CN1411369A - Biodegradable microparticles with novel erythropoietin stimulating protein - Google Patents

Abstract

The present invention relates to a composition for the sustained release of biologically active, novel erythropoietin stimulating protein (NESP), and improved methods of forming said composition. The composition comprises polymeric microparticles within which particles of NESP have been dispersed. The improved method utilizes a cosolvent mixture to effect more efficient and rapid removal of residual polymer solvents during any drying process.

Description

Novel biodegradable microparticles of erythropoietin-stimulating protein

field of invention

The present invention relates to a sustained release composition of a novel biologically active erythropoietin stimulating protein (NESP) and to an improved method of shaping said composition. The composition contains polymeric microparticles in which NESP particles have been dispersed. The improved method utilizes a co-solvent mixture to achieve more efficient and faster removal of residual polymer solvent during any drying process.

Background of the invention

Over the past several years, extensive efforts have been made to develop effective sustained-release formulations that can provide a means of controlling blood levels of active ingredients while also offering greater efficacy, safety, and patient convenience and patient compliance. Unfortunately, the instability of most proteins (such as time-varying and loss of biological activity when exposed to heat, organic solvents, etc.) greatly limits the development and evaluation of sustained-release formulations.

Both patent and scientific literature have described methods of microparticle preparation in the prior art. In particular, various methods for the preparation of poly(lactic acid) (PLA) and poly(lactic-co-glycolic acid) (PLGA) biodegradable microparticles for the controlled release of water-soluble drugs are described; see for example Wise et al., Contraception, 8 : 227-234 (1973); Hutchinson et al., Biochem. Soc. Trans., 13 : 520-523 (1985); and Jalil et al., J. Microencapsul., 7 : 297-325 (1990); Putney et al., Nature Biotech ., 16 :153-157 (1998); Burke, P., Handbook of Pharmaceutical Controlled Release Technology, Klibanov et al. (eds.) (in press). These methods include those involving emulsification (phase separation, solvent extraction and solvent evaporation) and atomization (spray drying, spray freezing).

In some cases, the main disadvantages associated with the above-cited methods include: 1) protein inactivation due to high temperature; 2) protein inactivation due to exposure to organic solvents; 3) loss of drug in the aqueous phase used for extraction of organic solvent 4) Large amounts of organic solvents are usually required during the process, and these organic solvents cannot be sufficiently removed from the final product, i.e., the residual solvent level in the final product is high; 5) Protein loading efficiency low; 6) low overall yield; 7) problems with drug leakage and/or high initial release of drug upon administration; and 8) costly and complicated scale-up of production. Point 4) For details, the problems associated with high levels of solvents in the spray drying process have been described; see e.g. Clarke et al., Drug Devel. And Industrial Pharmacy, 24 : 169-175 (1998); Bitz and Doelker, Inter. Journal of Pharm ., 131 :171-181 (1996); and Takada et al., Journ. Of Controlled Release, 32 :79-85 (1994).

There have been numerous reports of improved microparticle preparation methods both in the patent and scientific literature. For example, US Patent No. 5,019,400 to Gombotz et al. discloses a microparticle preparation method in which very cold temperatures are used to freeze a polymer-bioactive mixture in polymeric microspheres with high retention of bioactivity and material. US Patent No. 5,650,173 to Ramstack et al. discloses a method of microparticle preparation in which the polymer and the active substance are dissolved using a mixture of at least two halogenated hydrocarbon-free non-toxic solvents. The resulting microparticles, while free of residual toxic solvents, still had residual amounts of benzyl alcohol and ethyl acetate, which could have adverse effects on product integrity. US Patent No. 5,792,477 to Rickey et al. describes a method that alleviates the residual solvent problem reported by Ramstack et al. by adding an additional washing step to the process to substantially remove the solvent.

While the above reports and other research efforts have undoubtedly contributed to technological advancements, there remains a need for more efficient, economical, and widely applicable microparticle preparation methods for proteins, peptides, and small molecules that are amenable to aseptic processing and are scalable. .

Among various proteins, the one for which effective sustained release compositions have been reported is erythropoietin. Erythropoietin (EPO) is a glycoprotein hormone involved in the maturation of erythroid progenitor cells into erythrocytes. EPO is produced in the kidneys and is necessary to regulate circulating red blood cell levels. Erythropoietin, which stimulates erythropoiesis, is increased in conditions marked by low tissue oxygen signaling. Loss of renal function, such as that seen in chronic renal failure (CRF), often results in decreased production of erythropoietin, with a concomitant decrease in red blood cells.

Administration of recombinant human erythropoietin (rHuEPO) effectively increased red blood cell levels in anemia patients with end-stage renal disease; Eschbach et al., New Eng. J. Med., 316 :73-38 (1987). Subsequent studies have shown that treatment with rHuEPO can cure anemia associated with various other conditions; Fischl et al., New Eng. J. Med., 322 : 1488-1493 (1990); Laupacis, Lancet, 341 : 1228-1232 (1993 ). Use recombinant human erythropoietin to treat CRF anemia, treat anemia associated with HIV-infected patients with AZT (azidethymidine), anemia in patients with nonmyeloid malignancies receiving chemotherapy, and anemia in patients undergoing surgical procedures to reduce heterogeneity Source blood transfusion required, official approval has been obtained. Current treatment for all approved indications (except for surgical indications) consists of a starting dose of 50-150 units/Kg given three times per week (TIW) by intravenous (IV) or subcutaneous (SC) injection to achieve 30% -36% of the suggested target hematocrit range. For surgical indications, rHuEPO ( ^EPOGEN® Package Insert, 12/23/96) was administered daily for 10 days before surgery, the day of surgery, and 4 days after surgery. In general, the currently recommended starting dose of rHuEPO raises the hematocrit to the target range within about 6-8 weeks. Once the target hematocrit range is achieved, a maintenance dosing regimen that varies according to the patient is instituted, but typically three times per week for patients with CRF anemia. Administration of rHuEPO as described above is an effective and well-tolerated regimen for the treatment of anemia.

US Patent No. 5,674,534 to Zale et al. describes sustained release compositions of non-aggregated bioactive EPO. The composition comprises a polymeric matrix of a biocompatible polymer and bioactive aggregation-stabilized EPO particles, wherein the particles are dispersed in the biocompatible polymer, and wherein the microparticles are prepared using the method described in U.S. Patent No. 5,019,400 . The described and claimed method stabilizes EPO using a salting-out excipient. Advantages of this formulation are said to include longer duration, more constant in vivo blood EPO levels, lower initial burst release of EPO, and enhanced therapeutic effect by eliminating fluctuations in serum EPO levels.

Recombinant human erythropoietin expressed by mammalian cells contains three N-linked oligosaccharide chains and one O-linked oligosaccharide chain, which together account for about 40% of the total molecular weight of the glycoprotein. N-linked glycosylation occurs at asparagine residues at positions 24, 38, and 83, while O-linked glycosylation occurs at a serine residue at position 126; Lai et al., J. Biol. Chem., 261 : 3116 (1986); Broudy et al., Arch. Biochem. Biophys., 265 : 329 (1988). It has been shown that the oligosaccharide chains are modified with terminal sialic acid residues. Enzymatic treatment of glycosylated erythropoietin to remove sialic acid residues results in loss of in vivo activity but does not affect in vitro activity; Lowy et al., Nature, 185 :102 (1960); Goldwasser et al., J.Biol.Chem., 249 : 4202 (1974). This property is explained by the rapid clearance of asialo-erythropoietin from circulation upon interaction with hepatic asialoglycoprotein binding protein; Morrell et al., J. Biol. Chem., 243 :155 (1968); Briggs et al., Am. J. Physiol., 227 :1385 (1974); Ashwell et al., Methods Enzymol., 50 :287 (1978).

Novel erythropoietin-stimulating protein (NESP) is a hyperglycosylated erythropoietin analog with five changes in the rHuEPO amino acid sequence, which provide two additional sugar chains. More specifically, NESP contains two additional N-linked sugar chains at amino acids 30 and 88 (numbering corresponding to the human EPO sequence) (see PCT Application No. US94/02957, incorporated herein by reference in its entirety). NESP differs biochemically from EPO in that NESP has a longer serum half-life and higher biological activity in vivo; Egrie et al., ASH97, Blood, 90 :56a (1997). The serum half-life of NESP has been shown to increase approximately 3-fold in mice, rats, dogs and humans; supra. In mice, the longer serum half-life and higher in vivo activity of NESP allowed for less frequent dosing (weekly or every other week) compared to rHuEPO to achieve the same biological response; supra.

Pharmacokinetic studies demonstrated that NESP had a significantly longer serum half-life than rHuEPO in patients with chronic renal failure, consistent with animal studies, suggesting that a less frequent dosing regimen could also be used in humans; MacDougall et al, J American Society of Nephrology, 8 :268A (1997). Less frequent dosing regimens are more convenient for both physicians and patients, and are especially helpful for those who self-administer. Other advantages of less frequent dosing may include less drug entering the patient's body, lessening the nature or severity of some of the side effects observed with rHuEPO administration, and increasing compliance.

The present invention is based on the discovery that NESP can be encapsulated in microparticles to provide an even more pronounced pharmaceutical composition with a sustained release profile allowing administration every 4-6 weeks to increase hematocrit and treat anemia , and thus have a great therapeutic advantage. In addition, for the NESP/PLGA system, the PLGA component is cleared (biodegraded) at least one week prior to cessation of therapeutic effect, thus allowing repeated dosing with less problem of polymer and drug accumulation from dose to dose. Such a system is not feasible for eg EPO/PLGA microparticles.

In addition, the present invention provides an improved, economical, and scalable method for the preparation of microparticles that can be broadly applied to non-NESP biologically active substances, including peptides and small molecules.

Summary of the invention

Accordingly, one aspect of the present invention is a sustained release active ingredient pharmaceutical composition comprising a biologically active ingredient contained in polymeric microparticles. Importantly, the sustained release composition of the present invention maintains the activity, integrity and safety of the active ingredient during encapsulation and release, which contributes to a longer time constant release.

A second aspect of the present invention relates to a new and improved process for the preparation of sustained release compositions containing the active ingredient contained in polymeric microparticles. The method is economical, amenable to aseptic processing, scalable and broadly applicable to proteins, peptides and small molecules. The improved process generally comprises the steps of: (a) obtaining a dry powder of the specified active ingredient or formulated active ingredient; (b) preparing a polymer solution comprising the polymer dissolved in a co-solvent mixture; (c) converting the The dry powder is dispersed in the polymer solution to produce an active ingredient/polymer mixture; (d) preparing active ingredient-containing microparticles from the mixture; (e) collecting the microparticles; and (f) passing a second Secondary drying finally yields granules. Importantly, the drying times of this method are significantly reduced compared to prior art methods.

Brief description of the drawings

Figure 1 is a schematic diagram of the process according to the invention for the preparation of active ingredient-containing granules.

Figure 2 shows chromatograms depicting the size exclusion chromatography results of NESP preparations before spray drying (_...), spray-dried NESP protein powders (__), and typical NESP after microencapsulation (__) Result chromatogram.

Figure 3 graphically illustrates NESP serum levels of various microparticle preparations injected subcutaneously (360 μg/kg NESP peptide dose) into rats. Serum concentrations are plotted against time (days).

Figure 4 graphically illustrates the hematocrit of rats injected subcutaneously (360 μg/kg NESP peptide dose) with various microparticle preparations. Hematocrit (%) is plotted against time (days).

Figure 5 graphically illustrates NESP serum levels of various microparticle preparations injected subcutaneously (360 μg/kg NESP peptide dose) into rats. Serum concentrations are plotted against time (days).

Figure 6 graphically illustrates NESP serum levels of various microparticle preparations injected subcutaneously (360 μg/kg NESP peptide dose) into rats. Serum concentrations are plotted against time (days).

Figure 7 graphically illustrates rat hemoglobin levels compared with a single subcutaneous bolus of 10,000 μg/kg NESP versus 100 μg/kg (20 mg microparticles, injected) of a NESP microparticle preparation (50:50, intrinsic viscosity 0.4 dL/g). Hemoglobin levels (g/dL) are plotted against time (days).

Figure 8 graphically illustrates NESP serum levels of various microparticle preparations injected subcutaneously (360 μg/kg NESP peptide dose) into rats. Serum concentrations are plotted against time (days).

Figure 9 graphically illustrates leptin serum levels in rats injected with various leptin preparations. Serum concentrations are plotted against time (hours).

Figure 10 graphically illustrates the percent body weight loss of rats injected with various leptin preparations. Body weight (mg) is plotted against time (days).

Figure 11 graphically illustrates NESP serum levels of microparticle preparations injected subcutaneously (360 μg/kg NESP peptide dose) into rats. Serum concentrations are plotted against time (days).

Figure 12 graphically illustrates rat hematocrit for subcutaneous injection (360 μg/kg NESP peptide dose) of microparticle preparations. Hematocrit (%) is plotted against time (days).

Figure 13 graphically illustrates NESP serum levels of microparticle preparations injected subcutaneously (360 μg/kg NESP peptide dose) into rats. Serum concentrations are plotted against time (days).

Figure 14 graphically illustrates rat hematocrit for subcutaneous injection (360 μg/kg NESP peptide dose) of microparticle preparations. Hematocrit (%) is plotted against time (days).

Detailed description of the invention

Unless otherwise stated, the term microparticle may be used to include microparticles, microspheres and microcapsules.

As fully described below, the present invention provides a sustained release active ingredient pharmaceutical composition comprising a biologically active ingredient contained within polymeric microparticles. A sustained release composition is defined as a release of a biologically active substance that results in the maintenance of detectable serum levels of said substance for a period longer than that obtained after direct administration of the aqueous bioactive substance. Sustained release can be continuous or discontinuous, linear or non-linear, and can be achieved using one or more polymer compositions, drug loading, selection of excipients, or other modifications. In one embodiment of the present invention, said sustained release composition comprises said bioactive ingredient NESP.

NESP of the present invention is a hyperglycosylated EPO analogue containing two additional glycosylation sites and additional sugar chains attached to each site. NESPs were constructed using site-directed mutagenesis and expressed in mammalian host cells. Co-owned PCT Application No. US94/02957 provides details of the production of NESP. A new rHuEPO N-linked glycosylation site was introduced by DNA sequence alteration to encode the amino acids Asn-X-Ser/Thr in the polypeptide chain. The NESP-encoding DNA was transfected into Chinese hamster ovary (CHO) host cells, and the presence of other sugar chains of the expressed polypeptide was analyzed. In a preferred embodiment, NESP has two additional N-linked sugar chains at residues 30 and 88. The numbering of the amino acid sequence is the amino acid sequence numbering of human erythropoietin (EPO). The amino acid sequence of EPO is shown in SEQ ID NO:1. The amino acid sequence of NESP is shown in SEQ ID NO:2. Apparently, NESP has the normal addition of N-linked and O-linked glycosylation sites in addition to the new sites.

The NESPs of the invention may also include conservative amino acid changes of one or more residues in SEQ ID NO:2. These changes do not generate additional sugar chains and have little effect on the biological activity of the analogs.

Other active ingredients incorporated into the microparticles of the invention are synthetic or natural compounds introduced into living organisms to exhibit biological effects. Contemplated active substances include peptides, small molecules, sugars, nucleic acids, lipids, proteins, and the like. Proteins to consider include potent cytokines, including various hematopoietic factors such as G-CSF, GM-CSF, M-CSF, MGDF, interferons (α, β, and γ), interferon consensus sequences, interleukins (1- 12), erythropoietin (EPO), fibroblast growth factor, KGF, TNF, TNFbp, IL-1ra, stem cell factor, nerve growth factor, GDNF, BDNF, NT3, platelet-derived growth factor and tumor growth factor (α , β), osteoprotegerin (OPG), NESP and OB proteins. OB protein is also called lean protein.

It is also contemplated to incorporate derivatives, fusion proteins, conjugates, analogs or modified forms of the native active ingredients into the compositions of the invention. Chemically modified biologically active proteins have been found to have other advantages in certain circumstances, such as increased stability and circulation time of therapeutic proteins and reduced immunogenicity. For example, US Patent No. 4,179,337 to Davis et al. discloses conjugates of water-soluble polypeptides such as enzymes and insulin with polyethylene glycol (PEG); see also US Patent No. 5,824,784 to Kinstler et al.

In general, the pharmaceutical compositions included in the present invention contain an effective amount of the protein or derivative products of the present invention and pharmaceutically acceptable diluents, stabilizers, preservatives, solubilizers, emulsifiers, adjuvants and/or carriers. Such compositions include various buffer components (e.g. Tris-HCl, phosphoric acid), diluents for pH and ionic strength; additives such as detergents and solubilizers (e.g. polysorbate 20, polysorbate 80), Antioxidants (eg, ascorbic acid, sodium metabisulfite), preservatives (eg, thimerosal, benzyl alcohol), and bulking substances (eg, lactose, mannitol); see, eg, Remington's Pharmaceutical Sciences, 18th ed. (1990, Mack Publishing Co. ., Easton, PA 18042), pp. 1435-1712, the contents of which are incorporated herein by reference. An effective amount of active ingredient is a therapeutically, prophylactically or diagnostically effective amount which can be readily determined by one skilled in the art by taking into account factors such as body weight, age, therapeutic or prophylactic or diagnostic purpose and desired rate of release.

As used herein and in the design of NESP-containing microparticles, the term "therapeutically effective amount" refers to an amount that produces an increase in hematocrit that provides benefit to the patient. Said amount will vary between individuals, depending on many factors including the general condition of the patient and the underlying cause of the anemia. For example, the therapeutically effective dose of rHuEPO for patients with chronic renal failure is 50-150 units/kg three times a week. The amount of rHuEPO used for treatment produces an acceptable rate of increase in hematocrit and maintains hematocrit at a beneficial level (usually at least about 30%, typically in the range of 30%-36%). A therapeutically effective amount of a composition of the invention can be readily determined by one skilled in the art using publicly available materials and methods.

The invention provides that NESP-containing microparticles are administered less frequently than NESP and/or EPO. The frequency of dosing will vary depending on the condition being treated, but will generally be about every 4-6 weeks. Obviously, due to differences in individual responses to NESP-containing microparticles, the actual dosing frequency used may vary slightly from that disclosed herein; the term "about" is used to reflect such differences.

The invention is therefore useful for stimulating red blood cell production and treating reduced red blood cell levels. The most common cause of decreased red blood cell levels is anemia. Conditions treatable by the present invention include anemia associated with decreased or lost kidney function (chronic renal failure), anemia associated with myelosuppressive therapy such as chemotherapy or antiviral drugs such as AZT, anemia associated with the development of non-myeloid cancer and anemia associated with viral infections such as HIV. Conditions that can lead to anemia in an otherwise healthy individual, such as the expected loss of blood during a surgical procedure, can also be treated. In general, any condition treatable with rHuEPO can also be treated with the NESP-containing microparticles of the invention.

The invention also provides for the administration of a therapeutically effective amount of iron to maintain increased erythropoiesis during treatment. The amount of iron administration can be readily determined by those skilled in the art based on rHuEPO treatment.

The present invention also provides an improved method of preparing active ingredient-containing polymeric microparticles comprising the use of co-solvent mixtures for more rapid removal of organic solvents during drying. The secondary organic solvent component remaining in the mixture during drying accelerates the removal of residual polymer solvent during any drying process. This improved method offers several significant advantages over the methods described in the art, including, for example, 1) reduction of residual solvent levels in the polymer system after the initial microparticle formation step; 2) reduction in the number of drying cycles of the polymer system; and 3 ) to remove toxic solvents to acceptable levels for use in human medicine. Importantly, these advantages help to make such an approach industrially feasible.

Main embodiments of an improved method for preparing protein-loaded microparticles include: (a) obtaining a dry powder of an active ingredient or formulated active ingredient; (b) dissolving a polymer in a co-solvent mixture to produce a polymer solution; ( c) adding the dry powder to the polymer solution to produce an active ingredient/polymer mixture; (d) spray drying the mixture to produce microparticles loaded with the desired active ingredient; (e) collecting the said microparticles; and (f) finally obtaining said granules by a second drying. The method is shown diagrammatically in FIG. 1 .

In one embodiment of the invention, the active ingredient or formulated active ingredient is in the form of a spray-dried powder. Spray drying nebulizes a solution to form a fine mist and dries by direct contact with a hot carrier gas. For a detailed review on spray drying, see for example Masters, K., "Spary Drying Handbook" (ed. John Wiley & Sons, New York 1984). The improved industrial-scale spray-dried protein powder preparation method provided herein provides significantly higher yields and improved collection of the microparticles.

Polymer solvents contemplated for use in step b) of the process of the invention include, for example, chloroform, ethyl acetate, acetone, dichloromethane and dimethylsulfoxide. In one embodiment of the invention, the polymer solvent used is dichloromethane. Non-solvents considered include ethanol, ethyl formate, and heptane.

Microparticle preparation step (d) may optionally include emulsification-type preparation or spray freezing. For the emulsions produced in the process of the invention it is contemplated to use organic:water ratios of 1:1 to 12:1. In general, microparticles prepared by the methods of the invention typically comprise 0.001-60% protein by weight.

Secondary drying methods contemplated for step (f) include gas bleeddrying, fluid bed drying, freeze drying, vacuum drying and tray drying. In one embodiment of the present invention, step (f) uses gas suction drying.

The polymers contemplated for use may be selected from biocompatible and/or biodegradable polymers. Biodegradation is defined herein to mean erosion or degradation of the composition in vivo to form smaller biocompatible chemical species. For example, it can be degraded by enzymatic, chemical or physical processes. Suitable biodegradable polymers contemplated for use in the present invention include poly(lactide), poly(glycolide), poly(lactic acid), poly(glycolic acid), polyanhydrides, polyorthoesters, polyetheresters, poly( Caprolactones, polyesteramides, polyphosphazenes, polyphosphates, pseudopolyaminoacids, mixtures and copolymers thereof; Langer, Nature, 392:5-10 (1998).

Those skilled in the art can readily determine the molecular weight range of polymers contemplated for use in the process of the invention, depending on factors such as the desired rate of degradation of the polymer. Typically, the molecular weight range is 2000-2,000,000 Daltons. Almost any type of polymer can be used as long as a suitable solvent or co-solvent system is found.

As used herein, the term "PLGA" refers to a polymer of lactic acid alone, a polymer of glycolic acid alone, a blend of said polymers, a copolymer of glycolic acid and lactic acid, a blend of said copolymers, or said polymers and copolymers. mixture of substances. PLGA can be used in the free acid ("uncapped") form or in terminal ester ("capped") form. The biodegradable polymer is preferably polylactide-co-glycolide (PLGA). The polymer concentration contemplated for use in the process of the invention is in the range of 5-70 g/100 mL (g%).

In general, the active substance in aqueous, suspension or solid form can be mixed with an organic solvent containing the polymer. When an aqueous solution of the active ingredient is used, an emulsion or a water-in-oil emulsion of the aqueous active ingredient in a polymer solution is formed (the aqueous phase contains the active ingredient and the organic phase contains the polymer), which is used to prepare microparticles . When using the active ingredient in suspension or solid form, a suspension of the solid active ingredient in the polymer solution is formed which is used to prepare the microparticles. Alternatively, a single phase solution of active ingredient and polymer may be used. In one embodiment of the invention, the active ingredient is in the form of a spray-dried powder, the protein powder having a particle size range of less than 10 μm. Protein concentrations (in emulsion or suspension) in the range of 0.001-500 mg/mL are contemplated for use in the methods of the invention.

Solutions, suspensions, emulsions or solid forms of the active ingredient may also be formulated, i.e. including buffers, surfactants or excipients for stabilizing the active ingredient during drying, e.g. trehalose, ammonium sulfate, 2-hydroxy Propyl beta-cyclodextrin, sucrose, or other protein-stabilizing sugars or excipients.

Protein-loaded microparticle suspensions prepared according to the invention are preferably administered by intraperitoneal, subcutaneous or intramuscular injection. However, it will be clear to those skilled in the art that other routes of delivery may also be used effectively with the compositions of the present invention.

The following examples are provided to more fully illustrate the invention but should not be construed as limiting the scope of the invention. Example 1 describes a method for preparing protein-loaded microparticles using spray drying to prepare microparticles. NESP (in the form of spray-dried protein powder) was used as the experimental protein. Example 2 describes various characterization experiments performed on the NESP-containing microparticles of Example 1. Example 3 demonstrates the ability of the NESP-containing microparticles of Example 1 to sustainably release NESP in vivo. Example 4 describes a new method of preparing polymeric microparticles in which a co-solvent is used to allow for faster and more efficient removal of residual solvent. Example 5 describes the preparation of NESP-containing microparticles using spray freezing for the microparticle preparation step. Example 6 describes various characterization experiments performed on the NESP-containing microparticles of Example 5. Example 7 demonstrates the ability of the NESP-containing microparticles of Example 5 to sustainably release NESP in vivo. Example 8 describes the double emulsion/solvent extraction and evaporation method used to prepare NESP-containing PLGA microparticles and demonstrates the ability of PLGA microparticles to release NESP sustainably in vivo. Example 9 describes the preparation of leptin-containing microparticles using the method of Example 1 and shows the ability of leptin-containing microparticles to provide sustained release of leptin in vivo. Example 10 describes the preparation of protein loaded microparticles using spray drying for microparticle preparation. Example 11 describes various characterization experiments performed on the NESP-containing microparticles of Example 10. Example 12 demonstrates the ability of the NESP-containing microparticles of Example 10 to sustainably release NESP in vivo. Example 13 demonstrates the ability of the NESP-containing microparticles of Example 10 to sustainably release NESP in vivo.

Example 1

This example describes the preparation of protein-loaded microparticles using spray drying for the microparticle preparation step; specifically, the preparation of NESP-containing poly(D,L-lactide-co-glycolide) microspheres.

NESP was formulated with 46% NESP, 29% sodium phosphate salt, 25% trehalose (w/w/w), and spray dried by a laboratory scale spray dryer (BUCHI190) using the following conditions: feed rate 2.0ml/min, The atomization is 500NL/hour, the inlet temperature is 135°C, the outlet temperature is 99°C, and the drying gas flow rate is 800SLPM. Protein powder was collected and characterized as described in Example 2 below.

The protein powder was added to a polymer solution (40% w/v) of PLGA in dichloromethane (intrinsic viscosity 0.18, 11 kD) and the resulting suspension was sprayed through a pilot-scale spray dryer (Niro Mobile Minor ^™ ) using the following conditions Drying: feed rate 50ml/min, atomization flow rate (two-flow nozzle) 3.0kg/hour, inlet temperature 50°C, outlet temperature 30°C, drying gas flow rate 93kg/hour. The resulting NESP-containing microparticles (0.53% NESP) were then collected and sieved (mesh size 125 μm) for characterization.

Various PLGA compositions containing NESP microparticles were produced using this method. The lactide:glycolide copolymer ratio of the PLGA composition is between 50:50 and 100% lactide. The PLGA polymer used has free acid polymeric chain end groups.

Example 2

This example describes various characterization experiments performed on the spray-dried NESP/trehalose protein powder and NESP-containing microparticles described in Example 1.

The NESP/Trehalose protein powder was characterized by size exclusion chromatography under native conditions; the percent monomer was unchanged (>99.8%) after spray drying (see Figure 2). No other aggregation was observed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) using silver staining. The protein powder was analyzed by reverse phase high performance liquid chromatography (RP-HPLC); no changes were observed different from raw NESP. Protein powders were also characterized by HPLC glycoform determination and isoelectric focusing gel electrophoresis (IEF) and IEF Western blot characterization; no changes in glycoform distribution were observed. Radioimmunoassays performed on replica powders showed full antibody recognition, while trypsin profiling showed no oxidative changes distinct from the unprocessed material. The particle size distribution of the protein powder was determined by Fraunhoffer diffraction with an average volume distribution of 4.7 μm.

The particle size of the NESP-containing microparticles was determined by Fraunhoffer diffraction and averaged over 5 batches, the average volume distribution was 58±8 μm. The integrity of NESP encapsulation in these microparticles was assessed by extracting proteins and analyzing the extracts by anion exchange and size exclusion HPLC under native conditions. To extract NESP from microparticles, approximately 20 mg of microparticles were placed in a test tube containing 1 mL of acetonitrile. Samples were vortexed for 10-20 seconds and centrifuged at 14,000 rpm for 2 minutes at 4°C to pellet proteins and excipients. Remove the supernatant and resuspend the pellet with 1 mL of acetonitrile. Repeat the above steps three more times. After the last removal of the supernatant, the samples were dried in a vacuum oven at room temperature for 2-3 hours. The NESP pellet was resuspended in 20 mM sodium phosphate pH 6.0 with or without 0.005% Tween80. After gentle flicking of the tube, the samples were incubated at room temperature for 2 hours to achieve complete dissolution. Proteins were quantified and integrity was determined by anion exchange followed by size exclusion HPLC. Quantitative analysis of protein recovery (>99%), the average of 5 batches was taken by size exclusion HPLC, the protein integrity was 98.2±1.0% monomer, and each batch was characterized in triplicate (see Figure 2). Extracts from the 75% lactide preparation were additionally characterized by radioimmunoassay, capillary electrophoresis, and peptide mapping. The protein recovery obtained by RIA is consistent with the SEC results, indicating that the antibody can recognize the protein. Capillary electrophoresis confirmed that the glycoform distribution was identical to that of unprocessed NESP. The degree of oxidation as determined by peptide mapping was equivalent to that of unprocessed NESP.

Example 3

This example demonstrates the ability of NESP-containing microparticles prepared as described in Example 1 to sustainably release NESP in vivo.

Male Sprague Dawley rats (385±14g) were subcutaneously injected with NESP-containing microparticles (360μg/kg NESP peptide dose) in the back of the neck. A single subcutaneous bolus injection of NESP equal to the dose of the microparticles was included as a control. Blood samples were taken from the tail vein at various times after injection up to week 8. Rat serum NESP concentrations at weeks 2-4 were determined by ELISA. Whole blood was analyzed for hematocrit, hemoglobin, and reticulocyte count.

With a single injection of NESP-containing microparticles formulated from a 50:50 lactide:glycolide polymer, serum levels of NESP were greater than 1 ng/mL for 15 days in all three batches studied (see Figure 3). A single bolus administration of NESP alone increased serum levels for 11 days (see Figure 3). The NESP-containing microparticles raised hemoglobin and hematocrit above baseline for 25 and 28 days, respectively (see Figure 4). NESP Concentrate bolus increases hemoglobin and hematocrit for 25 days (see Figure 4).

With a single injection of NESP-containing microparticles formulated with a 75:25 lactide:glycolide polymer, NESP serum levels were greater than 1 ng/mL for 20 days (see Figure 3). The NESP-containing microparticles increased hemoglobin and hematocrit above baseline for more than 40 days (see Figure 4).

NESP-containing microparticles were formulated using a 50:50 and 100% lactide polymer solution mixture to yield a total average of 75% lactide polymer with NESP serum levels greater than 1 ng/mL for 18 days after a single injection ( See Figure 3). The NESP-containing microparticles increased hemoglobin and hematocrit above baseline for 35 days (see Figure 4).

Example 4

This example describes a new method for the preparation of microparticles using a co-solvent for more rapid and efficient removal of residual solvent; specifically, the preparation of poly(D,L-lactide co-ethylene lactide) microspheres.

Two batches of 50:50 PLGA (11 kD) microparticles were produced by spray drying. For batch 1, pure dichloromethane was used to dissolve PLGA with a mass equal to 26% of the solvent mass. For batch 2, a co-solvent of dichloromethane (86.4% by mass) and ethanol (13.6% by mass) was used to dissolve PLGA having a mass equal to the mass of 26% of the co-solvent. The obtained solution was spray-dried by a pilot-scale spray dryer (Niro Mobile Minor ^TM ) using the following conditions: feed rate 50 ± 5ml/min, atomization flow rate (two-flow nozzle) 60SLPM, inlet temperature 55°C, outlet temperature 33- 36°C, drying gas flow rate 2.1 lbs/min. The resulting microparticles were collected and characterized.

The residual solvent concentration of batch 1 after spray drying was 18550 ppm dichloromethane. The residual solvent concentration of the second batch after spray drying was 6190 ppm dichloromethane and 3330 ppm ethanol. Both batches were dried a second time as follows: Place the granules in a 1.5" diameter dryer over a retarder screen. Seal the dryer and flow nitrogen gas through the bed at 4.4 L/min. Submerge the dryer In a temperature-controlled heating bath. After drying batch 1 for 73 hours (starting at 20°C and ending at 41°C), the residual methylene chloride level was 750ppm. Batch 2 was dried for 40 hours, starting at 18°C, at End at 30° C. The microparticles achieved residual solvent levels of 487 ppm dichloromethane and 455 ppm ethanol.

Thus, the use of co-solvents reduces the length of drying time necessary and improves the overall residual solvent level of the final microparticles, thus making processes utilizing these solvents more commercially viable.

Example 5

This example describes the preparation of protein-loaded microparticles; specifically, poly(D,L-lactide-co-glycolide) microspheres containing NESP were prepared using spray freezing for the microparticle preparation step.

Using the conditions described in Example 1, two NESP formulations (the trehalose and ammonium sulfate formulations of Example 1) were spray dried via a laboratory scale spray dryer. The ammonium sulfate formulation of NESP was 11% NESP, 10% phosphate, 79% ammonium sulfate (w/w/w).

Microparticles containing NESP/trehalose protein powder were prepared from unblocked PLGA from Alkermes/Medisorb Wilmington, Ohio or Boehringer Ingelheim Chemicals, Inc., Montvale, N.J. or from bocked PLGA from Boehringer Ingelheim. The polymer intrinsic viscosity is in the range of 0.14 dL/g to 0.5 dL/g (11-47 kD molecular weight) and the lactide content is 50%-100%. NESP/ammonium sulfate protein powder was encapsulated into uncapped PLGA (50:50) with an intrinsic viscosity of 0.18 dL/g (11 kD).

The spray-dried NESP powder was encapsulated into PLGA using the method described by Gombotz et al. (US Patent No. 5,019,400). Protein powder was added to a polymer solution of PLGA in dichloromethane (5-20% w/v) such that the protein solids content in the microparticles was in the range of 1-5% (w/w). The ethanol container was frozen by immersion in liquid nitrogen and covered with a layer of liquid nitrogen prior to the spray freezing step. A suspension of protein powder in polymer solution was pumped via a syringe pump into an ultrasonic nozzle placed above an ethanol cooling bath. The suspension was atomized into droplets, which were frozen on contact with liquid nitrogen and immobilized on the surface of frozen ethanol to form microparticles. The freezing bath was turned to -80°C for 72 hours to allow the ethanol to melt and extract the polymer solvent from the microparticles. The resulting slurry of microparticles in ethanol was freeze-filtered (0.65 μm PTFE) and the collected microparticles were lyophilized. After lyophilization, the microparticles (0.53% NESP peptide content) were sieved (125 [mu]m mesh size) and then characterized.

Example 6

This example describes various characterization experiments performed on the spray-frozen NESP-containing microparticles described in Example 5.

The NESP protein powder was characterized as described in Example 2, which has given the results for NESP/trehalose. NESP/Ammonium Sulfate (AS) was characterized by size exclusion chromatography under native conditions; the percent monomer was significantly reduced by 2.5% after spray drying. Dimers were confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and irreducible using silver staining. The particle size distribution of the NESP/AS protein powder was determined by Fraunhoffer diffraction with an average volume distribution of 4.3 μm.

The particle size of NESP-containing microparticles formulated by spray freezing 28 different batches of each formulation was determined and the average volume distribution ranged from 20-45 μm. The residual dichloromethane levels for the 10 batches evaluated were all less than 500 ppm. The integrity of NESP in these microparticles after encapsulation was assessed by extracting the protein and analyzing the extract as described in Example 2. Quantitative analysis of protein recovery rate, each batch was characterized by at least two parallel copies, the integrity (monomer %) was 97.2±2%. Extracts from nine selected preparations were additionally characterized by radioimmunoassay, capillary electrophoresis, and peptide mapping. The protein recovery obtained by RIA is consistent with the SEC results, indicating that the antibody can recognize the protein. Capillary electrophoresis confirmed that the glycoform distribution was identical to that of unprocessed NESP. The degree of oxidation, as determined by peptide mapping, ranged from 7-12%, typically 8±2% for unprocessed material.

Example 7

This example shows the ability of the NESP-containing microparticles prepared in Example 5 to sustainably release NESP in vivo.

Male Sprague Dawley rats were treated as described in Example 3 with different NESP-containing microparticle formulations prepared by the spray freezing method described in Example 6. Serum NESP concentrations and whole blood at 4-6 weeks were analyzed as described in Example 3.

Figure 5 shows the results of serum levels of NESP obtained using various copolymer formulations of Example 5. All copolymer formulations showed an initial burst phase followed by a zero-order release phase which then dropped below the quantitative limit of the assay. Increased lactide content decreased serum levels of NESP by more than 100-fold through the zero-order phase. Increases in intrinsic viscosity (molecular weight) also decreased NESP serum levels, but only by a factor of 4 (see Figure 6). Increasing both the intrinsic viscosity and the lactide content of either the copolymer composition or the polymer solution blend increased the duration of quantifiable serum NESP concentrations from 18 days to as long as 35 days.

Pharmacodynamic effects were detected by increasing hematocrit above baseline levels, which was similar to the trend observed for serum NESP concentrations. Preliminary data on increases in NESP serum concentrations and reticulocyte counts suggest that effective serum levels in rats treated with NESP microparticles approached 0.4 ng/mL.

In the study of pharmacodynamics in mice, NESP/PLGA microparticles (50:50, intrinsic viscosity 0.4dL/g) were injected subcutaneously into the back neck of mice with a single dose of NESP of 6, 30 and 100 μg/kg, and the treatment Male BDF1 mice (body weight 22 g). A single dose of NESP at 100, 1,000 and 10,000 μg/kg was injected subcutaneously into the back of the mouse's neck quickly, and administered to the NESP solution test group. The study design was to collect blood for whole blood analysis every 2-4 days for 35 days, but no individual animal was bled more than twice within 7 days.

The dose-response of the microparticle-treated groups was observed. The 100 μg/kg microparticle dose increased hemoglobin levels above baseline over 4 weeks. The duration of action after treatment with the NESP concentrated bolus solution was equal to that observed with the microparticles only when administered at a dose 100-fold higher than that of the microparticles (see Figure 7).

Example 8

This example describes the double emulsion/solvent extraction and evaporation method used to prepare NESP-containing PLGA microparticles and demonstrates their ability to sustainably release NESP in vivo.

Prepare two kinds of NESP aqueous solvents: preparation 1 is 5.5mg/mL NESP (peptide concentration) formulated with 20mM sodium phosphate pH6.0; .0 formulated 5.2 mg/mL NESP (peptide concentration).

Microparticles containing the above NESP formulation were prepared from unblocked PLGA (50:50, intrinsic viscosity 0.2, 11 kD) obtained from Boehringer Ingelheim Chemicals, Inc., Montvale, N.J. Solvent extraction and evaporation after double emulsification was used as described below.

Approximately 2 g of polymer was dissolved in 6 ml of dichloromethane and homogenized on ice in a 18 x 150 mm glass test tube at 25 krpm. Protein solution (1.0 ml) was added during homogenization, and homogenization was continued on ice for 30 seconds to form an initial emulsion. For the second emulsification, 40 ml of an aqueous external phase (18 mM sodium phosphate, 0.5% polyvinyl alcohol, pH 6.0) in a 100 ml beaker of 4.5 cm inner diameter was precooled to 15° C. in a water bath. Dip the 1" Rushton impeller blade into the pre-cooled outer phase and start mixing at 1480 rpm. Add the primary emulsion quickly to form the secondary emulsion and continue mixing at the same speed for a total of 40 minutes.

The solution was transferred to two 50ml test tubes, centrifuged at 500g for 30 seconds, decanted to a volume of 10ml and the above steps were repeated, thereby washing the solidified emulsion twice with water. The final suspension was transferred to vials and lyophilized. The final dry powder was sieved (180 μm mesh size) and characterized as described in Example 2.

The method achieves quantifiable encapsulation efficiency, low product yield (50%), large particle size (120 μm) and acceptable residual dichloromethane levels (approximately 500 ppm). The post-encapsulation integrity of NESP in these microparticles was assessed by extracting the protein and analyzing the extract as described in Example 2. Quantitative analysis of protein recovery showed that the integrity (monomer %) of preparation (1) was 97.5%±0.01%, while the integrity of preparation (2) was 96.7%±0.3%; each batch was characterized in triplicate.

As described in Example 3, male Sprague Dawley rats were treated with two formulations of NESP-containing microparticles formulated by the double emulsion method. Serum NESP levels showed an initial burst release phase followed by a zero order release phase, which then fell below the limit of quantitation of the assay after 18 days for Formulation 1 and below the limit of quantitation for Formulation 2 after 22 days. Formulation (1 ) and Formulation (2) continued for 25 days and over 28 days (end of study), respectively, based on the measured pharmacodynamic effects of hemoglobin elevation above baseline levels.

Example 9

This example describes the preparation of leptin-containing microparticles and demonstrates the ability of the leptin-containing microparticles to release leptin sustainably in vivo.

Leptin-containing microparticles were prepared using the method described in Example 1 and then characterized as described in Example 2. After determination, the protein recovery rate is greater than 95%, while the integrity is greater than 98%. This method provides high encapsulation yield (85-95%), good product yield (75-85%), low initial burst release (<15%), acceptable particle size (~35 μm) and acceptable residual solvent level. The formulations were also shown to have good storage stability.

The "in vivo" bioactivity of leptin-containing microparticles was evaluated in normal rats. A total leptin dose of 50 mg/kg (equivalent to approximately 150 mg microparticles/kg) was given as a single injection on day 0. In addition, the following control groups were included: daily bolus leptin injection (5mg/kg/day×10 days); one-time high-dose control (dose dump control) (50mg/kg on day 0); placebo microparticles; and daily placebo injection controls. Animals were weighed daily and serum leptin concentrations were assessed in periodically collected serum samples.

Serum leptin levels are shown in FIG. 9 . Serum leptin concentrations remained above baseline for approximately 5 days. Figure 10 lists the weight loss. Percent body weight loss was determined at 30 days relative to the buffer control. Daily injections of the leptin solution produced a 4-6% body weight loss relative to placebo controls. A single injection of leptin-containing microparticles produced a 9-10% reduction in body weight relative to placebo microparticles and resulted in a 25-day sustained weight loss in rats.

After 10 days, some animals were sacrificed and the injection sites were examined histologically. Histological examination of the injection site revealed a localized minimal to mild inflammatory response, which was completely reversed over time as the microparticles biodegraded.

Example 10

This example describes a method for preparing protein-loaded microparticles; specifically, NESP-containing poly(D,L-lactide-co-glycolide) microspheres were prepared using spray drying for the microparticle preparation step.

NESP was formulated with 46% NESP, 29% sodium phosphate salt, 25% trehalose (w/w/w) and spray dried via a pilot scale spray dryer (Niro Mobile Minor ^™ ) using the following conditions: Feed rate 8.0ml /min, atomizing gas 0.33lbs/min (two-flow nozzle), inlet temperature 200°C, outlet temperature 100°C, drying gas flow rate 2.0lbs/min. Protein powder was collected and characterized as described in Example 11 below.

Add protein powder to PLGA (high molecular weight, (intrinsic viscosity 0.49 dL/g), 50% lactide) in dichloromethane/ethanol polymer solution (10% w/v) and obtain a suspension using the following conditions Spray drying by pilot-scale spray dryer (Niro Mobile Minor ^TM ): feed rate 12ml/min, atomization (ultrasonic nozzle) 1.3 watts, inlet temperature 55°C, outlet temperature 28°C, drying gas flow rate 1.2lbs/min . The resulting NESP-containing microparticles (0.53% NESP) were then collected and dried a second time as described in Example 4 to reduce the residual solvent concentration to less than 1000 ppm. The microparticles obtained were characterized after sieving (mesh size 125 μm).

Example 11

This example describes various characterization experiments performed on the spray-dried NESP/trehalose protein powder and NESP-containing microparticles described in Example 10.

The NESP/Trehalose protein powder was characterized by size exclusion chromatography under native conditions; the percentage of monomer after spray drying was 99.8% (100% for raw material). No other aggregation was observed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) using silver staining. The protein powder was analyzed by reverse phase high performance liquid chromatography (RP-HPLC); no changes were observed that differed from raw NESP. The protein powder was also characterized by HPLC glycoform determination and isoelectric focusing gel electrophoresis (IEF), and no change in glycoform distribution was observed. The particle size distribution of the protein powder was determined by Fraunhoffer diffraction with an average size of 2.5 μm (volume distribution).

The average size of the NESP-containing microparticles was determined by Fraunhoffer diffraction to be 45±1 μm (volume distribution). The residual solvent concentrations of dichloromethane and ethanol were determined by headspace gas chromatography to be 638 ppm and less than 100 ppm, respectively.

The integrity of NESP in these microparticles after encapsulation was assessed by extracting the protein as described in Example 2 and analyzing the extract by anion exchange and size exclusion HPLC under native conditions. Proteins were quantified and integrity was determined by anion exchange followed by size exclusion HPLC. Quantitative analysis of protein recovery (>99%), protein integrity determined by size exclusion HPLC was 98.6±0.3% monomer. No other aggregation was observed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) using silver staining. The NESP particulate protein extract was analyzed by reversed-phase high-performance liquid chromatography (RP-HPLC); no changes were observed in the NESP protein powder. NESP microparticle protein extracts were also characterized by HPLC glycoform determination and isoelectric focusing gel electrophoresis (IEF), and no changes in glycoform distribution were observed.

Example 12

This example shows the ability of the NESP-containing microparticles prepared in Example 10 to sustainably release NESP in vivo.

Male Sprague Dawley rats were treated as described in Example 3 with NESP-containing microparticles formulated as described in Example 10. Serum NESP concentrations and whole blood at 4-8 weeks were analyzed as described in Example 3.

Serum levels of NESP were greater than 1 ng/mL for 20 days with a single injection of NESP microparticles (see Figure 11). A single bolus administration of NESP alone increased serum levels for 11 days (see Figure 11). Microparticles containing NESP increased hematocrit above baseline for 34 days (see Figure 12). The NESP concentrated bolus increased hematocrit for up to 20 days (see Figure 12).

Example 13

This example shows the ability of the NESP-containing microparticles prepared in Example 10 to sustainably release NESP in vivo.

Male NIHRNU-M athymic rats were treated as described in Example 3 with NESP-containing microparticles formulated as described in Example 10. Serum NESP concentrations and whole blood at 4-8 weeks were analyzed as described in Example 3.

Serum levels of NESP were greater than 1 ng/mL for 21 days with a single injection of NESP microparticles (see Figure 13). A single bolus administration of NESP alone increased serum levels for less than 11 days (see Figure 13). Microparticles containing NESP increased hematocrit above baseline for up to 44 days (see Figure 14). The NESP concentrate bolus increased hematocrit for 18 days (see Figure 14). Materials and methods

The NESPs of the present invention may be prepared according to PCT Application No. US94/02957, incorporated herein by reference above.

The recombinant methionyl-human-OB protein (leptin) of the present invention can be prepared as described in PCT Publication WO96/05309151-159, incorporated herein by reference above. For the embodiments of the present invention, position 35 of the human OB protein used (compared to the amino acid sequence on page 158) is replaced by lysine for arginine, and position 74 is replaced by isoleucine for isoleucine . Other recombinant human OB proteins can be prepared using recombinant DNA techniques according to methods generally known in the art of protein expression.

While the invention has been described in terms of certain preferred embodiments, it is evident that alterations and modifications will occur to those skilled in the art. Accordingly, the appended claims cover all such equivalent changes which fall within the scope of the present invention.

sequence listing

Sequence Listing <110> AMGEN INC. <120> Biodegradable Microparticles for Sustained Delivery of Novel Erythropoietin Stimulating Protein <130> A-626 <140> Assignment <141> 1999-10- 22 <160> 2 <170> Patentin Ver.2.1 <210> 1 <211> 165 <212> PRT <213> People <400> 1ALA Pro Pro ARG Leu Ile Cys ARG Val Leu Glu ARG Tyr Leu1 5 10 15LEU Glu Ala Lys Glu Ala Glu Asn Ile Thr Thr Gly Cys Ala Glu His

20 25 25 30Cys Ser Leu Asn Glu Asn Ile Thr Val Pro Asp Thr Lys Val Asn Phe

35 40 45Tyr Ala Trp Lys Arg Met Glu Val Gly Gln Gln Ala Val Glu Val Trp

50 55 60GLN GLY Leu Ala Leu Leu Serg Gln GLN Ala Leu65 70 80leu Val Gln Pro Tru Leu GLN Leu His Val Val Val Val Val Val Val Val Val Val Val Val Val

85 90 95Lys Ala Val Ser Gly Leu Arg Ser Leu Thr Thr Leu Leu Arg Ala Leu

100 105 110Gly Ala Gln Lys Glu Ala Ile Ser Pro Pro Asp Ala Ala Ser Ala Ala

115 120 125Pro Leu Arg Thr Ile Thr Ala Asp Thr Phe Arg Lys Leu Phe Arg Val

130 135 140tyr Serg GLY LEU LEU LEU THR THR GLY GLU ALA145 150 160 16CY THR GLY ASP

165 <210> 2 <211> 165 <212> PRT <213> People <400> 2ALA Pro Pro ARG Leu Ile Cys ARG Val Leu Glu ARG TYR Leu1 5 10 15LEU Ala Lysn Glu Ale Thr Thr Thr Thr Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Cys Asn Glu Thr

20 25 25 30Cys Ser Leu Asn Glu Asn Ile Thr Val Pro Asp Thr Lys Val Asn Phe

35 40 45Tyr Ala Trp Lys Arg Met Glu Val Gly Gln Gln Ala Val Glu Val Trp

50 55 60GLN GLY Leu Ala Leu Leu Serg Gln Gln Ala Leu65 70 80leu Val Gln Val asn Gl Leu His Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val

85 90 95Lys Ala Val Ser Gly Leu Arg Ser Leu Thr Thr Leu Leu Arg Ala Leu

100 105 110Gly Ala Gln Lys Glu Ala Ile Ser Pro Pro Asp Ala Ala Ser Ala Ala

115 120 125Pro Leu Arg Thr Ile Thr Ala Asp Thr Phe Arg Lys Leu Phe Arg Val

130 135 140tyr Serg GLY LEU LEU LEU THR THR GLY GLU ALA145 150 160 16CY THR GLY ASP

165

Claims (12)

1. the Pharmaceutical composition of a lasting release of active ingredients, its bioactive ingredients that contains is included in the polymer particle, and described compositions is with the method preparation that comprises following steps:

(a) dried powder of the active component of described active component of acquisition or preparation;

(b) preparation comprises the polymer solution that is dissolved in the polymer in the cosolvent mixture;

(c) described dried powder is dispersed in the described polymer solution, to produce active component/mixture of polymers;

(d) prepare the microgranule that contains active component by described mixture;

(e) collect described microgranule;

(f) by the second time drying make described microgranule at last.

2. according to the compositions of claim 1, wherein said active component is selected from peptide, micromolecule, sugar, nucleic acid, lipid, albumen and analog thereof.

3. according to the compositions of claim 2, wherein said active component is a kind of albumen.

4. according to the compositions of claim 3, wherein said albumen is NESP or its chemical modification form.

5. according to the compositions of claim 4, the aminoacid sequence of wherein said NESP is shown in SEQ ID NO:2.

6. according to the compositions of claim 3, wherein said albumen is leptin or its chemical modification form.

7. according to the compositions of claim 1, wherein said polymer is selected from poly-(lactide), poly-(Acetic acid, hydroxy-, bimol. cyclic ester), poly-(lactic acid), poly-(glycolic), polyanhydride, poe, polyether ester, polycaprolactone, polyesteramide, polyphosphazene, poly phosphate, false polyamino acid, their mixture and copolymer.

8. one kind prepares lasting release medicinal method for compositions, and the active component that described Pharmaceutical composition contains is included in the polymer particle, and described method comprises:

(a) dried powder of the active component of described active component of acquisition or preparation;

(b) preparation comprises the polymer solution that is dissolved in the polymer in the cosolvent mixture;

(c) described dried powder is dispersed in the described polymer solution, to produce active component/mixture of polymers;

(d) prepare the microgranule that contains active component by described mixture;

(e) collect described microgranule;

(f) by the second time drying make described microgranule at last.

9. according to the method for claim 8, wherein said polymer is selected from poly-(lactide), poly-(Acetic acid, hydroxy-, bimol. cyclic ester), poly-(lactic acid), poly-(glycolic), polyanhydride, poe, polyether ester, polycaprolactone, polyesteramide, polyphosphazene, poly phosphate, false polyamino acid, their mixture and copolymer.

10. according to the method for claim 8, wherein said cosolvent mixture is made up of polymer solvent and non-solvent.

11. according to the method for claim 10, wherein said polymer solvent is a dichloromethane, and wherein said non-solvent is an ethanol.

12. a Pharmaceutical composition that contains the NESP of NESP or preparation, the NESP of wherein said NESP or preparation are included in the biodegradation biocompatibility polymerizing microballoons, described compositions discharges described NESP at least one month after giving the patient.

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