part of the normal range and shorter duration in the normal range. Significant improvement in IGF-1 levels, as evidenced by duration within the normal range, was observed with the higher dose (55% of the weekly total human GH dose).

[00355] The dose-dependent response of IGF-1, compared to baseline, is shown in Figure 12. The results presented in Figure 12 support the notion that IGF-1 levels increase with dose of MOD-4023, allowing adjustment weekly IGF-1 profile. In addition, the mean change in IGF-1 concentrations from baseline returned to baseline 120-168 hours post-dose, indicating that in this stable population of growth hormone deficient (GHD) adult patients, residual levels of IGF-1 are stable without deterioration (figure 12).

[00356] The Cavg (AUC/time), which is the average exposure to IGF-1, of stable patients receiving daily injections was compared with that of the same patients after weekly treatment with MOD-4023 (AUC _0-24/24h according to compared with AUC _0-168/168h, respectively). Weekly doses of 55-123.4% of the total weekly dose of human GH provided a comparable amount of IGF-1 exposure, as evidenced by Cavg, while patients receiving 37% of the weekly dose had a reduced Cavg value, which was in line with expectations in association with a relatively low weekly dose (Tables 12 and 13).

[00357]

Table 12
Summary data on pharmacodynamic parameters for IGF-1 after subcutaneous administration of recombinant human growth hormone in adult patients with growth hormone deficiency before the introduction of MOD-4023 (stage I; 4 weeks) Parameter Cohort* (optimized human GH treatment) 2 3 1a _Cavg (ng/ml) 174±57.0(11) 178±43.1(11) 154±28.5(11) *Mean value ± standard deviation (N).

[00358] Table 13
Summary data on pharmacodynamic parameters for IGF-1 after subcutaneous administration of MOD-4023 in adult patients with growth hormone deficiency (stage I; 4 weeks) Parameter Cohort* (MOD-4023 treatment) 2 (37%) 3 (55.5%) 1a (123.4%) _Cavg (ng/ml) 117±32.5(11) 147±50.9(11) 50.4±132(11)

[00359] Based on the study of PD during Phase I of the Phase II clinical study, the following conclusions were made: 1) although the aim of this study was not to optimize the levels of IGF-1 of patients by reducing the SDS value of IGF-1 to 0, (because optimization of IGF-1 SDS requires a relatively long titration period), a therapeutic dose range for weekly administration of MOD-4023 can still be established: approximately 56%-123% of the weekly cumulative dose of human GH administered daily. 2) The profile of IGF-1 after weekly administration of MOD-4023 is relatively flat, as evidenced by the rather small difference between C _max and C _trough . 3) The Cavg value (AUC _0-168/168h ), which is the average weekly exposure to IGF-1, correlates with day 4 values. Accordingly, day 4 after MOD-4023 administration was chosen as the day to monitor IGF-1 levels.

Results and prospects of the phase II clinical trial phase II (4-month follow-up study):

[00360] The possibility of maintaining IGF-1 within the normal range at the optimal dose and for a longer period of time with weekly administration of MOD-4023 was considered during the second part of the study (stage II - 4-month additional period; figure 10). In this study, this same population of patients from the first phase received 61.7% of their weekly dose of human GH, and IGF-1 levels were monitored every two weeks. Most patients maintained the SDS value for IGF-1 levels within the normal range throughout the study, as measured on day 4 post-injection. The dose of patients whose IGF-1 levels were below the normal range was additionally titrated, and the dose of MOD-4023 was increased in such patients (in accordance with clinical practice).

[00361] A minority of patients with IGF-1 SDS values below the normal range required additional titration but showed a significant improvement in IGF-1 SDS, indicating that the IGF-1 profile can be optimized with positive/negative dose increments MOD -4023. Required high susceptibility and minimal dose modification, as presented in figure 13 and summarized in table 14 below.

[00362] Table 14
Summary data on necessary dose modifications during phase II Number of dose modifications Men Women No dose modifications 22 (out of 34) 3 (of 8) 1 dose modification 6 (out of 34) 1 (of 8) 2 dose modifications 3 (out of 34) 3 (of 8) 3 dose modifications 3 (out of 34) 1 (of 8)

[00363] Based on day 4 IGF-1 SDS values (correlated with Cavg), a significant improvement in IGF-1 levels compared to Phase I of the study was observed in selected patients. This observation further supported the notion that an adjustment period is necessary to reach the optimal level and profile of IGF-1. Women are known to be less responsive to human GH replacement therapy (also MOD-4023), and tend to require higher doses and a longer titration period. In addition, IGF-1 SDS levels measured on day 4 were consistently maintained at similar values within the normal range for a 4-month additional period, indicating that MOD-4023 can be administered weekly. After sequential injections of MOD-4023, no significant decrease in IGF-1 levels was observed on day 4, indicating that C _max and C _{trough of} the "sinusoidal" IGF-1 profile persisted throughout the study, once again confirming the feasibility of a weekly dosing regimen. MOD-4023.

[00364] In conclusion, MOD-4023 should eliminate the need for multiple injections currently required to treat GH deficiency. The results of this study showed that MOD-4023 can be administered by injection once a week and achieve the clinical efficacy endpoints assessed in this study while maintaining a favorable safety profile. A GH regimen that requires less frequent injections may improve patient adherence and potential clinical outcomes.

[00365] Therefore, based on the achieved IGF-1 profile and the results of the phase II safety and tolerability assessment, the recommended frequency of injections and dosing for the phase III study is as follows: a single weekly injection of MOD-4023 containing 61.7% of the total weekly dose Human growth hormone, individually selected for each patient.

EXAMPLE 10

Administration of a CTP-modified variant of human growth hormone (HGH-CTP) improved pharmacokinetics in prepubertal children with growth hormone deficiency (GHD)

[00366] A randomized, open-label, phase II clinical trial was conducted to evaluate the safety, tolerability, pharmacokinetics, and pharmacodynamic parameters of three doses of MOD-4023 compared to commercially available standard daily recombinant human growth hormone. The study included a 6-month screening period and two active treatment periods: a 6-month treatment period including PK/PD sampling followed by an additional 6-month continuous multiple dosing period as shown in Figure 14. Secondary goals were to evaluating the pharmacokinetic (PK) and pharmacodynamic (PD) profiles of three different doses of MOD-4023 in prepubertal children with growth hormone deficiency (GHD) and in selecting the optimal dose of MOD-4023 for a subsequent phase III study based on safety and efficacy data.

[00367] In order to start gradually introducing the prescribed doses of MOD-4023 to naive patients (table 15), a stepwise increase in dose was carried out. All patients randomized to receive one of three doses of MOD-4023 began treatment for 2 weeks with a low dose of MOD-4023 (0.25 mg/kg). Based on the patient's prescribed dose, the dose was increased to the next dose level every two weeks until the final prescribed dose was reached.

Table 15
Dosing cohorts Cohort Dose MOD-4023/genotropin 1 0.25 mg MOD-4023 protein/kg/week equivalent to a weekly injection of 0.18 mg/kg human GH 2 0.48 mg MOD-4023 protein/kg/week equivalent to a weekly injection of 0.35 mg/kg human GH 3 0.66 mg MOD-4023 protein/kg/week equivalent to a weekly injection of 0.48 mg/kg human GH 4 Genotropin: 0.034 mg/kg/day.

[00368] After the second target dose, limited sampling (based on populations) was performed to determine PK/PD parameters as described in Table 16.

Table 16
Dose escalation schedule for MOD-4023 cohorts Cohort Dosing schedule Week 1 Week 2 Week 3 Week 4 Week 5 Week 6 Cohort 1 0.25 mg protein/kg/week sampling for PK/PD Cohort 2 0.25 mg protein/kg/week 0.48 mg protein/kg/week sampling for PK/PD Cohort 3 0.25 mg protein/kg/week 0.48 mg protein/kg/week 0.66 mg protein/kg/week sampling for PK/PD

[00369] Patients allocated to the MOD-4023 dosing cohort were randomized within the cohort to one of three blocks and subjected to limited PK/PD sampling (4 samples per patient in one week) according to Table 17 below.

[00370] Patients assigned to cohort 1 underwent limited PK/PD imaging after the 2nd dose of MOD-4023 (V2a-g - 2nd week) and returned to health centers for a single visit 4 days after dosing during the 6th week (V4h).

[00371] Patients allocated to Cohort 2 visited health centers for a single visit 4 days after dosing during week 2 (V2h), underwent limited PK/PD sampling after 4th dose of MOD-4023 ( 4th week: second dose at the prescribed dose level; V3a-g) and returned to the medical centers for one visit 4 days after dosing during the 6th week (V4h).

[00372] Patients allocated to cohort 3 visited health centers for a single visit 4 days post-dosing for weeks 2 and 4 (V2h and V3h) and underwent limited PK/PD sampling after 6 doses of MOD-4023 ( week 6: second dose at the prescribed dose level, V4a-g).

[00373] During Visits 2a and 2h, 3a and 3h, and 4a and 4h, patients underwent the following examinations: physical examination, assessment of vital signs, AEs, local tolerability, concomitant medications, glucose metabolism parameters (fasting glucose and insulin; HbA1c only in V4), levels of other hormones (TSH, fT4, T3, cortisol), standard biochemical and complete blood count for clinical safety assessment (visits 2a and 2h, 4a and 4h), assessment of the patient's height and weight, lipid metabolism parameters, and also serum levels of IGF-1 and IGF-1-BS-3.

[00374] Patients allocated to the genotropin cohort (cohort 4) visited health centers for visits 2 and 4 during weeks 2 and 6 of treatment. The following studies were performed: physical examination, assessment of vital signs, AEs, local drug tolerance, concomitant medications, glucose metabolism parameters (fasting glucose and insulin, HbA1c in V4 only), other hormone levels (TSH, fT4, T3, cortisol), standard biochemical and complete blood count to assess clinical safety, assessment of patient height and weight, lipid metabolism, as well as serum levels of IGF-1 and IGF-1-BS-3 (figure 17).

[00375] Also after the 8th dose of genotropin (beginning of the 2nd week of dosing the drug), patients assigned to the genotropin cohort were randomized to one of three blocks and underwent limited sampling for PK / PD determination (4 samples for one patient within 24 hours), according to Table 18 below:

[00376] After the first 6 weeks of the study, all patients attended the hospital on a monthly basis (weeks 10, 14, 18, 22 and 26). Patients assigned to the MOD-4023 dosing cohort (cohorts 1-3) were asked to return 4 days after MOD-4023 administration to determine MOD-4023, IGF-1 and IGF-1-BS-3 levels and perform routine safety assessments. In addition, after 5 months of dosing, patients allocated to the MOD-4023 dosing cohort were asked to return to the medical center in the morning prior to dosing in order to determine the residual level of MOD-4023 and obtain samples for the determination of PD (IGF-1 and IGF-1- BS-3). Patients allocated to the genotropin dosing cohort (cohort 4) were asked to return on any day during the respective dosing week.

Results:

[00377] Demographic characteristics in the study are presented for all patients in Table 19 below.

[00378]

Table 19
Baseline Demographic Characteristics of the Phase II Study Cohort 1 Cohort 2 Cohort 3 Cohort 4 Dose 0.25 mg/kg/wk MOD-4023 0.48 mg/kg/wk MOD-4023 0.66 mg/kg/wk MOD-4023 0.034 mcg/kg/day genotropin N 9 9 10 7 Average SD Average SD Average SD Average SD Age (g) 6.44 2.3 6.33 2.1 6.10 2.2 5.43 1.9 STG Peak
(ng/ml)* 2.84 2.9 3.58 1.7 4.41 3.2 2.92 2.4 HVSDS -3.05 2.0 -2.82 1.1 -3.11 1.8 -3.36 2.0 HTSDS -3.99 0.9 -3.82 0.8 -3.91 1.1 -4.79 1.7 HtSDS-TH SDS -3.47 0.9 -3.23 0.7 -3.15 1.3 -4.20 1.8 Screening for IGF-1 SDS -2.48 0.8 -2.28 0.7 -1.81 0.7 -2.34 1.2 Floor (%) AND
1 (11.1) M
8 (88.8) AND
4 (44.4) M
5 (55.6) AND
3 (30) M
7 (70) AND
3 (42.9) M
4 (57.1)

[00379] The mean PK profile of MOD-4023 administered to naive GHD patients at their final dose level is shown in Figure 15 and the PK parameters are presented in Table 20 below.

Table 20
Comparative average FC parameters Units MOD-4023 human growth hormone C1 (n=13) C2 (n=12) C3 (n=13) C4 (n=11) T _1/2 h 36.1 29.2 29.1 3.6 _Tmax h 12 12 12 2 _Cmax ng/ml 459.9 810.2 795.5 17.3 AUC 0-inf_obs ng/ml⋅h 10943.4 20050.3 25503.1 135.7 Cl/F_obs (mg/kg)/(ng/ml)/h 2.28×10 ^-5 2.39×10 ^-5 2.59×10 ^-5 2.51×10 ^-5

[00380] As expected, MOD-4023, when administered once a week, showed a long half-life, which was shown to be 8 times higher than that of human GH when administered daily. In addition, a dose-response relationship was observed, as evidenced by the AUC values for each dose of MOD-4023.

[00381] A dose-dependent response was maintained during the first 6 months of weekly administration of MOD-4023 (data not shown).

[00382] Concentrations of IGF-1, which is an approved surrogate marker of human GH activity, also increased in a dose-dependent manner (Figure 23), maintaining target values (above -2 SD Growth Index [SDS]) for most weeks without excess levels (>2SDS, figure 21). IGF-1 SDS levels continued to rise moderately in a dose-dependent manner during the first 6 months of the study, without reaching excessively high levels above 2SDS (Figure 16).

[00383] The human GH group (cohort 4) also showed an increase in IGF-1 SDS values, which were very similar to the trend observed for the two cohorts receiving the highest doses of MOD-4023. In addition, the levels of IGF-1 BS-3 also increased in a dose-dependent manner with the introduction of MOD-4023, reaching stationary values at approximately 15 weeks (figure 17 and 18, respectively). In total, two pharmacodynamic profiles of IGF-1 and IGF-1-BS-3 confirm that a single weekly injection of MOD-4023 can replace 7 daily injections in a similar dose range.

[00384] Growth rate was monitored before dosing and 6 months after weekly administration of MOD-4023 or daily administration of growth hormone. For all cohorts, an excellent growth response was obtained with no statistically significant differences between different cohorts (Table 21 and Figure 19), further confirming that a weekly injection of MOD-4023 can provide adequate growth, as well as daily injections of human GH.

Table 21
Average annual growth rate with the introduction of MOD-4023 for 6 months - in all patients who completed the 6-month treatment period Cohort Dose N Mean (CM) Standard deviation Cohort 1 0.25 mg/kg/week MOD-4023 9 13.48 2.71 Cohort 2 0.48 mg/kg/week MOD-4023 9 12.25 2.64 Cohort 3 0.66 mg/kg/week MOD-4023 10 14.37 5.26 Cohort 4 0.034 mcg/kg/day of genotropin 7 15.46 2.68 Historical Data - Daily Human GH Injections ~10

[00385] Simultaneously, a very favorable increase in SDS growth rate was also obtained (table 22, table 23 and figure 20). Finally, the growth rate delta SDS correlated well with the catch-up growth of patients (Figure 22).

[00386]

Table 22
Pediatric Phase 2 Clinical Study MOD-4023 - Results of a Preliminary Growth Rate SDS Study Cohort Dose N Average Standard off Standard err. avg. Cohort 1 0.25 mg/kg/week MOD-4023 8 -3.21 2.05 0.72 Cohort 2 0.48 mg/kg/week MOD-4023 8 -2.94 1.14 0.40 Cohort 3 0.66 mg/kg/week MOD-4023 10 -3.11 1.79 0.56 Cohort 4 0.034 mcg/kg/day of genotropin 7 -3.36 1.98 0.75

[00387]

Table 23
Pediatric Phase 2 Clinical Study MOD-4023 - Results of SDS Growth Rate Study Over 6 Months of Treatment Cohort Dose N Average Standard off Standard err. avg. Cohort 1 0.25 mg/kg/week
MOD-4023 8 5.03 1.23 0.44 Cohort 2 0.48 mg/kg/week
MOD-4023 8 3.23 1.88 0.67 Cohort 3 0.66 mg/kg/week
MOD-4023 10 5.73 3.72 1.18 Cohort 4 0.034 mcg/kg/day of genotropin 7 5.67 1.53 0.58

Conclusion

[00388] All doses provided a good catch-up growth response. Preliminary statistical analysis indicates that there are no statistically significant differences between cohorts, but there are some limitations regarding the small number of patients in the cohort and regarding patients with severe GHD.

EXAMPLE 11

Development of the composition MOD-4023

[00389] Protein: MOD-4023 is a long-acting recombinant human growth hormone (HGH) for subcutaneous administration. MOD-4023 consists of human GH hybridized to three copies of the C-terminal peptide (CTP) of the human chorionic gonadotropin (hCG) beta chain; CTP contains four O-glycosylation sites and therefore this protein is a single chain containing 275 amino acids and up to twelve O-linked carbohydrates. The protein is produced in CHO cells from a producing clone.

[00390] Producing clone: Clone #2 was the original clone used for early toxicological studies, phase I and phase II (adults). Stability data for DS and DP for this clone is available for up to 2 years at -20°C and 5°C. The transition to a new producing clone (clone # 28) was carried out in order to improve the performance and stability of the clone. Clone #28 DP supported long-term toxicological and phase II studies in children and will support all future clinical activities and commercial production. Stability data for DP for this clone is available for up to 1 year at -20°C and 5°C.

[00391] Manufacturing of CMO: Manufacturing of MOD-4023 was performed by Xcellerex (USA) in the early stages and supported pre-clinical studies up to phase II. The method was transferred to Rentschler Biotechnologie (RB), (Germany). Two batches in accordance with the requirements of the GMP have already been produced in RB.

Additional Information

[00392] Physical and chemical properties -

[00393] Highly glycosylated and negatively charged with IEP = 3-4.5

[00394] Density: 1.0216 g/ml

[00395] Soluble in aqueous solution

[00396] Liquid composition for DS and DP: 10 mM citrate, 147 mM NaCl, pH=6.

[00397] DS final concentration: 40 mg/ml

[00398] Final DP concentration: 5, 10, 20 and 40 mg/ml

[00399] Primary packaging -

[00400] 2R vials (Schott)

[00401] Plugs (West)

[00402] Aluminum caps (West)

[00403] Future primary packaging - syringe pen

Substantiation of composition optimization

[00404] Development of a stable liquid formulation for MOD-4023:

[00405] 1. First target: Stability for 2 years at 5°C in ampoules

[00406] 2. Second Goal: Stability for 2 years at 5°C in cartridges

[00407] Necessary analytical studies:

[00408] RP-HPLC (validated method)

[00409] HPLC with size exclusion chromatography (validated method)

[00410] Capillary Zone Electrophoresis (TBD) (established method)

[00411] Graph:

[00412] Based on the stability data, a 2-week test period at 25°C can be used to predict the stability of the product at 5°C to provide an initial estimate for comprehensive matrix formulation studies.

Stability Data

[00413] Data are shown for batches produced within the GMP and outside the GMP, made in 10 mm citrate, 147 mm NaCl, pH=6 (figure 24). The data also show that clone 2 MOD-4023 is stable at 5°C for 24 months at a concentration of 20 mg/ml, and that the stability profile is similar for 5 mg/ml and 20 mg/ml (see figure 25A and 25B) . In addition, clone 2 MOD-4023 is stable for 3 months at room temperature (see figure 26). Clone 28 MOD-4023 is stable at 5°C for 12 months at 20 mg/mL or 40 mg/mL; characteristic of the main peak DP>88% (figure 27A and figure 27B). Clone 28 MOD-4023 is stable for at least one month at room temperature. (figure 28A).

[00414] Based on the similarity between clone 28 and clone 2, which is stable at 5°C for 24 months, Xcellerex (XC) made clone 28 is expected to be stable for 24 months at 5°C; characteristic of the main peak DP>88% (figure 28B).

Production of MOD-4023

[00415] The profile at T=0 is comparable between Xcellerex (XC) and Rentschler (RB) DS (Figure 29). Figures 30-34 show the differences in stability between XC and RB. The results of isoelectric focusing (IEF) indicate that there is a similar band structure in the range of IEF values from 3.5 to 4.2. In one batch of XC, there are weaker isoforms at the upper limit of the IET, and in batch RB, there are weaker isoforms at the lower limit of the IET (figure 35A). In addition, there are more diffuse bands in the XC sample compared to the RB sample (Figure 35B).

[00416] Additional observations showed that the formation of both peaks (3 and 5 - peak definitions below) is temperature dependent and accelerates at high temperature (Figure 36A-D.). In addition, there was no change in % peak 3 after incubation for 5 days at pH=4 and up to 2 hours at pH=12 (Figure 37B, 37D), and there was also no change in % peak after incubation for up to 6 hours at pH=4. However, after 6 hours, a sharp increase in % peak was observed; when incubated for no more than 2 hours at pH=12 - peak disappears (figure 37A-D).

Forced degradation studies in RB (clone 28)

[00417] A stressed MOD-4023 active ingredient sample was prepared (65° C. for approximately three days) to test the related Form 5 in MOD-4023 active ingredient because the peak is below the quantitation limit for the non-stressed sample.

[00418] In order to test the effect of pH, three samples were examined by reverse-phase HPLC related forms:

[00419] RB - 40 mg/ml, pH=5.9.

[00420] RB - 10 mg/ml, pH=6.2.

[00421] XC - 40 mg/ml, pH=6.2.

[00422] The results are shown in Figures 38 and 39.

Isolation and characterization of peaks of related forms (1-7) - M-Scan

[00423] Peak 1 - oxidation of deamidated MOD-4023

[00424] Peak 2 - deamidation of MOD-4023

[00425] Peak 3 - partial oxidation of MOD-4023

[00426] Peak 5 - Cleavage of the peptide bond between amino acid residues 167 and 168 and between amino acid residues 171 and 172 of MOD-4023. disulfide

[00427] Peak 6 and Peak 7 Truncated Shapes

Conclusions (see figures 24-39)

Stability

[00428] The product obtained from clone 2 is stable for a period of not more than 2 years at 5°C.

[00429] The product obtained from clone 28 produced in XC is stable for at least 1 year at 5°C with a stability profile similar to that of clone 2.

[00430] The product derived from clone 28 produced in RB has an altered stability profile with accelerated formation of related forms (mainly peak 5) and the formation of a new peak (peak 7) that was not previously observed.

[00431] Previous studies have shown that peak 3 and 5 have a similar molecular weight as the main peak and react with an anti-GH antibody to form a band corresponding to the band of MOD-4023.

[00432] Both peaks 3 and 5 are temperature dependent (% of peaks increases with temperature).

[00433] Did not observe changes in the proportion of high molecular weight forms during incubation at -20°C and 5°C.

[00434] Stability of the RB GMP1 product (after 2 weeks incubation) at various temperatures, 5, 25, 37, 50 and 65°C, revealed that peak 7 formation is accelerated at 25°C and 37°C, but not observed at 50°C and 65°C.

[00435] Incubation of RB samples for 10 min at 65°C followed by incubation at 25°C eliminated the formation of peak 7 (after 2 weeks at 25°C).

[00436] The preferred embodiments of the present invention have been described above with reference to the accompanying drawings, it should be understood that the present invention is not limited to the exact embodiments, and various changes and modifications can be made to the present invention by a person skilled in the art without departing from the scope or spirit of the present invention as defined in the appended claims.

--->

SEQUENCE LIST

<110> OPKO BIOLOGICS LTD.

FARES, Fuad

FIMA, Udi Eyal

<120> LONG-LASTING POLYPEPTIDES AND METHODS OF THEIR PRODUCTION AND ADMINISTRATION

<130> P-9520-PC6

<150> 14/059.134

<151> 2013-10-21

<150> 14/309.496

<151> 2014-06-19

<160> 50

<170> PatentIn version 3.5

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<400> 21

tctagaggtc atcatggggg tgcacgaatg tcctgcctgg ctgtggcttc tcctgtccct 60

tctgtcgctc cctctgggcc tcccagtcct gggctcctct tcctcaaagg cccctccccc 120

gagccttcca agtccatccc gactcccggg gccctcggac accccaatat taccacaagc 180

cccaccacgc ctcatctgtg acagccgagt cctggagagg tacctcttgg aggccaagga 240

ggccgagaat atcacgacgg gctgtgctga acactgcagc ttgaatgaga atatcactgt 300

cccagacacc aaagttaatt tctatgcctg gaagaggatg gaggtcgggc agcaggccgt 360

agaagtctgg cagggcctgg ccctgctgtc ggaagctgtc ctgcggggcc aggccctgtt 420

ggtcaactct tcccagccgt gggagcccct gcagctgcat gtggataaag ccgtcagtgg 480

ccttcgcagc ctcaccactc tgcttcgggc tctgggagcc cagaaggaag ccatctcccc 540

tccagatgcg gcctcagctg ctccactccg aacaatcact gctgacactt tccgcaaact 600

cttccgagtc tactccaatt tcctccgggg aaagctgaag ctgtacacag gggaggcctg 660

caggacaggg gacagatcct cttcctcaaa ggcccctccc ccgagccttc caagtccatc 720

ccgactcccg gggccctccg acacaccaat cctgccacag agcagctcct ctaaggcccc 780

tcctccatcc ctgccatccc cctcccggct gcctggcccc tctgacaccc ctatcctgcc 840

tcagtgatga aggtcttctg gatccgcggc cgc 873

<210> 22

<211> 221

<212> PRT

<213> Homo sapiens

<400> 22

Met Gly Val His Glu Cys Pro Ala Trp Leu Trp Leu Leu Leu Ser Leu

1 5 10 15

Leu Ser Leu Pro Leu Gly Leu Pro Val Leu Gly Ala Pro Pro Arg Leu

20 25 30

Ile Cys Asp Ser Arg Val Leu Glu Arg Tyr Leu Leu Glu Ala Lys Glu

35 40 45

Ala Glu Asn Ile Thr Thr Gly Cys Ala Glu His Cys Ser Leu Asn Glu

50 55 60

Asn Ile Thr Val Pro Asp Thr Lys Val Asn Phe Tyr Ala Trp Lys Arg

65 70 75 80

Met Glu Val Gly Gln Gln Ala Val Glu Val Trp Gln Gly Leu Ala Leu

85 90 95

Leu Ser Glu Ala Val Leu Arg Ser Gln Ala Leu Leu Val Asn Ser Ser

100 105 110

Gln Pro Trp Glu Pro Leu Gln Leu His Val Asp Lys Ala Val Ser Gly

115 120 125

Leu Arg Ser Leu Thr Thr Leu Leu Arg Ala Leu Gly Ala Gln Lys Glu

130 135 140

Ala Ile Ser Pro Pro Asp Ala Ala Ser Ala Ala Pro Leu Arg Thr Ile

145 150 155 160

Thr Ala Asp Thr Phe Arg Lys Leu Phe Arg Val Tyr Ser Asn Phe Leu

165 170 175

Arg Gly Lys Leu Lys Leu Tyr Thr Gly Glu Ala Cys Arg Thr Gly Asp

180 185 190

Arg Ser Ser Ser Ser Lys Ala Pro Pro Pro Ser Leu Pro Ser Pro Ser

195 200 205

Arg Leu Pro Gly Pro Ser Asp Thr Pro Ile Leu Pro Gln

210 215 220

<210> 23

<211> 217

<212> PRT

<213> Homo sapiens

<400> 23

Met Ala Thr Gly Ser Arg Thr Ser Leu Leu Leu Ala Phe Gly Leu Leu

1 5 10 15

Cys Leu Pro Trp Leu Gln Glu Gly Ser Ala Phe Pro Thr Ile Pro Leu

20 25 30

Ser Arg Leu Phe Asp Asn Ala Met Leu Arg Ala His Arg Leu His Gln

35 40 45

Leu Ala Phe Asp Thr Tyr Gln Glu Phe Glu Glu Ala Tyr Ile Pro Lys

50 55 60

Val Gln Lys Tyr Ser Phe Leu Gln Asn Pro Gln Thr Ser Leu Cys Phe

65 70 75 80

Ser Glu Ser Ile Pro Thr Pro Ser Asn Arg Glu Glu Thr Gln Gln Lys

85 90 95

Ser Asn Leu Glu Leu Leu Arg Ile Ser Leu Leu Leu Ile Gln Ser Trp

100 105 110

Leu Glu Pro Val Gln Phe Leu Arg Ser Val Phe Ala Asn Ser Leu Val

115 120 125

Tyr Gly Ala Ser Asp Ser Asn Val Tyr Asp Leu Leu Lys Asp Leu Glu

130 135 140

Glu Gly Ile Gln Thr Leu Met Gly Arg Leu Glu Asp Gly Ser Pro Arg

145 150 155 160

Thr Gly Gln Ile Phe Lys Gln Thr Tyr Ser Lys Phe Asp Thr Asn Ser

165 170 175

His Asn Asp Asp Ala Leu Leu Lys Asn Tyr Gly Leu Leu Tyr Cys Phe

180 185 190

Arg Lys Asp Met Asp Lys Val Glu Thr Phe Leu Arg Ile Val Gln Cys

195 200 205

Arg Ser Val Glu Gly Ser Cys Gly Phe

210 215

<210> 24

<211> 166

<212> PRT

<213> Homo sapiens

<400> 24

Met Ser Tyr Asn Leu Leu Gly Phe Leu Gln Arg Ser Ser Asn Phe Gln

1 5 10 15

Ser Gln Lys Leu Leu Trp Gln Leu Asn Gly Arg Leu Glu Tyr Cys Leu

20 25 30

Lys Asp Arg Met Asn Phe Asp Ile Pro Glu Glu Ile Lys Gln Leu Gln

35 40 45

Gln Phe Gln Lys Glu Asp Ala Ala Leu Thr Ile Tyr Glu Met Leu Gln

50 55 60

Asn Ile Phe Ala Ile Phe Arg Gln Asp Ser Ser Ser Thr Gly Trp Asn

65 70 75 80

Glu Thr Ile Val Glu Asn Leu Leu Ala Asn Val Tyr His Gln Ile Asn

85 90 95

His Leu Lys Thr Val Leu Glu Glu Lys Leu Glu Lys Glu Asp Phe Thr

100 105 110

Arg Gly Lys Leu Met Ser Ser Leu His Leu Lys Arg Tyr Tyr Gly Arg

115 120 125

Ile Leu His Tyr Leu Lys Ala Lys Glu Tyr Ser His Cys Ala Trp Thr

130 135 140

Ile Val Arg Val Glu Ile Leu Arg Asn Phe Tyr Phe Ile Asn Arg Leu

145 150 155 160

Thr Gly Tyr Leu Arg Asn

165

<210> 25

<211> 30

<212> PRT

<213> Homo sapiens

<400> 25

His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly

1 5 10 15

Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg

20 25 30

<210> 26

<211> 21

<212> PRT

<213> Homo sapiens

<400> 26

Met Thr Asn Lys Cys Leu Leu Gln Ile Ala Leu Leu Leu Cys Phe Ser

1 5 10 15

Thr Thr Ala Leu Ser

20

<210> 27

<211> 19

<212> DNA

<213> Artificial

<220>

<223> XbaI forward primer for HGH-CTP constructs

<400> 27

ctctagagga catggccac 19

<210> 28

<211> 24

<212> DNA

<213> Artificial

<220>

<223> Reverse primer for HGH-CTP constructs

<400> 28

acagggaggt ctgggggttc tgca 24

<210> 29

<211> 26

<212> DNA

<213> Artificial

<220>

<223> Forward primer for HGH-CTP constructs

<400> 29

tgcagaaccc ccagacctcc ctgtgc 26

<210> 30

<211> 22

<212> DNA

<213> Artificial

<220>

<223> Reverse primer for HGH-CTP constructs

<400> 30

ccaaactcat caatgtatct ta 22

<210> 31

<211> 19

<212> DNA

<213> Artificial

<220>

<223> XbaI forward primer for HGH-CTP constructs

<400> 31

ctctagagga catggccac 19

<210> 32

<211> 25

<212> DNA

<213> Artificial

<220>

<223> Reverse primer for HGH-CTP constructs

<400> 32

cgaactcctg gtaggtgtca aaggc 25

<210> 33

<211> 25

<212> DNA

<213> Artificial

<220>

<223> Forward primer for HGH-CTP constructs

<400> 33

gcctttgaca cctacgga gttcg 25

<210> 34

<211> 33

<212> DNA

<213> Artificial

<220>

<223> NotI reverse primer for HGH-CTP constructs

<400> 34

acgcggccgc atccagacct tcatcactga ggc 33

<210> 35

<211> 34

<212> DNA

<213> Artificial

<220>

<223> Reverse primer for HGH-CTP constructs

<400> 35

gcggccgcgg actcatcaga agccgcagct gccc 34

<210> 36

<211> 217

<212> PRT

<213> Homo sapiens

<400> 36

Met Ala Thr Gly Ser Arg Thr Ser Leu Leu Leu Ala Phe Gly Leu Leu

1 5 10 15

Cys Leu Pro Trp Leu Gln Glu Gly Ser Ala Phe Pro Thr Ile Pro Leu

20 25 30

Ser Arg Leu Phe Asp Asn Ala Met Leu Arg Ala His Arg Leu His Gln

35 40 45

Leu Ala Phe Asp Thr Tyr Gln Glu Phe Glu Glu Ala Tyr Ile Pro Lys

50 55 60

Glu Gln Lys Tyr Ser Phe Leu Gln Asn Pro Gln Thr Ser Leu Cys Phe

65 70 75 80

Ser Glu Ser Ile Pro Thr Pro Ser Asn Arg Glu Glu Thr Gln Gln Lys

85 90 95

Ser Asn Leu Glu Leu Leu Arg Ile Ser Leu Leu Leu Ile Gln Ser Trp

100 105 110

Leu Glu Pro Val Gln Phe Leu Arg Ser Val Phe Ala Asn Ser Leu Val

115 120 125

Tyr Gly Ala Ser Asp Ser Asn Val Tyr Asp Leu Leu Lys Asp Leu Glu

130 135 140

Glu Gly Ile Gln Thr Leu Met Gly Arg Leu Glu Asp Gly Ser Pro Arg

145 150 155 160

Thr Gly Gln Ile Phe Lys Gln Thr Tyr Ser Lys Phe Asp Thr Asn Ser

165 170 175

His Asn Asp Asp Ala Leu Leu Lys Asn Tyr Gly Leu Leu Tyr Cys Phe

180 185 190

Arg Lys Asp Met Asp Lys Val Glu Thr Phe Leu Arg Ile Val Gln Cys

195 200 205

Arg Ser Val Glu Gly Ser Cys Gly Phe

210 215

<210> 37

<211> 245

<212> PRT

<213> Homo sapiens

<400> 37

Met Ala Thr Gly Ser Arg Thr Ser Leu Leu Leu Ala Phe Gly Leu Leu

1 5 10 15

Cys Leu Pro Trp Leu Gln Glu Gly Ser Ala Phe Pro Thr Ile Pro Leu

20 25 30

Ser Arg Leu Phe Asp Asn Ala Met Leu Arg Ala His Arg Leu His Gln

35 40 45

Leu Ala Phe Asp Thr Tyr Gln Glu Phe Glu Glu Ala Tyr Ile Pro Lys

50 55 60

Glu Gln Lys Tyr Ser Phe Leu Gln Asn Pro Gln Thr Ser Leu Cys Phe

65 70 75 80

Ser Glu Ser Ile Pro Thr Pro Ser Asn Arg Glu Glu Thr Gln Gln Lys

85 90 95

Ser Asn Leu Glu Leu Leu Arg Ile Ser Leu Leu Leu Ile Gln Ser Trp

100 105 110

Leu Glu Pro Val Gln Phe Leu Arg Ser Val Phe Ala Asn Ser Leu Val

115 120 125

Tyr Gly Ala Ser Asp Ser Asn Val Tyr Asp Leu Leu Lys Asp Leu Glu

130 135 140

Glu Gly Ile Gln Thr Leu Met Gly Arg Leu Glu Asp Gly Ser Pro Arg

145 150 155 160

Thr Gly Gln Ile Phe Lys Gln Thr Tyr Ser Lys Phe Asp Thr Asn Ser

165 170 175

His Asn Asp Asp Ala Leu Leu Lys Asn Tyr Gly Leu Leu Tyr Cys Phe

180 185 190

Arg Lys Asp Met Asp Lys Val Glu Thr Phe Leu Arg Ile Val Gln Cys

195 200 205

Arg Ser Val Glu Gly Ser Cys Gly Phe Ser Ser Ser Ser Lys Ala Pro

210 215 220

Pro Pro Ser Leu Pro Ser Pro Ser Arg Leu Pro Gly Pro Ser Asp Thr

225 230 235 240

Pro Ile Leu Pro Gln

245

<210> 38

<211> 273

<212> PRT

<213> Homo sapiens

<400> 38

Met Ala Thr Gly Ser Arg Thr Ser Leu Leu Leu Ala Phe Gly Leu Leu

1 5 10 15

Cys Leu Pro Trp Leu Gln Glu Gly Ser Ala Phe Pro Thr Ile Pro Leu

20 25 30

Ser Arg Leu Phe Asp Asn Ala Met Leu Arg Ala His Arg Leu His Gln

35 40 45

Leu Ala Phe Asp Thr Tyr Gln Glu Phe Glu Glu Ala Tyr Ile Pro Lys

50 55 60

Glu Gln Lys Tyr Ser Phe Leu Gln Asn Pro Gln Thr Ser Leu Cys Phe

65 70 75 80

Ser Glu Ser Ile Pro Thr Pro Ser Asn Arg Glu Glu Thr Gln Gln Lys

85 90 95

Ser Asn Leu Glu Leu Leu Arg Ile Ser Leu Leu Leu Ile Gln Ser Trp

100 105 110

Leu Glu Pro Val Gln Phe Leu Arg Ser Val Phe Ala Asn Ser Leu Val

115 120 125

Tyr Gly Ala Ser Asp Ser Asn Val Tyr Asp Leu Leu Lys Asp Leu Glu

130 135 140

Glu Gly Ile Gln Thr Leu Met Gly Arg Leu Glu Asp Gly Ser Pro Arg

145 150 155 160

Thr Gly Gln Ile Phe Lys Gln Thr Tyr Ser Lys Phe Asp Thr Asn Ser

165 170 175

His Asn Asp Asp Ala Leu Leu Lys Asn Tyr Gly Leu Leu Tyr Cys Phe

180 185 190

Arg Lys Asp Met Asp Lys Val Glu Thr Phe Leu Arg Ile Val Gln Cys

195 200 205

Arg Ser Val Glu Gly Ser Cys Gly Phe Ser Ser Ser Ser Lys Ala Pro

210 215 220

Pro Pro Ser Leu Pro Ser Pro Ser Arg Leu Pro Gly Pro Ser Asp Thr

225 230 235 240

Pro Ile Leu Pro Gln Ser Ser Ser Ser Lys Ala Pro Pro Pro Ser Leu

245 250 255

Pro Ser Pro Ser Arg Leu Pro Gly Pro Ser Asp Thr Pro Ile Leu Pro

260 265 270

Gln

<210> 39

<211> 301

<212> PRT

<213> Homo sapiens

<400> 39

Met Ala Thr Gly Ser Arg Thr Ser Leu Leu Leu Ala Phe Gly Leu Leu

1 5 10 15

Cys Leu Pro Trp Leu Gln Glu Gly Ser Ala Ser Ser Ser Ser Lys Ala

20 25 30

Pro Pro Pro Ser Leu Pro Ser Pro Ser Arg Leu Pro Gly Pro Ser Asp

35 40 45

Thr Pro Ile Leu Pro Gln Phe Pro Thr Ile Pro Leu Ser Arg Leu Phe

50 55 60

Asp Asn Ala Met Leu Arg Ala His Arg Leu His Gln Leu Ala Phe Asp

65 70 75 80

Thr Tyr Gln Glu Phe Glu Glu Ala Tyr Ile Pro Lys Glu Gln Lys Tyr

85 90 95

Ser Phe Leu Gln Asn Pro Gln Thr Ser Leu Cys Phe Ser Glu Ser Ile

100 105 110

Pro Thr Pro Ser Asn Arg Glu Glu Thr Gln Gln Lys Ser Asn Leu Glu

115 120 125

Leu Leu Arg Ile Ser Leu Leu Leu Ile Gln Ser Trp Leu Glu Pro Val

130 135 140

Gln Phe Leu Arg Ser Val Phe Ala Asn Ser Leu Val Tyr Gly Ala Ser

145 150 155 160

Asp Ser Asn Val Tyr Asp Leu Leu Lys Asp Leu Glu Glu Gly Ile Gln

165 170 175

Thr Leu Met Gly Arg Leu Glu Asp Gly Ser Pro Arg Thr Gly Gln Ile

180 185 190

Phe Lys Gln Thr Tyr Ser Lys Phe Asp Thr Asn Ser His Asn Asp Asp

195 200 205

Ala Leu Leu Lys Asn Tyr Gly Leu Leu Tyr Cys Phe Arg Lys Asp Met

210 215 220

Asp Lys Val Glu Thr Phe Leu Arg Ile Val Gln Cys Arg Ser Val Glu

225 230 235 240

Gly Ser Cys Gly Phe Ser Ser Ser Ser Lys Ala Pro Pro Pro Ser Leu

245 250 255

Pro Ser Pro Ser Arg Leu Pro Gly Pro Ser Asp Thr Pro Ile Leu Pro

260 265 270

Gln Ser Ser Ser Ser Lys Ala Pro Pro Pro Ser Leu Pro Ser Pro Ser

275 280 285

Arg Leu Pro Gly Pro Ser Asp Thr Pro Ile Leu Pro Gln

290 295 300

<210> 40

<211> 285

<212> PRT

<213> Homo sapiens

<400> 40

Met Ala Thr Gly Ser Arg Thr Ser Leu Leu Leu Ala Phe Gly Leu Leu

1 5 10 15

Cys Leu Pro Trp Leu Gln Glu Gly Ser Ala Ser Ser Ser Ser Lys Ala

20 25 30

Pro Pro Pro Ser Leu Pro Phe Pro Thr Ile Pro Leu Ser Arg Leu Phe

35 40 45

Asp Asn Ala Met Leu Arg Ala His Arg Leu His Gln Leu Ala Phe Asp

50 55 60

Thr Tyr Gln Glu Phe Glu Glu Ala Tyr Ile Pro Lys Glu Gln Lys Tyr

65 70 75 80

Ser Phe Leu Gln Asn Pro Gln Thr Ser Leu Cys Phe Ser Glu Ser Ile

85 90 95

Pro Thr Pro Ser Asn Arg Glu Glu Thr Gln Gln Lys Ser Asn Leu Glu

100 105 110

Leu Leu Arg Ile Ser Leu Leu Leu Ile Gln Ser Trp Leu Glu Pro Val

115 120 125

Gln Phe Leu Arg Ser Val Phe Ala Asn Ser Leu Val Tyr Gly Ala Ser

130 135 140

Asp Ser Asn Val Tyr Asp Leu Leu Lys Asp Leu Glu Glu Gly Ile Gln

145 150 155 160

Thr Leu Met Gly Arg Leu Glu Asp Gly Ser Pro Arg Thr Gly Gln Ile

165 170 175

Phe Lys Gln Thr Tyr Ser Lys Phe Asp Thr Asn Ser His Asn Asp Asp

180 185 190

Ala Leu Leu Lys Asn Tyr Gly Leu Leu Tyr Cys Phe Arg Lys Asp Met

195 200 205

Asp Lys Val Glu Thr Phe Leu Arg Ile Val Gln Cys Arg Ser Val Glu

210 215 220

Gly Ser Cys Gly Phe Ser Ser Ser Ser Lys Ala Pro Pro Pro Ser Leu

225 230 235 240

Pro Ser Pro Ser Arg Leu Pro Gly Pro Ser Asp Thr Pro Ile Leu Pro

245 250 255

Gln Ser Ser Ser Ser Lys Ala Pro Pro Pro Ser Leu Pro Ser Pro Ser

260 265 270

Arg Leu Pro Gly Pro Ser Asp Thr Pro Ile Leu Pro Gln

275 280 285

<210> 41

<211> 273

<212> PRT

<213> Homo sapiens

<400> 41

Met Ala Thr Gly Ser Arg Thr Ser Leu Leu Leu Ala Phe Gly Leu Leu

1 5 10 15

Cys Leu Pro Trp Leu Gln Glu Gly Ser Ala Ser Ser Ser Ser Lys Ala

20 25 30

Pro Pro Pro Ser Leu Pro Ser Pro Ser Arg Leu Pro Gly Pro Ser Asp

35 40 45

Thr Pro Ile Leu Pro Gln Phe Pro Thr Ile Pro Leu Ser Arg Leu Phe

50 55 60

Asp Asn Ala Met Leu Arg Ala His Arg Leu His Gln Leu Ala Phe Asp

65 70 75 80

Thr Tyr Gln Glu Phe Glu Glu Ala Tyr Ile Pro Lys Glu Gln Lys Tyr

85 90 95

Ser Phe Leu Gln Asn Pro Gln Thr Ser Leu Cys Phe Ser Glu Ser Ile

100 105 110

Pro Thr Pro Ser Asn Arg Glu Glu Thr Gln Gln Lys Ser Asn Leu Glu

115 120 125

Leu Leu Arg Ile Ser Leu Leu Leu Ile Gln Ser Trp Leu Glu Pro Val

130 135 140

Gln Phe Leu Arg Ser Val Phe Ala Asn Ser Leu Val Tyr Gly Ala Ser

145 150 155 160

Asp Ser Asn Val Tyr Asp Leu Leu Lys Asp Leu Glu Glu Gly Ile Gln

165 170 175

Thr Leu Met Gly Arg Leu Glu Asp Gly Ser Pro Arg Thr Gly Gln Ile

180 185 190

Phe Lys Gln Thr Tyr Ser Lys Phe Asp Thr Asn Ser His Asn Asp Asp

195 200 205

Ala Leu Leu Lys Asn Tyr Gly Leu Leu Tyr Cys Phe Arg Lys Asp Met

210 215 220

Asp Lys Val Glu Thr Phe Leu Arg Ile Val Gln Cys Arg Ser Val Glu

225 230 235 240

Gly Ser Cys Gly Phe Ser Ser Ser Ser Lys Ala Pro Pro Pro Ser Leu

245 250 255

Pro Ser Pro Ser Arg Leu Pro Gly Pro Ser Asp Thr Pro Ile Leu Pro

260 265 270

Gln

<210> 42

<211> 245

<212> PRT

<213> Homo sapiens

<400> 42

Met Ala Thr Gly Ser Arg Thr Ser Leu Leu Leu Ala Phe Gly Leu Leu

1 5 10 15

Cys Leu Pro Trp Leu Gln Glu Gly Ser Ala Ser Ser Ser Ser Lys Ala

20 25 30

Pro Pro Pro Ser Leu Pro Ser Pro Ser Arg Leu Pro Gly Pro Ser Asp

35 40 45

Thr Pro Ile Leu Pro Gln Phe Pro Thr Ile Pro Leu Ser Arg Leu Phe

50 55 60

Asp Asn Ala Met Leu Arg Ala His Arg Leu His Gln Leu Ala Phe Asp

65 70 75 80

Thr Tyr Gln Glu Phe Glu Glu Ala Tyr Ile Pro Lys Glu Gln Lys Tyr

85 90 95

Ser Phe Leu Gln Asn Pro Gln Thr Ser Leu Cys Phe Ser Glu Ser Ile

100 105 110

Pro Thr Pro Ser Asn Arg Glu Glu Thr Gln Gln Lys Ser Asn Leu Glu

115 120 125

Leu Leu Arg Ile Ser Leu Leu Leu Ile Gln Ser Trp Leu Glu Pro Val

130 135 140

Gln Phe Leu Arg Ser Val Phe Ala Asn Ser Leu Val Tyr Gly Ala Ser

145 150 155 160

Asp Ser Asn Val Tyr Asp Leu Leu Lys Asp Leu Glu Glu Gly Ile Gln

165 170 175

Thr Leu Met Gly Arg Leu Glu Asp Gly Ser Pro Arg Thr Gly Gln Ile

180 185 190

Phe Lys Gln Thr Tyr Ser Lys Phe Asp Thr Asn Ser His Asn Asp Asp

195 200 205

Ala Leu Leu Lys Asn Tyr Gly Leu Leu Tyr Cys Phe Arg Lys Asp Met

210 215 220

Asp Lys Val Glu Thr Phe Leu Arg Ile Val Gln Cys Arg Ser Val Glu

225 230 235 240

Gly Ser Cys Gly Phe

245

<210> 43

<211> 12

<212> PRT

<213> Artificial

<220>

<223> CTP amino acid sequence

<400> 43

Ser Ser Ser Ser Lys Ala Pro Pro Pro Ser Leu Pro

1 5 10

<210> 44

<211> 853

<212> DNA

<213> Homo sapiens

<400> 44

tctagaggac atggccaccg gcagcaggac cagcctgctg ctggccttcg gcctgctgtg 60

cctgccatgg ctgcaggagg gcagcgccag ctcttcttct aaggctccac ccccatctct 120

gcccagcccc agcagactgc cgggccccag cgacacaccc attctgcccc agttccccac 180

catccccctg agcaggctgt tcgacaacgc catgctgagg gctcacaggc tgcaccagct 240

ggcctttgac acctaccagg agttcgagga agcctacatc cccaaggagc agaagtacag 300

cttcctgcag aacccccaga cctccctgtg cttcagcgag agcatcccca cccccagcaa 360

cagagaggag acccagcaga agagcaacct ggagctgctg aggatctccc tgctgctgat 420

ccagagctgg ctggagcccg tgcagttcct gagaagcgtg ttcgccaaca gcctggtgta 480

cggcgccagc gacagcaacg tgtacgacct gctgaaggac ctggaggagg gcatccagac 540

cctgatgggc cggctggagg acggcagccc caggaccggc cagatcttca agcagaccta 600

cagcaagttc gacaccaaca gccacaacga cgacgccctg ctgaagaact acgggctgct 660

gtactgcttc agaaaggaca tggacaaggt ggagaccttc ctgaggatcg tgcagtgcag 720

aagcgtggag ggcagctgcg gcttcagctc cagcagcaag gcccctcccc cgagcctgcc 780

ctccccaagc aggctgcctg ggccctccga cacaccaatc ctgcctcagt gatgaaggtc 840

tggatgcggc cgc 853

<210> 45

<211> 937

<212> DNA

<213> Homo sapiens

<400> 45

tctagaggac atggccaccg gcagcaggac cagcctgctg ctggccttcg gcctgctgtg 60

cctgccatgg ctgcaggagg gcagcgccag ctcttcttct aaggctccac ccccatctct 120

gcccagcccc agcagactgc cgggccccag cgacacaccc attctgcccc agttccccac 180

catccccctg agcaggctgt tcgacaacgc catgctgagg gctcacaggc tgcaccagct 240

ggcctttgac acctaccagg agttcgagga agcctacatc cccaaggagc agaagtacag 300

cttcctgcag aacccccaga cctccctgtg cttcagcgag agcatcccca cccccagcaa 360

cagagaggag acccagcaga agagcaacct ggagctgctg aggatctccc tgctgctgat 420

ccagagctgg ctggagcccg tgcagttcct gagaagcgtg ttcgccaaca gcctggtgta 480

cggcgccagc gacagcaacg tgtacgacct gctgaaggac ctggaggagg gcatccagac 540

cctgatgggc cggctggagg acggcagccc caggaccggc cagatcttca agcagaccta 600

cagcaagttc gacaccaaca gccacaacga cgacgccctg ctgaagaact acgggctgct 660

gtactgcttc agaaaggaca tggacaaggt ggagaccttc ctgaggatcg tgcagtgcag 720

aagcgtggag ggcagctgcg gcttcagctc cagcagcaag gcccctcccc cgagcctgcc 780

ctccccaagc aggctgcctg ggccctccga cacaccaatc ctgccacaga gcagctcctc 840

taaggcccct cctccatccc tgccatcccc ctcccggctg cctggcccct ctgacacccc 900

tatcctgcct cagtgatgaa ggtctggatg cggccgc 937

<210> 46

<211> 889

<212> DNA

<213> Homo sapiens

<400> 46

tctagaggac atggccaccg gcagcaggac cagcctgctg ctggccttcg gcctgctgtg 60

cctgccatgg ctgcaggagg gcagcgccag ctcttcttct aaggctccac ccccgagcct 120

gcccttcccc accatccccc tgagcaggct gttcgacaac gccatgctga gggctcacag 180

gctgcaccag ctggcctttg acacctacca ggagttcgag gaagcctaca tccccaagga 240

gcagaagtac agcttcctgc agaaccccca gacctccctg tgcttcagcg agagcatccc 300

cacccccagc aacagagagg agacccagca gaagagcaac ctggagctgc tgaggatctc 360

cctgctgctg atccagagct ggctggagcc cgtgcagttc ctgagaagcg tgttcgccaa 420

cagcctggtg tacggcgcca gcgacagcaa cgtgtacgac ctgctgaagg acctggagga 480

gggcatccag accctgatgg gccggctgga ggacggcagc cccaggaccg gccagatctt 540

caagcagacc tacagcaagt tcgacaccaa cagccacaac gacgacgccc tgctgaagaa 600

ctacgggctg ctgtactgct tcagaaagga catggacaag gtggagacct tcctgaggat 660

cgtgcagtgc agaagcgtgg agggcagctg cggcttcagc tccagcagca aggcccctcc 720

cccgagcctg ccctccccaa gcaggctgcc tgggccctcc gacacaccaa tcctgccaca 780

gagcagctcc tctaaggccc ctcctccatc cctgccatcc ccctcccggc tgcctggccc 840

ctctgacacc cctatcctgc ctcagtgatg aaggtctgga tgcggccgc 889

<210> 47

<211> 217

<212> PRT

<213> Homo sapiens

<400> 47

Met Ala Thr Gly Ser Arg Thr Ser Leu Leu Leu Ala Phe Gly Leu Leu

1 5 10 15

Cys Leu Pro Trp Leu Gln Glu Gly Ser Ala Phe Pro Thr Ile Pro Leu

20 25 30

Ser Arg Leu Phe Asp Asn Ala Met Leu Arg Ala His Arg Leu His Gln

35 40 45

Leu Ala Phe Asp Thr Tyr Gln Glu Phe Glu Glu Ala Tyr Ile Pro Lys

50 55 60

Glu Gln Lys Tyr Ser Phe Leu Gln Asn Pro Gln Thr Ser Leu Cys Phe

65 70 75 80

Ser Glu Ser Ile Pro Thr Pro Ser Asn Arg Glu Glu Thr Gln Gln Lys

85 90 95

Ser Asn Leu Glu Leu Leu Arg Ile Ser Leu Leu Leu Ile Gln Ser Trp

100 105 110

Leu Glu Pro Val Gln Phe Leu Arg Ser Val Phe Ala Asn Ser Leu Val

115 120 125

Tyr Gly Ala Ser Asp Ser Asn Val Tyr Asp Leu Leu Lys Asp Leu Glu

130 135 140

Glu Gly Ile Gln Thr Leu Met Gly Arg Leu Glu Asp Gly Ser Pro Arg

145 150 155 160

Thr Gly Gln Ile Phe Lys Gln Thr Tyr Ser Lys Phe Asp Thr Asn Ser

165 170 175

His Asn Asp Asp Ala Leu Leu Lys Asn Tyr Gly Leu Leu Tyr Cys Phe

180 185 190

Arg Lys Asp Met Asp Lys Val Glu Thr Phe Leu Arg Ile Val Gln Cys

195 200 205

Arg Ser Val Glu Gly Ser Cys Gly Phe

210 215

<210> 48

<211> 33

<212> PRT

<213> Homo sapiens

<400> 48

Asp Pro Arg Phe Gln Asp Ser Ser Ser Ser Lys Ala Pro Pro Pro Ser

1 5 10 15

Leu Pro Ser Pro Ser Arg Leu Pro Gly Pro Ser Asp Thr Pro Ile Leu

20 25 30

Gln

<210> 49

<211> 26

<212> PRT

<213> Homo sapiens

<400> 49

Met Ala Thr Gly Ser Arg Thr Ser Leu Leu Leu Ala Phe Gly Leu Leu

1 5 10 15

Cys Leu Pro Trp Leu Gln Glu Gly Ser Ala

20 25

<210> 50

<211> 17

<212> DNA

<213> Artificial sequence

<220>

<223> Reverse primer for EPO-CTP constructs

<400> 50

gcttccgaca gcagggc 17

<---

Claims (37)

1. A pharmaceutical composition comprising a buffer, a tonicity agent, and a therapeutically effective amount of a modified human chorionic gonadotropin (CTP) carboxy-terminal peptide polypeptide consisting of growth hormone having one human chorionic gonadotropin CTP attached to the amino terminus of the growth hormone and two human chorionic gonadotropin CTPs attached to the carboxy terminus of growth hormone and formulated to maintain insulin-like growth factor (IGF-1) levels in the normal range with standard deviation index (SDS) values within ±2 in a human subject, with CTP- the modified polypeptide is CTP-modified growth hormone, and where the buffer is 10 mM citrate, the tonicity agent is 147 mM sodium chloride, where the amino acid sequence of the CTP-modified growth hormone corresponds to amino acids 27 to 301 in SEQ ID NO: 39 or amino acids 1 to 301 in SEQ ID NO: 39, and wherein the CTP-modified polypeptide is stable at -20°C and 5°C for 24 months. 2. Pharmaceutical composition according to claim 1, characterized in that the composition has a pH of approximately 6.2-6.6. 3. Pharmaceutical composition according to claim. 1 or 2, characterized in that the composition is a liquid composition. 4. Pharmaceutical composition according to any one of paragraphs. 1-3, characterized in that at least one CTP of the CTP-modified polypeptide consists of the amino acid sequence of SEQ ID NO: 18. 5. Pharmaceutical composition according to any one of paragraphs. 1-4, characterized in that at least one CTP of the CTP-modified polypeptide is glycosylated. 6. Pharmaceutical composition according to any one of paragraphs. 1-5, characterized in that the CTP-modified polypeptide additionally consists of a signal peptide attached to the amino-terminus of one of the CTPs. 7. Pharmaceutical composition according to claim 6, characterized in that the signal peptide sequence is shown in SEQ ID NO: 49. 8. Pharmaceutical composition according to any one of paragraphs. 1-7, characterized in that at least one CTP of the CTP-modified polypeptide is optionally attached to the specified growth hormone via a covalent linker. 9. Pharmaceutical composition according to claim 8, characterized in that the covalent linker is a peptide bond. 10. Pharmaceutical composition according to any one of paragraphs. 1-9, characterized in that the CTP-modified growth hormone is encoded by the nucleic acid sequence represented by nucleotides 89-913 of SEQ ID NO: 45 or nucleotides 11-913 of SEQ ID NO: 45. 11. Pharmaceutical composition according to any one of paragraphs. 1-10, characterized in that the growth hormone is human growth hormone (hGH). 12. Pharmaceutical composition according to any one of paragraphs. 1-11 formulated for intravenous or subcutaneous administration. 13. Pharmaceutical composition according to any one of paragraphs. 1-12 formulated for administration once a week to a human subject. 14. Pharmaceutical composition according to any one of paragraphs. 1-13, wherein the human subject is deficient in growth hormone. 15. Pharmaceutical composition according to any one of paragraphs. 1-14, characterized in that the normal range in a human subject is ±1.5 index standard deviation (SDS). 16. Pharmaceutical composition according to any one of paragraphs. 13-15, characterized in that the human subject is an adult or a child. 17. Pharmaceutical composition according to any one of paragraphs. 13-16, characterized in that the CTP-modified growth hormone is administered to the specified subject at a dose of 0.25 milligrams per kilogram per week (mg/kg/week), 0.48 mg/kg/week, 0.66 mg/kg/ week or a combination. 18. Dosage form for administration once a week, containing a pharmaceutical composition according to any one of paragraphs. 1-17 to maintain insulin-like growth factor (IGF-1) levels in the normal range with Standard Deviation Index (SDS) values within ±2 in a human subject. 19. A method of preparing a syringe containing a pharmaceutical composition according to any one of paragraphs. 1-17 for once-weekly administration to a human subject who is deficient in growth hormone to maintain insulin-like growth factor (IGF-1) levels within the normal ±2 index standard deviation (SDS) range in a human subject who is deficient growth hormone, the method comprising the steps of: (a) providing a syringe; (b) modifying growth hormone by attaching one carboxy-terminal peptide (CTP) of human chorionic gonadotropin to the amino terminus of growth hormone and two CTPs of human chorionic gonadotropin to the carboxy-terminus of growth hormone; (c) mixing the modified growth hormone in step (b) with a buffer and a tonicity agent at pH 6.2-6.6; And (d) prefilling the syringe with the pharmaceutical composition. 20. A method of filling a syringe with a composition according to any one of paragraphs. 1-17, including stages: (a) providing a syringe; (b) making a dosage form of CTP-modified growth hormone for administration once a week, having a predetermined amount of CTP-modified growth hormone; And (c) filling the syringe with the pharmaceutical composition. 21. A pharmaceutical composition comprising a buffer, a tonicity regulating agent, and a therapeutically effective amount of a modified human chorionic gonadotropin carboxy-terminal peptide (CTP) polypeptide consisting of growth hormone having one human chorionic gonadotropin CTP attached to the amino terminus of the growth hormone and two Human Chorionic Gonadotropin CTPs attached to the carboxy-terminus of growth hormone and formulated to maintain insulin-like growth factor (IGF-1) levels in the normal range with IGF-1 Standard Deviation Index (SDS) values within ±2 in a human subject at In this CTP-modified polypeptide is a CTP-modified growth hormone, where the buffer is 10 mM citrate, the tonicity agent is 147 mM sodium chloride, where the therapeutically effective amount is in the dose range of 1 to 90 milligrams per week (mg /week), where the amino acid sequence of the CTP-modified growth hormone is represented by amino acids 27 to 301 in SEQ ID NO: 39, and where the CTP-modified polypeptide is stable at -20°C and 5°C for 24 months. 22. The method of claim 19, wherein the normal range in a human subject is ±1.5 index standard deviation (SDS). 23. Pharmaceutical composition according to claim 21, characterized in that the normal range in a human subject is ±1.5 standard deviation index (SDS). 24. The pharmaceutical composition according to claim 21, characterized in that the dose range from 1 to 90 mg/week includes a dose of 0.25 milligrams per kilogram per week (mg/kg/week), 0.48 mg/kg/week, 0 .66 mg/kg/week or combinations thereof. 25. Pharmaceutical composition according to claim 21, characterized in that the dose range from 1 to 90 mg/week includes a dose from 1 mg/week to 50 mg/week. 26. A pharmaceutical composition according to claim 21, wherein the human subject is an adult or a child. 27. Pharmaceutical composition according to any one of paragraphs. 21 and 23-26, characterized in that the dose of a therapeutically effective amount of the CTP-modified polypeptide can be increased or decreased in subsequent doses if the levels of IGF-1 obtained as a result of monitoring on the 4th day after administration of the indicated dose are outside the normal range . 28. Pharmaceutical composition according to any one of paragraphs. 21 and 23-26, characterized in that the levels of IGF-1 include the levels of IGF-1 obtained as a result of monitoring on the 4th day after administration of the pharmaceutical composition. 29. The pharmaceutical composition according to claim 25, characterized in that the dose range from 1 to 50 mg/week includes a dose selected from the group consisting of doses from 1 to 5 mg/week, from 5 to 10 mg/week, from 10 up to 20 mg/week and 15 to 25 mg/week. 30. A pharmaceutical composition according to claim 26, wherein the human subject is deficient in growth hormone.

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