JP2007523840A - Combined use of keratinocyte growth factor agonist and castrin compound - Google Patents

Abstract

The present invention generally relates to compositions, conjugates and methods comprising a KGF agonist and a gastrin compound. The composition comprises a condition in which either a KGF agonist or a gastrin compound exhibits a therapeutic effect, including but not limited to diabetes, hypertension, chronic heart failure, fluid retention, metabolic syndrome and related diseases and disorders, and obesity. Can be used in treatment and / or prevention. The compositions or combination therapies of the present invention can be used at reduced doses of each compound, reducing the harmful toxic effects of each compound.

Description

(Field of Invention)
The present invention relates generally to compositions, conjugates and methods comprising keratinocyte growth factor agonists and gastrin compounds, and uses thereof.

(Background of the Invention)
Fibroblast growth factor (FGF) consists of at least 22 structurally related polypeptides (FGF-1 to FGF-22). Members of the FGF family are typically in the molecular weight range of 17-34 kDa and share 13-71% amino acid identity. FGF is highly conserved among vertebrate species in both gene structure and amino acid sequence. Various FGF molecules exhibit a wide range of biological activities in normal and abnormal states. These activities include angiogenesis, mitogenesis, cell differentiation and wound healing (Baird, A. et al., Cancer Cells 3: 239-243 (1991), Burgess, WH et al. Annu. Rev. Biochem. 58: 575-606 (1989)). When expressed improperly, some FGFs may be involved in cancer pathogenesis.

  Keratinocyte growth factor (KGF, KGF-1 or KGF-7) is a member of the FGF family, but unlike other FGF family members, it has little activity on mesenchymal tissue-derived cells but stimulates epithelial cells To do. KGF has been reported to provide changes in hair follicle morphogenesis, hepatocyte proliferation and epithelial cell proliferation in the lung, milk, pancreas, stomach, small intestine and large intestine (Panos et al., J. Clin. Invest 92: 969-977 (1993), Ulich et al., Am J. Path 144: 862-868 1994, Yi et al., Am J Path 145: 80-85, 1994, and Ulich et al., J. Clin Invest. 93: 1298-1306, 1994). Keratinocyte growth factor 2 (KGF-2, FGF-10) is a 208 amino acid glycoprotein having high protein sequence similarity to FGF-7, and both have mitotic activity against keratinocytes (Emoto et al. J. Biol. Chem. 272, 23191-23194). (For a review of fibroblast growth factors, see Powers CJ et al., Endocrine-Related Cancer 7: 165-197, 2000).

  Systemically administered KGF has been reported to partially reduce the increase in blood glucose in streptozotocin-induced diabetic rats, however, this effect is within 1-2 days of streptozotocin (STZ). Limited to administered KGF (US Pat. No. 5,858,977, filed Jan. 12, 1999). The improvement in diabetes may be due to KGF protecting β cells from the toxicity of streptozotocin. Therefore, this method can be limited in use because the beta cell deficiency for most diabetes occurs well before the start of treatment. Furthermore, KGF is effective in reducing blood glucose only in moderately diabetic rats. KGF administration results in a long-term decrease in blood glucose only when nonfasting blood glucose is less than 15 mM. More severe STZ diabetic rats with blood glucose levels above 15 mM respond in the form of a transient decrease in their blood glucose, and their pancreatic insulin content only increases gently. Since no increase in plasma insulin was shown, the observed increase in pancreatic insulin may be the result of low blood glucose-decreasing insulin release from the pancreas. Quantitative histological data showing an increase in β cell weight in response to administration of KGF has not been reported. Thus, improved glucose tolerance in STZ diabetic rats after KGF treatment has not been finally shown for the results of stimulation of β-cell regeneration. Thus, KGF is less effective in causing an antidiabetic response. High doses of KGF are necessary to improve glucose tolerance, ie doses of 1 mg / kg or less have no effect on blood glucose levels. The dose of KGF required to reduce blood glucose in diabetic rats further caused significant growth of the liver and other tissues.

  Citation of a reference herein is not an admission that such reference is available as prior art to the present invention.

(Summary of the Invention)
The present invention relates to any keratinocyte growth factor (KGF) agonist or gastrin compound, including but not limited to diabetes, hypertension, chronic heart failure, fluid retentive state, metabolic syndrome and related diseases and disorders, and obesity. However, the subject is a combination of a KGF agonist and a gastrin compound that provides a beneficial effect in the treatment of a condition that has been shown to have a therapeutic effect. A combination of a KGF agonist and a gastrin compound can be selected to provide an unexpected additive effect, or an effect that is greater than the additive effect, ie, a synergistic effect.

  Higher doses or longer periods of monotherapy (ie, treatment with each compound alone) with compositions, conjugates, or combination therapies, including KGF agonists and gastrin compounds, using different mechanisms to achieve maximal therapeutic effect Can reduce the risk of side effects and improve tolerance to treatment. The compositions or combination therapies of the present invention can be used at reduced doses of each compound, reducing the harmful toxic effects of each compound. Sub-optimal doses can increase safety margins and reduce the cost of drugs required to achieve prevention and treatment. Furthermore, treatment using a single combination dosage unit can increase convenience and, as a result, enhance compliance.

  The present invention contemplates a composition, preferably a pharmaceutical composition, comprising a KGF agonist and a gastrin compound. The pharmaceutical composition can optionally include a pharmaceutically acceptable carrier, excipient or vehicle.

  In one aspect, the composition provides a beneficial effect compared to each compound alone. In another aspect, the present invention contemplates a pharmaceutical composition comprising a KGF agonist and a gastrin compound that provides maintenance of beneficial effects, preferably sustained beneficial effects after treatment.

  The beneficial effects provided by the compositions of the present invention may include increased absorption, distribution, metabolism and / or elimination of KGF agonists and / or gastrin compounds. The composition can enhance bioavailability (absorbed more quickly and to a greater extent) and can enhance the therapeutic effect.

  The present invention also provides a pharmaceutical composition for the treatment of a disease or condition comprising a therapeutically effective amount of a KGF agonist and a gastrin compound in a pharmaceutically acceptable carrier, excipient, or vehicle. To do.

  In another aspect, the invention features a composition that includes a gastrin compound and a KGF agonist. The composition is an effective dose to induce proliferation of islet precursor cells into large amounts of mature insulin secreting cells. Furthermore, the composition is an effective dose to induce differentiation of islet precursor cells into mature insulin secreting cells. The composition can be in a pharmaceutically acceptable carrier.

  The present invention provides a conjugate comprising a KGF agonist linked to a gastrin compound. The conjugates can provide the beneficial effects described herein.

  The present invention also provides methods for preparing the compositions and conjugates of the invention that result in compositions and conjugates having beneficial effects.

  In one aspect, the present invention provides KGF agonists, gastrin compounds, and pharmaceutically acceptable carriers, excipients, or (s) effective to physically stabilize KGF agonists and / or gastrin compounds. A method of preparing a stable pharmaceutical composition of a KGF agonist adapted to provide a beneficial effect after treatment, comprising preparing a composition comprising a vehicle).

  The invention also provides a composition or conjugate of the invention for preventing and / or ameliorating disease severity, disease symptoms and / or the recurrence cycle of a disease or condition described herein, or The use of the therapeutic combinations of the present invention is contemplated. The present invention also relates to the prevention and treatment of a condition or disease in a subject using the compositions, therapeutic combinations, and conjugates of the present invention.

  The present invention treats a condition or disease in a subject comprising administering to the subject a therapeutically effective amount of at least one KGF agonist and at least one gastrin compound to obtain a beneficial effect. And / or methods for prevention are provided. In one aspect, the present invention provides a treatment method that provides a beneficial effect continuously after treatment.

  The invention comprises administering a KGF agonist and a gastrin compound, a composition or conjugate of the invention, together with a plurality of cells, to a subject in need thereof, thereby producing a beneficial effect. Alternatively, a method for treating a disease is provided. In one embodiment, the compound / composition / conjugate is administered systemically.

  The invention comprises contacting a stem cell or progenitor cell with a GF agonist and a gastrin compound, or a composition or conjugate of the invention, in an amount sufficient to proliferate and differentiate the stem cell or progenitor cell. Alternatively, a method for proliferating and differentiating progenitor cells into insulin secreting cells is provided. The amount of proliferation and differentiation can be significantly different compared to those performed in the absence of the compound, composition or conjugate. In one embodiment, stem or progenitor cells are contacted with the compound, composition or conjugate in culture. In other embodiments, stem or progenitor cells are contacted with a compound, composition or conjugate in a subject. The compound, composition or conjugate may be administered to the subject before, during or after implantation of the stem cells in the subject to proliferate and differentiate the stem cells in the subject.

  The invention also provides insulin secreting cells in a culture medium comprising contacting the cells with a KGF agonist and a gastrin compound, or a composition or conjugate of the invention, in an amount sufficient to enhance cell proliferation. Relates to a method for enhancing the growth of The amount of proliferation can be significantly different compared to that performed in the absence of the compound, composition or conjugate.

  The present invention also includes culturing the cells in the presence of the KGF agonist and gastrin compound, or composition or conjugate of the present invention, in the amount necessary to maintain the cells in the culture. In particular, it relates to a method for maintaining islet cells or precursor cells. The cells can be maintained in the culture for a significantly longer period of time compared to cells cultured in the absence of the compound, composition or conjugate. Culturing cells in the presence of a KGF agonist and gastrin compound, or a composition or conjugate of the invention can be particularly beneficial in preparing and maintaining cells for transplantation.

  The present invention further provides for contacting a plurality of cells ex vivo with a KGF agonist and a gastrin compound, or a composition or conjugate of the present invention, optionally culturing the cells and cells in need thereof. And a method for treating a subject suffering from a condition or disease described herein.

  The present invention still further comprises islet precursor cells in an amount sufficient to increase islet precursor cell proliferation in a subject, thereby inducing islet neogenesis, a KGF agonist and gastrin compound, composition of the invention Inducing islet neogenesis in a subject, including contacting with an object or conjugate.

  The present invention provides a gastrin compound and an amount sufficient to affect differentiation of a patient's islet progenitor cells into mature insulin-secreting cells and / or stimulate insulin synthesis in existing islet cells. A method for treating diabetes in a patient in need is provided by administering a composition comprising a KGF agonist. The composition is administered systemically or the nucleic acid structure comprises a coding sequence for a gastrin compound and / or a coding sequence for a KGF agonist, together with transcriptional and translational regulatory regions functional in islet precursor cells. By including the nucleic acid structure therein in the host cell, it can be expressed in situ.

  In addition, in order to increase the number of pancreatic beta cells in the islet cells, a sufficient amount of a gastrin compound and a KGF agonist are transplanted into the islet cells of a diabetic patient exposed in the culture medium, so that Methods and compositions for treating diabetes are also provided, and optionally a population of pancreatic beta cells can be grown in culture for the time required to expand the population of β-cells prior to transplantation.

  The present invention also contemplates the use of a composition comprising a combination of at least one KGF agonist and at least one gastrin compound for the preparation of a medicament for preventing and / or treating a condition or disease. In one embodiment, the present invention provides a synergistically effective combination of at least one KGF agonist and at least one gastrin compound for the preparation of a medicament for preventing and / or treating a condition or disease. Related to using the correct amount. The present invention further provides the use of the pharmaceutical compositions and conjugates of the present invention in the preparation of a medicament for the prevention and / or treatment of conditions and diseases. The medicament provides a beneficial effect, preferably a sustained beneficial effect after treatment.

  In one embodiment, the present invention provides a method for preventing and / or treating diabetes, in an amount sufficient to increase the number of pancreatic insulin secreting beta cells in a mammal, KGF or FGF. A composition comprising a combination of a KGFR ligand, such as -7 or KGF-2 or FGF-10, and a gastrin compound (eg, a gastrin / CCK receptor ligand) to a mammal in need thereof, thereby Methods are provided that include preventing and / or treating. The composition is administered systemically. The mammal is a diabetic mammal, for example, diabetic for a period of about 1 percent of its lifetime. The amount of KGF in the composition is significantly less than the minimum effective amount of KGF agonist required to reduce blood glucose in a diabetic mammal in the absence of a gastrin compound. The KGF agonist and gastrin compound are provided in an amount sufficient to induce differentiation of islet precursor cells into glucose-responsive insulin secreting islet cells.

  Another embodiment of the present invention is a method for preventing and / or treating diabetes, wherein the KGF agonist and gastrin are in an amount sufficient to proliferate proliferation of islet precursor cells in pancreatic tissue. Methods are provided that include administering a compound comprising a compound (eg, a gastrin / CCK receptor ligand) combination to a mammal in need thereof, and thereby preventing and / or treating diabetes.

  The method of the invention may further comprise administering at least one agent for suppression of the immune response, eg, the agent is selected from rapamycin, cyclosporine, ISAtx247 and FK506.

  Another embodiment of the present invention is a method for preventing and / or treating diabetes in an amount sufficient to increase the proliferation of islet precursor cells and the amount of insulin secreting islet cells, Contacting a plurality of cells ex vivo with a composition comprising a KGF agonist and a gastrin compound (eg, a gastrin / CCK receptor ligand), and administering the contacted cells to a mammal in need; And thereby providing a method comprising preventing and / or treating diabetes. The cell can be autologous. The composition is provided in an amount sufficient to affect stem cell differentiation, eg, to affect differentiation of islet precursor cells in pancreatic tissue into mature insulin secreting islet cells. The composition is provided in an amount sufficient to increase the proliferation of islet stem cells, eg, islet precursor cells. Stem cells can be obtained either from pancreatic tissue or from non-pancreatic cells, such as liver or bone marrow.

  In another aspect, embodiments of the present invention provide a method for proliferating and differentiating stem cells in a diabetic recipient of cells into insulin secreting cells, said method comprising transplanting the cells into the recipient And administering an effective dose of each gastrin compound and a composition comprising a KGF agonist. For example, transplanted cells are obtained from humans, such as from human islets, human liver, human bone marrow, human umbilical cord, or human embryos. The transplantation of cells into the recipient can be by a route such as direct injection into the tissue, eg, the pancreas, kidney, or liver. Alternatively, cell transplantation can be administered by intravenous injection, eg, into the portal vein or hepatic vein. In certain embodiments, prior to transplantation, the cells are treated ex vivo with a composition comprising a gastrin compound and a KGF agonist.

  In another aspect, the present invention provides a method for preventing and / or treating diabetes, said method being in an amount sufficient to increase the number of pancreatic insulin secreting beta cells in a mammal. Administering a composition comprising a combination of a KGF agonist and a gastrin compound (eg, a gastrin / CCK receptor ligand) to a mammal in need thereof, and measuring the amount of islet neogenesis, thereby preventing diabetes; And / or treating. Determination of the amount of islet neogenesis is measured by parameters selected from the group of blood glucose, serum glucose, glycosylated hemoglobin, pancreatic beta cell weight, serum insulin and pancreatic insulin content. Administration of the composition reduces blood glucose as compared to blood glucose assayed prior to administration of the composition. The glycosylated hemoglobin concentration is reduced compared to the glycosylated hemoglobin concentration in a mammal assayed prior to administration of the composition. Serum insulin concentration is increased compared to serum insulin concentration in mammals assayed prior to administration of the composition. Pancreatic insulin concentration is increased compared to pancreatic insulin concentration in mammals assayed prior to administration of the composition.

  In another aspect, the invention provides a method for inducing islet neogenesis in a mammal, said method comprising an amount sufficient to increase the proliferation of islet precursor cells in the pancreatic tissue. Administering a composition comprising a combination of a KGF agonist and a gastrin compound, thereby inducing islet neogenesis. The plurality of cells can be multicellular. Multiple cells are transmitted systemically to a mammal.

  In another aspect, the present invention provides a method for inducing islet neogenesis in a mammal, said method comprising an amount of KGF in an amount sufficient to increase the number of pancreatic insulin secreting beta cells in the mammal. Administration of a composition comprising a combination of an agonist and a gastrin compound is included.

  In another aspect, the present invention provides a method for inducing islet neogenesis therapy in an animal cell, wherein the cell is operatively linked to a regulatory element, such as an insulin promoter receptor ligand. It includes contacting a nucleic acid sequence encoding a compound and a nucleic acid sequence encoding a KGF agonist operably linked to a regulatory element, eg, a metallothionein promoter. For example, the cell is a germ cell, or the cell is an autologous cultured autologous cell.

  In another aspect, the invention provides a nucleic acid sequence encoding a mammalian KGF agonist operably linked to a heterologous promoter and a nucleic acid sequence encoding a mammalian gastrin compound operably linked to the heterologous promoter. Nucleic acid structures comprising are provided.

  In another aspect, the present invention provides a mammalian gastrin compound, wherein the germ cell is operably linked to a heterologous promoter, a nucleic acid sequence encoding a mammalian KGF agonist, and a mammalian gastrin compound operably linked to the heterologous promoter. A transgenic animal is provided comprising the encoding nucleic acid sequence.

  Since the present invention relates to a method of treatment comprising a combination of active agents that can be administered separately or administered as a conjugate or composition, the present invention also includes the KGF agonist and gastrin of the present invention in kit form. Also provided are kits comprising compounds, pharmaceutical compositions, conjugates.

  Accordingly, in one aspect, the present invention provides a kit for preventing and / or treating diabetes comprising a composition comprising a gastrin compound and a KGF agonist, a container and instructions. The composition of the kit further includes a pharmaceutically acceptable carrier.

  These and other aspects, features and advantages of the present invention should be apparent to those skilled in the art from the following drawings and detailed description.

  The invention can be better understood with reference to the drawings.

(Detailed description of implementation mode)
(the term)
In the evaluation items, the numerical ranges cited in this specification include all numbers and rational numbers included in the range (for example, 1 to 5 is 1, 1.5, 2, 2.75, 3, 3 .90, 4 and 5). It should be understood that all numbers and rational numbers are assumed to be altered by the phrase “about”. The phrase “about” is a plus or minus 0.1-50%, 5-50%, or 10-40%, preferably 10-20%, more preferably 10% or 15% of the referenced number. is there. Further, “a”, “an” and “the” should be understood to include the plural unless the content clearly dictates otherwise. Thus, for example, reference to a composition containing “a compound” includes a mixture of two or more compounds.

  The compounds described herein have one or more asymmetric centers and are enantiomers, diastereomers, and other stereoisomers that can be defined as absolute stereochemistry as (R)-or (S)-. May include body form. The present invention therefore includes all such possible diastereomers and enantiomers, as well as their racemic and optically pure forms. Optically active (R)-and (S) -isomers can be prepared using chiral synthons or chiral reagents, or can be separated using conventional techniques. When a compound described herein contains a geometrically asymmetric center, it is intended that the compound contain both E and A geometric isomers, unless otherwise stated. All tautomeric forms are intended to be included within the scope of the present invention.

  The terms “subject”, “individual”, “recipient” or “patient” mean an animal, including a warm-blooded animal such as a mammal, Suffering from or suspected of having or predisposing to the disease or condition described in. Mammals include, but are not limited to, mammalian members. In general, this phrase means human. The phrase also includes food, including horses, cows, sheep, poultry, fish, pigs, cats, dogs and zoo animals, rats, goats, apes (eg gorillas or chimpanzees), and rodents such as rats and mice. Including livestock raised for or as pets. The methods herein for use in a subject / individual / patient are intended for prophylactic as well as therapeutic use. Exemplary subjects for treatment include individuals suspected, suffering from, or suffering from the conditions or diseases described herein. In an embodiment of the invention, the subject can be non-diabetic, pre-diabetic or diabetic.

  The phrase “pharmaceutically acceptable carrier, excipient, or vehicle” does not interfere with the effect or activity of the active ingredient and is not toxic to the administered host. Means medium. Carriers, excipients, vehicles or diluents are required to prepare binders, adhesives, lubricants, disintegrants, swelling agents, wetting or emulsifying agents, pH buffering agents, and certain compositions Various materials are included such as the resulting absorbent. Examples of carriers and the like include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol and mixtures thereof. The use of such media and agents for active substrates is well known in the art.

  “Pharmaceutically acceptable salt (s)” includes salts of acidic or basic groups that may be present in compounds that are suitable for use in the present invention. . Examples of pharmaceutically acceptable salts include sodium, calcium, ammonium, iron hydroxide, isopropylamine, triethylamine, 2-ethylamine, 2-ethylamino, ethanol, histidine, procalin, and potassium salts and amino acids of carboxylic acid groups. The group hydrochloride is included. Other pharmaceutically acceptable salts of amino groups are hydrobromide, sulfate, hydrogen sulfate, phosphoric acid, acetic acid, oxalic acid, hydrogen phosphate, phosphoric anhydride, acetic acid, succinic acid, citric acid, tartaric acid, butyric acid , Mandelic acid, methanesulfonic acid (mesylate) and p-toluenesulfonic acid (tosylate) salts.

  The phrase “preventing and / or treating” means administering a composition or conjugate of the invention to a subject either before or after the onset of a condition or disease. To do. Treatment can be urgent or long-term.

  “Beneficial effect” means the effect of a combination of a KGF agonist and a gastrin compound, or a composition or conjugate thereof, in particular greater than the effect of either compound alone. Beneficial effects include favorable pharmacological and / or therapeutic effects, and improved pharmacokinetic properties and biological activity. The beneficial effect can be an additive effect or a synergistic effect.

  In embodiments of the invention, beneficial effects include, but are not limited to, the following: reduce or eliminate islet inflammation, reduce or prevent disease progression, increase survival, or disease or condition. Includes treating or disabling.

  In one embodiment, the beneficial effect is one or more of the following: (a) a decrease in fasting blood glucose level, particularly if the blood glucose level is greater than 7-10 mM; It can be demonstrated in diabetes by decreasing hemoglobin, (c) increasing serum insulin levels, (d) increasing pancreatic insulin production or content, and / or (e) preventing disease progression. In certain embodiments, beneficial effects include (a), (b) and (c), or (a), (c) and (d).

  In a preferred embodiment, the beneficial effect is a “sustained beneficial effect” in which the beneficial effect is maintained for a long time after the treatment is completed. The beneficial effect can be maintained after treatment for at least about 2-4 weeks, 2-8 weeks, 2-12 weeks, 2-24 weeks, 2-12 months, and 2-18 weeks. The period during which the beneficial effect is maintained can be proportional to the duration and timing of the treatment. Subjects are treated continuously for about 2-8 weeks, 2-12 weeks, 2-16 weeks, 2 weeks-6 months, 2 weeks-12 months, or on a regular basis. The sustained beneficial effect manifests as one or more of an increase in C-peptide output, an increase in pancreatic insulin output or concentration, and a near normal or low blood glucose level for a long period after treatment.

  The beneficial effect can be a statistically significant effect with respect to the statistical analysis of the effect of the two compounds versus the effect of each compound. Compared to each compound alone, the “statistically significant” or “significantly different” effect or level of the two compounds is higher or lower than the standard Represents a level. In embodiments of the present invention, the difference may be 1.5, 2, 3, 4, 5 or 6 times higher or lower compared to the effect obtained with each compound alone.

  The “additive effect” of a KGF agonist and a gastrin compound means an effect equal to the sum of the effects of the two individual compounds.

  A “synergistic effect” of a KGF agonist and a gastrin compound means an effect that is greater than the additive effect resulting from the sum of the effects of the two individual compounds.

  “Combination treatment” or “administering in combination” means that the active ingredients are administered simultaneously to the patient being treated. When administered in combination, each component may be administered at the same time or sequentially in any order at different points in time. Thus, each component may be administered separately, but may be administered at sufficiently close time intervals to obtain the desired effect, especially useful, additive or synergistic effects. The first compound may be administered on a dosing schedule that further includes treatment with the second compound.

A “therapeutically effective amount” is an active compound of the invention (eg, a KGF agonist and a compound) that can lead to one or more desired effects, particularly one or more sustained beneficial effects. Gastrin compound), or the amount or dose of a composition or conjugate. The therapeutically effective amount of the substrate may vary according to factors such as the disease state, age, sex and individual weight, and the ability of the substrate to manifest the desired response in the individual. Dosage regimes may be adjusted to provide the optimum therapeutic response. For example, several separate doses may be administered daily, or may be reduced proportionally, as suggested by treatment requirements.

  “Suboptimal dose” or “suboptimal dose” is the dose or dose of an active compound that is less than the optimum dose or dose for a compound when used in monotherapy. means.

  A “native-sequence polypeptide” or “a native polypeptide” includes a polypeptide having the same amino acid sequence of a polypeptide derived from nature. Such native-sequence polypeptides can be isolated from nature or can be produced by recombinant or synthetic methods. The phrase specifically includes naturally occurring shortened or secreted forms of the polypeptide, polypeptide variants that include naturally occurring variant forms (eg, alternatively spliced forms or splice variants), and naturally occurring alleles. Includes genetic variants.

  The phrase “polypeptide variant” comprises at least about 70-80%, preferably at least about 85%, more preferably at least about 90%, most preferably at least about 95% of a native-sequence polypeptide. A poly having amino acid sequence identity, in particular at least 70-80%, 85%, 90%, 95%, 98% or 99% amino acid sequence identity with any arbitrarily identified sequence of SEQ ID NOs: 1-11 Means peptide. Such variants include, for example, one or more amino acid residues, including variants from other species, at the N- or C-terminus of the full-length or saturated sequence of SEQ ID NOs: 1-11. Or a missing polypeptide, except for native-sequence polypeptides.

  The percent identity of two amino acid sequences or two nucleic acid sequences identified herein is the sequence after aligning the sequences and, if necessary, introducing gaps to achieve maximum percent sequence identity. As part of identity, it is defined as the percentage of amino acid residues or nucleotides in a candidate sequence that are identical to amino acid residues in a polypeptide or nucleic acid sequence, without considering conservative substituents. Alignments for purposes of determining percent amino acid or nucleic acid sequence identity can be found in various conventional methods, such as the GCG program package (Devereux J. et al., Nucleic Acids Research 12 (1): 387, 1984), BLASTP. , BLASTN, and FASTA (Atschul, SF et al. J. Molec. Biol. 215: 403-410, 1990). The BLAST program is publicly available from NCBI and other sources (BLAST Manual, Altschul, S. et al. NCBI NLM NIH Bethesda, Md. 20894, Altschul, S. et al. J. Mol. Biol. 215: 403-410, 1990). One skilled in the art can determine appropriate parameters for measuring alignment, including the algorithms necessary to achieve maximal alignment over the full length of the sequences being compared. The methods for determining identity and similarity are organized by publicly available computer software.

  An “analog” is the inversion of one or more amino acid residues of a parent polypeptide, wherein one or more amino acid residues of the parent polypeptide are replaced by other amino acid residues. Means a polypeptide in which one or more amino acid residues of the parent polypeptide are deleted and / or one or more amino acid residues are added to the parent peptide. Such additions, substitutions, deletions and / or inversions can be either N-terminal or C-terminal, or within the parent polypeptide, or a combination thereof.

  Mutations can be introduced into polypeptides by standard methods, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative substitutions can be made at one or more predicted non-essential amino acid residues. A “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Amino acids with similar side chains are known in the art, amino acids with basic side chains (eg, Lys, Arg, His), amino acids with acidic side chains (eg, Asp, Glu), uncharged polarity Amino acids with side chains (eg Gly, Asp, Glu, Ser, Thr, Tyr and Cys), amino acids with non-polar side chains (eg Ala, Val, Leu, Iso, Pro, Trp), β-branched side chains (Eg, Thr, Val, Iso) and aromatic side chains (eg, Tyr, Phe, Trp, His). Mutations can also be randomly introduced into some or all of the natural sequence, eg, by saturation mutagenesis. Following mutagenesis, the mutant polypeptide can be expressed recombinantly.

  “Derivative” means a polypeptide in which one or more of the amino acid residues of the parent polypeptide is chemically modified. Chemical modification includes addition of chemical sites, creation of new bonds, and removal of chemical sites. Polypeptides can be chemically modified, for example, by alkylation, acylation, glycosylation, pegylation, ester formation, deamidation, or amide formation.

  A “chimeric polypeptide” is all or a portion (preferably biologically active) of a selected polypeptide operably linked to a heterologous polypeptide (ie, a polypeptide other than the selected polypeptide). Included). Within the fusion protein, the phrase “operably linked” is intended to suggest that the selected polypeptide and the heterologous polypeptide are fused in-frame to each other. The heterologous polypeptide can be fused to the N-terminus or C-terminus of the selected polypeptide. Chimeric and fusion proteins can be produced by standard recombinant DNA techniques.

  In this context, “keratinocyte growth factor agonist” or “KGF agonist” refers to the activity of keratinocyte growth factors or their receptors, either completely or partially, directly or indirectly. Is understood to mean any compound, including peptide and non-peptide compounds that enhance, induce, mimic or amplify. In one aspect, the KGF agonist fully or partially activates human keratinocyte growth factor receptor. Without being limited to a particular mechanism, full or partial activation of the human keratinocyte growth factor receptor thus modulates the FGF signaling pathway and therefore contributes to the condition or disease. Keratinocyte growth factor receptors include receptors for keratinocyte growth factors including, but not limited to, FGFR-2, IIIb and / or FGFR-1, IIIb.

In a preferred embodiment, a “keratinocyte growth factor agonist” or “KGF agonist” is preferably a selected affinity constant as measured by methods known in the art. Any peptide or non-peptide small molecule or compound related to, binding to, interacting with or stimulating the KGF receptor (“KGF receptor ligand”) (KD) or efficacy (EC ₅₀ ) It is.

  In one aspect, KGF agonists exhibit insulin secretagogue activity, where insulin secretagogue activity can be measured in in vivo or in vitro assays known to those skilled in the art. In another aspect, the KGF agonist stimulates keratinocyte proliferation. In one embodiment, the KGF agonist is selected to stimulate a proliferative metaplasia of the pancreatic duct, including reactivation of beta islet cell neogenesis.

  KGF agonists include natural-sequence or synthetic polypeptides, fragments, analogs (eg variants), derivatives, isoforms, chimeric polypeptides, polypeptides with sequence identity, peptidomimetics and their pharmaceutically acceptable Salts, and active metabolites and prodrugs.

  In particular, KGF agonists, for example, share amino acid sequence identity with a native-sequence KGF agonist (eg, KGF or KGF-2), eg, 60%, 70%, 80%, 90%, 95%, 98% Or active analogs, fragments and other modifications that share 99% sequence identity.

  In the context of the present invention, the KGF agonist is a KGF receptor ligand, in particular keratinocyte growth factor or KGF. Keratinocyte growth factor is a tyrosine kinase receptor growth factor that has biological activity similar to that of an EGF receptor ligand. KGF stimulates through a tyrosine kinase receptor that differs from the EGF receptor, which causes an increase in proliferation of epithelial precursors in many epithelial cell types (Krakowski, ML et al., Am. J). Pathol.154: 683-691, 1999). This has a unique target cell specificity. In general, KGF stimulates the proliferation and differentiation of various cell types derived from primary or secondary mesoderm and from neuroectodermal. KGF stimulates epithelial cell proliferation but, unlike other FGFs, does not stimulate endothelial or fibroblast proliferation (Finch PW et al., Science 245: 752-755, 1989). .

KGF contains 163 amino acids, except for the signal sequence, and has a potential N-glycosylation site at amino acid 14 of the consensus sequence for glycosylation extended from amino acids 14-16 at the N-terminus. A native-sequence full-length polypeptide, designated KGF ₁₆₃ (KGF or FGF-7) (Finch PW et al, Science 245: 752-755, 1989). fgf-7 encodes a 194 amino acid protein containing a signal sequence (Aarson SA, Bottaro DP, Miki T, Ron D, Finch PW, Fleming TP, Ahn J, Taylor WG and Rubin JF 1991, Analths fs. New York Academy of Sciences 638: 62-77).

The amino acid sequences for KGF are shown in SEQ ID NOs: 1, 2 and 19 (SEQ ID NO: 10 is 163 amino acids, starting with methionine), and interchangeably, the peptide sequence of native KGF, unless otherwise stated And with some or all of the remaining biological activity of natural KGF, particularly non-fibroblast epithelial cell proliferation (eg, more than NIH / 3T3 fibroblasts). Show at least about 500-fold greater stimulation in BALB / MK keratinocyte cells and more than against BS / 589 epithelial cells or against CC1209 / 8 epithelial cells, as determined by ³ H-thymidine incorporation Also exhibits at least about 50-fold stimulation in BALB / MK keratinocyte cells) Characterized Te, natural of KGF and KGF analogs (e.g. mutant) is.

  KGF also includes the natural sequence of KGF-2 (FGF-10), a 208 amino acid glycoprotein with a signal sequence (Emoto et al, 1997, supra, Yamazaki et al., J. Biol. Chem. 271: 15918-15921, 1996, review article Powers et al., Endocrine-Related Cancer 7: 165-197). The sequences for native KGF-2 are shown in SEQ ID NOs: 3, 4 and 11 (SEQ ID NO: 11 is 169 amino acids and starts from methionine).

  KGF agonists for use in the present invention include those of SEQ ID NO: 1, 2, 3 or 4 except the signal sequence (see for example the first about 30-40 amino acid residues, SEQ ID NO: 10 or 11). KGF is included.

  In a particular aspect of the present invention, KGF agonists for use in the present invention include repifermin (Human Genome Sciences, Rockville, MD) or parifermin (Amgen, Thousand Oaks, Cal. ).

  Examples of analogs and derivatives of KGF contemplated by the present invention are listed in Table 1, and references cited therein are incorporated herein by reference.

  Functional assays for use in selecting KGF agonists for use in the present invention are known in the art, such as Hsu et al, Protein Expression and Purification 12: 189, 1998, Rubin et al. . 1989, and Igarishi et al, J. MoI. Biol. Chem. 273: 13230, 1998, which includes the mitogen assay.

  KGF agonists can be prepared by conventional methods known in the art. In particular, the amino acid site of a KGF agonist can be prepared by various methods known in the art, such as solid phase synthesis, purification from natural sources of KGF agonist, recombinant techniques, or combinations of these techniques. It is. See, for example, U.S. Patent No. WO 90/08771, and Dugas and Penney 1981, Merrifield, 1962, Stewart and Young 1969, and references cited therein.

  Purification of KGF from conditioned medium of human embryo fibroblast cell line, partial amino acid sequencing of purified KGF, gene cloning, and production of biologically active recombinant KGF in bacterial cells Gene expression has been described (see, eg, International Patent No. WO 90/08771 and Ron et al., J Biol Chem. 268: 2984-2988, 1993. Also, Rubin, JJ et al, Proc. Natl. Acad, Sci. USA 86: 802-806, 1989, Hsu, Y-R, et al, Protein Expression and Purification, 12: 189-200, 1998). Cloning of recombinant KGF-2 has been described by Igarashi, M .; Et al, J. et al. Biol. Chem. 273: 13230-13235, 1998 and Yamazaki, M .; et al, 1996 (supra).

  KGF agonists, especially KGF, can be produced by recombinant methods well known to those skilled in the art. Accordingly, the present invention contemplates the use of KGF agonists, particularly nucleotide sequences encoding KGF, and host cells comprising the nucleotide sequences for the preparation of KGF agonists.

  “Gastrin compounds” are peptides and non-peptides that enhance, induce, mimic or amplify the activity of gastrin or gastrin / CCK receptor, completely or partially, directly or indirectly It is understood to mean any compound, including compounds. In particular, gastrin compounds can be used to fully and partially link and / or activate gastrin / CCK receptors. A gastrin / CCK receptor includes a receptor linked to gastrin.

In some applications of the invention, the gastrin compound is a ligand that binds, binds, interacts with or stimulates the gastrin / CCK receptor ("gastrin / CCK receptor ligand"). Gastrin compound, as measured by methods known in the art, suitable IC _50, for example, with an IC ₅₀ of about ～0.7NM, may be selected as a peptide or non-peptide small molecules (in vitro cell proliferation Singh et al (1995) J. Biol. Chem. 270: 8429-8438, and Kopin et al (1995) J. Biol. Chem. 270: 5019-5023, and Singh et al (1995) describing the assay. ) See J. Biol. Chem. 270: 8429-8438, Kopin et al. (1995) J. Biol.Chem. 270: 5019-5023).

  “Gastrin compounds” include natural-sequence or synthetic gastrin polypeptides, fragments, analogs (eg, variants), derivatives, isoforms, chimeric polypeptides, polypeptides with sequence identity, peptidomimetics and These pharmaceutically acceptable salts, and active metabolites and prodrugs are included. In particular, the phrase includes various types such as gastrin 34 (large gastrin), gastrin 17 (small gastrin), and gastrin 8 (minigastrin), pentagastrin, tetragastrin and fragments thereof, analogs and derivatives. Contains gastrin.

  Examples of gastrin compounds that can be used in the present invention include those disclosed in US Pat. No. 6,288,301. In some applications of the invention, the gastrin compound is a peptide or non-peptide agonist of a gastrin receptor, such as A71378 (Lin et al., Am. J. Physiol. 258 (4 Pt 1): G648, 1990), Or it can be selected to be a partial agonist. In other applications of the present invention, gastrin compounds include, but are not limited to, gastrin compounds, including gastrin compounds described herein, or cholecystokinin (CCK) such as CCK58, CCK33, CCK22, CCK12 and CCK8. / CCK receptor ligand.

  Gastrin compounds also share amino acid sequence identity with, for example, endogenous mammalian gastrin, or native-sequence gastrin, eg, 60%, 70%, 80%, 90%, 95%, 98% or 99% Also included are active analogs, fragments and other modifications that share sequence identity.

  Gastrin compounds also include substrates that increase secretion of endogenous gastrin, cholecystokinin, or similar active peptides from tissue storage sites. These examples include gastric release peptides, omeprazole that inhibits gastric acid secretion and increases plasma gastrin levels, soybean trypsin inhibitor that increases CCK stimulation, and gastrin release that stimulates gastrin secretion without binding to gastrin receptor It is a peptide.

  The sequences for gastrin, including large gastrin-34 (Bonato et al, 1986, Life Science 39: 959) and small gastrin-17 (Bentley et al (1966) Nature 209: 583) are shown in SEQ ID NOs: 5-9. . Large gastrin-34 essentially has an amino acid sequence extension at the N-terminus of small gastrin-17. Large gastrin is cleaved in vivo to release gastrin-17. The N-terminal Glp is pyroglutamic acid, which is naturally a cyclic form of glutamic acid. In various embodiments, where cysteine or lysine is added to the end of gastrin with pyroglutamic acid, pyroglutamic acid is replaced with glutamic acid or pyroglutamic acid is deficient. Furthermore, each of gastrin 34 and gastrin-17 can be used with methionine or leucine at position 15, as shown herein in SEQ ID NOs: 5-8.

  “Gastrin compound” includes modified forms of gastrin. Various gastrins can be modified including, but not limited to, gastrin 34 (large gastrin), gastrin 17 (small gastrin) and gastrin 8 (minigastrin), pentagastrin and tetragastrin. The sequences for gastrin, including large gastrin-34 (Bonato et al, 1986, Life Science 39: 959) and small gastrin-17 (Bentley et al (1966) Nature 209: 583) are shown in SEQ ID NOs: 5-9. . Modified gastrin compounds for use in the present invention are described in PCT / CA03 / 01778, US Patent No. 10 / 719,450, US Application No. 60/519, all of which are incorporated by reference. The modified gastrin compound described in No. 933 is included. Modified gastrin compounds may have increased activity upon administration to a subject compared to native-sequence gastrin. May have a longer half-life in the subject's circulation.

  The modified gastrin compound may comprise a minimal gastrin component comprising at least an amino acid at positions 29-34 of SEQ ID NO: 5, or positions 12-17 of SEQ ID NO: 6. These are located at the carboxyl terminus of gastrin generated in the circulation, and in various gastrin compounds contemplated herein, there may be additional amino acids from, for example, gastrin.

The group of modified gastrin compounds include compounds having an amino acid sequence consisting of the amino terminus _{Z-Y m -X n -AA 1} -AA 2 -AA 3 -AA 4 -AA 5 -AA 6, wherein gastrin AA ₁ is Tyr or Phe, AA ₂ is Gly, Ala or Ser, AA ₃ is Trp, Val or Ile, and AA ₄ is Met, provided that the compound binds to the gastrin / CCK receptor. Or Leu, AA ₅ is Asp or Glu, AA ₆ is Phe or Tyr that can be amidated, Z is a polymer or protein sequence (eg, a serum protein such as human serum albumin), and Y _m Is a small natural amino acid amino acid, including but not limited to serine, glycine and / or alanine. Any spacer region comprising a non-acid residue, wherein X is residues 1-28 of SEQ ID NO: 5, residues 1-28 of SEQ ID NO: 6, residues 1-11 of SEQ ID NO: 7, or SEQ ID NO: 8 is any conserved site of residues 1-11. Generally, m is 0 to about 20 residues.

Y _m can be an amino acid sequence comprising m residues with glycine alternating with alanine, eg, [Gly-Ala] ₅ , or with a random sequence of glycine and alanine. The gastrin compound may further have a cysteine residue at the amino terminus of Y (m is greater than 1) or at the amino terminus of X (m is 0). The gastrin compound may further comprise a bifunctional crosslinker for linkage to Z. Generally, m is 0 to about 20 residues. In particular embodiment, m is 0, compounds, for connection to Z, further comprising a bifunctional crosslinking _{agent, X n -AA 1 -AA 2 -AA} 3 -AA 4 -AA 5 - it is an AA _6.

  In certain embodiments, X is from position 1 to position 11 of SEQ ID NO: 5, position 1 to position 11 of SEQ ID NO: 6, position 2 to position 11 of SEQ ID NO: 7, and position 2 to position 11 of SEQ ID NO: 8. Selected from the sequence group. A gastrin compound in which Z is a protein can be produced recombinantly.

  In a preferred embodiment, X is one or more amino acid residues from position 18 to position 28 of SEQ ID NO: 5 or 6. Thus, a gastrin compound with X may have any gastrin sequence from positions 18-28, 19-28, 20-28, 21-28, etc. The gastrin compound optionally includes an amino acid spacer of length m, where m is 0 to about 20 residues.

A modified gastrin compound of formula X _n -AA ₁ -AA ₂ -AA ₃ -AA ₄ -AA ₅ -AA ₆ where there is no spacer and m is 0 is further bifunctional for linkage to Z Z further comprises a non-proteinaceous polymer.

  In some embodiments of the modified gastrin compounds described herein, the gastrin compound comprises at least amino acid residues 29-34 of SEQ ID NO: 5 or 6 and is associated with a polymer, lipid or carbohydrate.

  The polymer may be a synthetic or naturally occurring polymer. The phrase polymer includes amino acid protein polymers and is not limited to synthetic polymers. The polymer can be polyethylene glycol (PEG) or dextran. When the polymer is a protein, in various embodiments, it can be a serum protein, such as serum albumin, such as human serum albumin.

  The modified gastrin compound may be based on SEQ ID NO: 5 or 6, or may be “large” gastrin-34, and may have a residue at position 32 that is methionine or leucine, respectively.

Other preferred modified gastrin compounds have the structure Z—Y _m —X, where Z is Cys or Lys, Y _m is any spacer region containing m amino acid residues of small natural amino acids, and X Are at least 6 amino acid residues comprising at least positions 12-17 of gastrin-17 (SEQ ID NO: 7 or 8) or at least positions 29-34 of gastrin-34 (SEQ ID NO: 5 or 6). The modified gastrin compound further includes a bifunctional crosslinker, where one reactive site of the crosslinker is covalently bonded to Z and the other reactive site is covalently bonded to the polymer or protein.

  A modified gastrin can be a gastrin derivative or analog that contains a minimal sequence of 6 amino acids (from the C-terminus) and that has an addition of reactive groups that can undergo an additive reaction. Thus, the modified gastrin compound may further comprise at least one reactive amino acid, such as a cysteine or lysine residue. Reactive amino acids can be added or substituted at the N-terminus. The addition of reactive amino acids can be a terminal region, and the spacer region optionally overrides the added reactive residue. For example, a spacer can be synthesized biologically as part of a gastrin amino acid sequence or can be chemically linked to a gastrin amino acid sequence to form a structure with a gastrin sequence-spacer-cysteine / lysine. The spacer can be a series of several amino acids, such as alanine or glycine.

In one embodiment, the modified gastrin compound AA ₁ -AA ₂ -AA ₃ -AA ₄ -AA ₅ -AA ₆ is selected from Tyr-Gly-Trp-Met-Asp-Phe or Tyr-Gly-Trp-Leu-Asp- Phe.

  Modified gastrin compounds are related to larger molecules such as polymers, either noncovalently or as covalent valence conjugates, or as fusion proteins to other peptidic compounds with amino acid sequences. It may include a moiety that is a compound. The modified gastrin compound has a longer half-life in circulation within the subject animal or patient and / or maintains a higher concentration of the gastrin compound in vivo for an extended period of time compared to the natural form of gastrin.

  In one embodiment, the modified gastrin compound binds to a relatively large structure, or multiple structures in the blood, and still leaves the ability to bind to a target protein, namely gastrin / CCK receptor. Including. In general, gastrin compounds bind to carrier proteins resulting in long-term drug effects. Alternatively, the gastrin compound can be conjugated to a polymerized carrier such as polyethylene glycol (PEG) or dextran to achieve a similar purpose.

  In embodiments of the invention, chemical modifications of gastrin compounds are used to provide compounds that react covalently or non-covalently to carrier proteins or polymerized carriers, either in vitro (ex vivo) or in vivo. Is done. In certain embodiments, the non-covalent interaction is electrostatic or hydrophobic. In certain embodiments, conjugation of the gastrin compound to the carrier is performed prior to injection. In other embodiments, the gastrin compound is modified in a manner that enhances its affinity for the carrier in the bloodstream upon injection. In other embodiments, long-acting gastrin compounds are obtained via chemical modification, without the need for carrier proteins, either in vivo or ex vivo.

  In certain embodiments, the carrier protein is a plasma protein. In related embodiments, the plasma protein is albumin or immunoglobulin, or a component of an immunoglobulin. The immunoglobulin or immunoglobulin component may be a portion that has been modified or deleted prior to conjugation. In certain embodiments, the polymerized carrier is polyethylene glycol or dextran. For example, activated PEG can be coupled to a gastrin compound via the amino group of the gastrin compound (Vernonese, FM. Biomaterials 22 (2001) -405-417).

  In other embodiments, a gastrin compound that is an amino acid sequence is genetically fused to a carrier protein, which is also an amino acid sequence, using standard recombinant genetic techniques prior to injection. A gastrin compound can be recombinantly fused to a carrier protein, for example, with or without a linker / spacer comprising a sequence of small naturally occurring uncharged amino acids. Nucleic acid encoding a gastrin compound can be recombinantly fused or directly synthesized as a fusion to part or all of a carrier protein, where the nucleic acid structure or fusion protein contains a number of additional amino acids. It can encode or incorporate and serve as a spacer between the two proteins. The recombinant fusion protein can be expressed in the host cell. Modifications to the sequence of the gastrin compound polypeptide can be introduced, if necessary, during the construction of the fusion protein.

  In one embodiment, the gastrin compound is modified to introduce reactive groups, such as those present on amino acids such as lysine or cysteine, and in addition to other proteins such as carrier proteins or carrier non-proteinaceous polymers. When contacted with a compound, the reactive group can form a carrier protein or polymer covalent interaction. For example, a reactive thiol group can be removed via an amino group on lysine using, for example, succinimidyl 3-2-pyridyldithiopropionic acid (SPDP) followed by reduction with DTT to release the active thiol group. N-Succinimidyl 3- (2-pyridinthio), and a new heterofund. (New protein dihydrogen H, bionewr.), Can be added to a gastrin compound ("Protein thiolation and reversible protein-protein conjugation. 723-737 (1987)). In addition, the bifunctional group can also be added after adding cysteine or lysine, so that one reactive end of the crosslinker reacts with cysteine / lysine while the other end of the other ends. The reactive ends of either remain exposed or conjugated to the carrier.

  Thiols are incorporated into carboxylic acid groups by EDAC-mediated reaction with cystamine followed by reduction of the disulfide with DTT. ("Introduction of sulfhydryl groups into protein at carboxylic sites." Lin, Mihal KA, Kruger RJ. Biochem Biophys Acta 1038, 382-538). In a non-limiting example, the reaction of an amino group on a lysine residue in a gastrin compound with succinimidyl trans-4- (maleimidylmethyl) cyclohexane-1-carboxylate (“Conjugation of glucose from aspergillus niger” and Rabbit antioxidants using N-hydroxysuccinimide ester of N- (4-carboxyliccyclohexylmethyl) -M3eI-3E, and Ei. Compound Ami It was introduced to the site, its gastrin compound followed by reaction with free thiol groups of cysteine residues or activated polymer carrier protein.

  Gastrin compound-carrier complex is a spacer arm or element or other component that facilitates the preparation or isolation of a gastrin compound-carrier complex or that can amplify or maintain the functional activity of a gastrin compound It is possible to include further modular elements, including The spacer arm can be one or more amino acids, peptides, peptidomimetics, or small organic compounds, and can include homobifunctional or heterobifunctional bifunctional crosslinkers or chitin oligomers or polyethylene glycol or related polymers. .

  In other embodiments, the carrier and gastrin compound can be covalently cross-linked with or without spacer arms. Examples of non-spacer arms (zero-length bridges) include EDC. The homobifunctional cross-linker that produces the spacer arm can be, for example, disuccinimidyl suberate, and the heterobifunctional cross-linker that produces the spacer arm is, for example, 2-iminothiolate, succinimidyl 6- [3 -(2-pyridyldithio) propionylamido] hexanoate (LC-SPDP) and 4- (N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC).

  In various embodiments, the gastrin compound is bound to a larger carrier site such as a polymer, eg, a protein. Since binding can be covalent or non-covalent, the protein can be considered a carrier protein. The type of carrier protein can have the characteristics of being non-antigenic, i.e. it can be a natural human protein and can be sustained continuously in the circulation. An ideal carrier protein is one normally found in the human circulatory system.

  Gastrin compounds can be prepared using conventional processes. For example, a small form of gastrin, such as gastrin 17, is economically prepared by peptide synthesis and synthetic peptides are commercially available. Synthetic human gastrin 17 and derivatives such as human gastrin 17 substituted with leucine for methionine at position 15 are also available from Bachem AG, Bubendorf, Switzerland and Research Plus. .

  In particular, gastrin compounds can be synthesized by solid phase synthesis (Merifield, 1964, J. Am. Chem. Assoc. 85: 2149-2154) or in homogeneous solution (Houbenweyl, 1987, Methods of Organic Chemistry, ed. E. Wain). , Vol. 15 I and II, Thimeme, Stuttgart), and may be synthesized by chemical synthesis using techniques well known in protein chemistry. The synthesis can be performed using manual procedures or by automation. Automated synthesis can be performed, for example, using an Applied Biosystems 431A peptide synthesizer (Perkin Elmer). Gastrin compounds can also be obtained from commercial sources. For example, synthetic human gastrin 17 with methionine or leucine at position 15 is obtained from Bacchem AG (Bubedorf (Switzerland)) and Research Plus (New Jersey, USA).

  Gastrin compounds can be prepared by recombinant methods well known to those skilled in the art. Thus, the present invention contemplates the use of host cells containing nucleotide sequences for the preparation of gastrin compounds and nucleotide sequences encoding gastrin compounds.

  “Host cells” comprising a nucleotide sequence of a KGF agonist or gastrin compound include a variety of prokaryotic and eukaryotic host cells. For example, the polypeptide can be expressed in bacterial cells such as E. coli, Bacillus, or Streptomyces, insect cells (using baculovirus), yeast cells or mammalian cells. Good. Other suitable host cells can be found at Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1991). The host cell may also be selected to modulate the expression of the inserted nucleotide sequence or to modify (eg, glycosylate or phosphorylate) and process (eg, cleave) the polypeptide in the manner desired. Host systems or cell lines may be selected to have specific and characteristic mechanisms for post-translational processing and modification of proteins. For long-term, high-yield stable expression of proteins, cell lines and host systems that stably express gene products may be engineered.

  “Regulatory element” means a genetic element or elements that have a regulatory role in gene expression, such as promoters or enhancers. A number of suitable vectors and promoters are known to those of skill in the art and are commercially available to produce recombinant constructs encoding KGF agonists or gastrin compounds. The following vectors are presented as examples. Bacteria: pBs, phage script, PsiX174, pBluescript, SK, pBs KS, pNH8a, pNH16a, pNH18a, pNH46a (Stratagene), pTrc99A, pKK223-3, pKK233-3, pDR540m, RI . Eukaryotes: pWLneo, pSV2cat, pOG44, PXTL pSG (Stratagene), pSVK3, pBPV, pMSG, pSVL (Pharmacia). As defined herein, “operably linked” refers to a polypeptide by an isolated polynucleotide and a host cell into which a regulatory element has been transformed (transfected) with a ligand polynucleotide / regulatory element sequence. Means that it is located in the vector or cell in the manner in which it is expressed.

  “Condition (s)” and / or “disease (s)” are those in which either a KGF agonist or a gastrin compound, or both, provide a therapeutic effect, Or more pathological symptoms or syndromes. The condition or disease may require a reduction in blood glucose levels, inhibition of gastric acid secretion, inhibition of β-cell apoptosis, stimulation of β-cell proliferation or differentiation, and weight loss. Examples of conditions and diseases include, but are not limited to, dyslipidemia, hyperglycemia, severe hypoglycemic events, stroke, left ventricular hypertrophy, arrhythmia, bacteremia, sepsis, irritable bowel syndrome, functional dyspepsia, Diabetes, postoperative catabolic changes, stress-induced hyperglycemia, gastric ulcer, myocardial infarction, impaired glucose tolerance, hypertension, chronic heart failure, fluid retention, metabolic syndrome and related diseases and disorders, obesity, Alzheimer's disease, Parkinson's disease and other Diabetes complications and other disease symptoms that damage tissues due to elevated glucose levels, including age-related relationships, tissue degenerative diseases, and joint effects of elevated leptin, for example, in patients with impaired glucose tolerance and obese non-diabetic patients Is included.

  As used herein, the phrase “diabetes” includes experimental animal models, and type I and type II diabetes, early stage diabetes, and mild reductions in insulin or mild blood glucose levels. By an apparent symptom of diabetes in any mammal, including the human form, such as a prediabetic condition characterized by elevation. "Pre-diabetic condition" indicates symptoms of insulin or glucose levels and / or suspected diabetes or related conditions due to family history, genetics, or obesity in the case of type II diabetes Are subjects at risk of recurrence, including subjects who have previously had diabetes or related conditions.

  “Insulinotropic activity” is a substrate that stimulates insulin secretion in response to elevated glucose levels and produces or increases glucose uptake by cells and decreases in serum glucose or blood glucose levels Means ability. It is possible to assay for insulin secretagogue activity using methods known in the art. For example, in vitro and in vivo methods are described in KGF receptor binding activity or gastrin receptor binding activity, receptor activation (European Patent No. 619,322 and US Pat. No. 5,120,712 to Gelfand et al). See method) and can be used to measure insulin or C-peptide levels. The compounds, compositions or conjugates described herein are those in which the islet cells are above background levels in the presence of the compound, composition or conjugate, and in the absence of the compound, composition or conjugate. Insulin secretion promoting activity when secreting insulin at the ground level.

  A condition or disease includes applications that require stimulation of epithelial cells, especially keratinocytes. A condition can be characterized by a disorder or defect in these particular cell types.

  Recombinant KGF has been shown to increase reepithelialization and increase epithelial thickness when applied topically to surgically induced wounds in rabbit ears and pig skin (Pierce et al.,). J. Exp. Med. 179: 831-840, 1994, Staiano-Coico et al., J. Exp. Med. 178: 865-878, 1993). Thus, KGF agonists in combination with gastrin compounds can be used as wound healing agents for burns or to stimulate transplanted corneal tissue.

  The compounds, compositions and conjugates of the invention can be used to promote and / or enhance soft tissue growth and regeneration. In particular, to stimulate the proliferation and differentiation of appendage structures such as hair follicles, sweat glands and sebaceous wires, for example to regenerate epithelium and skin in patients with burns and other partial and complete thickness disorders Can be used for Accordingly, the conditions and diseases contemplated herein are also not limited to epidermolysis bullosa, chemotherapy-induced alopecia, male pattern baldness, gastric ulcer, duodenal ulcer, inflammatory bowel disease (eg, Crohn's disease and ulcers) Colitis), enterotoxicity in radiation and chemotherapy, hyaline in premature infants, airway injury, emphysema, cirrhosis (eg following viral hepatitis and chronic alcohol consumption), fulminant liver failure, acute viral hepatitis Toxic injury to the liver caused by acetaminophen, halothane, carbon tetrachloride and other toxins, and conditions requiring the production of mucosa in the gastrointestinal tract.

  The compounds, compositions and conjugates of the present invention may be used to stimulate gastrointestinal mucosal proliferation and promote healing after bowel injury (Playford, RJJ Pathol. 184: 316). 322, 1998). Thus, they may have particular application in the treatment of mucositis (deletion of intestinal epithelial tissue after chemotherapy or radiation therapy for cancer).

  “Islet neogenesis” refers to the formation of new beta cells by proliferation and differentiation and may or may not have the characteristics of stem cells with the ability to regenerate in an unrestricted manner.

(KGF agonist and gastrin compound)
The present invention relates to compositions, conjugates and methods using KGF agonists and gastrin compounds to provide particularly beneficial effects. The compositions, conjugates and methods of the present invention provide enhanced beneficial effects, in particular sustained beneficial effects compared to KGF agonists and / or gastrin compounds alone. In embodiments of the present invention, the beneficial effect can be an additive effect or a synergistic effect.

  In one embodiment, wherein the disease or condition is diabetes, the sustained beneficial effect of the composition, combination therapy, or conjugate of the present invention is manifested as one or more of the following items: It ’s okay.

  • Increase in pancreatic insulin levels after administration to a subject with diabetes symptoms in the absence of active compound or compared to levels measured with each compound alone. Preferably, the compounds together are at least about 0.05%, 0.1%, 0.5%, 1%, 2%, 5%, 10%, 15%, 20% at the pancreatic insulin level in the subject. , 30%, 33%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% increase.

  • Reduced lack of symptoms of islet inflammation after administration to subjects with diabetes symptoms.

  • Decrease in blood glucose levels in subjects with diabetes symptoms in the absence of active compound or compared to levels measured with each compound alone. Preferably, the compounds together are at least about 2%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% for blood glucose levels. Induces% reduction. Most preferably, the compound provides a blood glucose level that is about the same as or close to that common in normal subjects.

  ・ Improved glucose tolerance. In particular, at least about a 10-90% improvement in glucose tolerance.

  • Increase in C-peptide levels in subjects with diabetes symptoms in the absence of active compound or compared to levels measured with each compound alone. Preferably, the compounds together are at least about 0.05%, 0.1%, 0.5%, 1%, 2%, 5%, 10%, 15%, 20%, 30% for C-peptide levels. , 33%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% increase.

  • Maintenance of near normal blood glucose levels over a long period of time, especially at least 1 week, 4 weeks, 16 weeks, 52 weeks or 78 weeks.

  Reduce, prevent or delay the rate of disease progression in diabetic subjects.

  Reduce or prevent the development of severe hyperglycemia and ketoacidosis in diabetic symptoms.

  • Increased survival in subjects with symptoms of diabetes.

  To at least 10-90%, 10-80%, 10-70%, 10-60%, 10-50%, 10-40%, 10-30%, or 10-20% insulin injection / uptake Reduced need.

  One or more of these beneficial effects can be demonstrated in a diabetic subject or disease model, such as a non-obese (NOD) mouse with a symptom of diabetes.

  Gastrin compounds are identified for specific embodiments of the invention and based on properties including the ability to increase the activity of the KGF agonist and / or increase the physical and chemical stability of the KGF agonist Choose to provide the beneficial effect (s) of. A gastrin compound can also be selected based on its ability to stimulate beta cell proliferation / differentiation, its in vivo half-life, or its insulinotropic activity.

  In one embodiment of the invention, the gastrin compound is gastrin 17, and analogs and derivatives thereof. In a particular embodiment, the gastrin compound is synthetic human gastrin I having 17 amino acid residues with a Leu residue at amino acid position 15.

  A KGF agonist may be selected for a particular application in the present invention based on one or more of the following characteristics. Ability to bind to KGF receptor, beta cell proliferation and / or differentiation, or insulin secretagogue activity, ability to initiate signaling pathway, glucose level, ability to reduce insulin secretagogue activity, beta cell proliferation / differentiation In vivo half-life (eg, US 2003/2000) of at least about 15 minutes to 24 hours, preferably 2 to 10 hours, or 2 to 8 hours in humans using stimulation and / or conventional methods The method described in No. 0144206).

  In an embodiment of the invention, the KGF agonist is a shortened polypeptide, or an analog or derivative thereof. The sequences of these shortened KGF agonists are shown in US 20030109439, US Pat. No. 5,677,278, International Patent No. WO 3095637 and US Pat. No. 6,677,301.

  In other embodiments of the invention, the KGF agonist has less than 10 amino acid residues differing from those in KGF or KLGF-2, and less than 5 amino acid residues differing from those in KGF; Or less than 3 amino acid residues are different from those in KGF or KGF-2, preferably only one amino acid residue is an analog of KGF or KGF-2 that differs from the sequence of KGF or KGF-2 .

  KGF agonists that can be specifically used in the present invention include polypeptides in which one or more amino acids have been added to the N-terminus and / or C-terminus of KGF or KGF-2. Preferably, about 1-150 amino acids are added to the N-terminus and / or about 1-150 amino acids may be added to the C-terminus.

  In other embodiments of the invention, at least one amino acid of the KGF agonist has at least one substituent attached directly or indirectly (eg, via a spacer such as γ-Glu or β-Ala). And). Substituents are generally selected to prolong the activity profile of the parent KGF agonist, make the KGF agonist more metabolically and physically stable, and / or increase the solubility of the KGF agonist. Examples of specific substituents include, but are not limited to, alkyl groups, groups with α-carboxylic acid groups, linear or branched fatty acid acyl groups, or alkanes such as tetradecanoyl and hexadecanoyl. Affinity substituent.

  In an embodiment of the invention, the KGF agonist is selected from the group consisting of KGF-1 and KGF-2.

(Composition)
The present invention contemplates a composition, preferably a pharmaceutical composition, comprising a KGF agonist and a gastrin compound, especially a gastrin / CCK receptor ligand. The pharmaceutical composition may optionally include a pharmaceutically acceptable carrier, excipient, or vehicle. In one aspect, the composition provides a beneficial effect compared to each compound alone. In another aspect, the present invention contemplates a pharmaceutical composition comprising a KGF agonist and a gastrin compound, particularly a gastrin / CCK receptor ligand, that provides a beneficial effect after treatment, preferably a sustained beneficial effect. .

  The KGF agonist and gastrin compound in the composition of the invention may be present in a ratio selected to increase the activity of the agonist and / or gastrin compound to provide a beneficial effect.

  The present invention also provides pharmaceutical compositions in separate containers, including beneficial effects comprising a KGF agonist and a gastrin compound, optionally together with a pharmaceutically acceptable carrier, excipient, or vehicle. Are intended for simultaneous or sequential administration.

  The pharmaceutical composition of the present invention can be selected to have a statistically significant sustained beneficial effect, preferably a sustained beneficial effect, compared to the KGF agonist or gastrin compound alone.

  In one embodiment, the pharmaceutical composition having a statistically significant and beneficial effect is a KGF agonist selected from the group consisting of KGF and KGF-2, and gastrin 17, analogs and derivatives thereof, preferably A gastrin compound selected from the group consisting of synthetic human gastrin I having a Leu residue and 17 amino acid residues at amino acid position 15 is provided.

  In one embodiment, a pharmaceutical composition having a statistically significant and beneficial effect is provided comprising KGF and gastrin-17 (leu).

  In one embodiment, the present invention includes pharmaceutically acceptable salts of KGF agonists and / or pharmaceutically acceptable salts of gastrin compounds.

  In other embodiments, a pharmaceutical composition is provided that is adapted for administration to a subject to provide a sustained beneficial effect to treat a condition or disease, preferably diabetes. . In a preferred embodiment, after cessation of treatment, it will persist in the subject for an extended period of time in such a form that blood glucose levels become nearly normal upon administration to the subject.

  In one embodiment, a composition comprising a KGF agonist and a gastrin compound has greater sustained insulinotropic activity after treatment compared to the activity of a KGF agonist or gastrin compound alone or after KGF alone. Have.

(Conjugate)
The present invention provides a conjugate comprising a KGF agonist linked to a gastrin compound, wherein the linkage is, for example, an amino or carboxyl group. The invention also relates to an isolated covalent valence conjugate of the invention and a composition comprising the covalent valence conjugate of the invention.

  A KGF agonist can be conjugated to the species via an ester bond between the OH and COOH of the gastrin compound.

  The conjugate of the KGF agonist and gastrin compound may be conjugated or conjugated with an intermediate spacer or linker. Suitable spacers or linkers can be mono- or disaccharides, amino acids, sulfuric acid, succinic acid, acetic acid, or oligomeric polymerized spacers or linkers containing one or more such sites.

  The present invention also provides a method for preparing the above covalent valence conjugates, which have improved pharmacokinetic properties, biological activity, and become conjugates having beneficial effects. This method involves incubating or reacting a KGF agonist with a gastrin compound under conditions that form a covalent bond between the two compounds.

  The present invention thus provides a process for preparing a covalent valence conjugate comprising a KGF agonist covalently linked or linked to a gastrin compound, conditions sufficient to form a covalent bond or linkage between the KGF agonist and the gastrin compound, pH A step comprising incubating or reacting a KGF agonist with gastrin and at time and isolating the covalent valence conjugate.

  The above steps for preparing a conjugate comprising a KGF agonist and a gastrin compound can provide a conjugate with a sufficient amount of a KGF agonist covalently linked to the gastrin compound.

  An N-terminal or C-terminal fusion protein or chimeric protein comprising a KGF agonist conjugated to a gastrin compound, optionally with a spacer or linker, can also be obtained via recombinant technology via the N-terminal or C- It can also be prepared by fusing the terminal sequence with the sequence of the gastrin compound.

  The present invention also provides conjugates prepared by the processes described herein.

  The invention also relates to a pharmacological formulation comprising a conjugate of the invention and a pharmaceutically acceptable carrier, excipient, or vehicle.

  The present invention further includes an essentially pure covalent valence conjugate comprising a KGF agonist covalently linked to a gastrin compound that provides a useful effect, preferably a sustained beneficial effect, compared to the KGF agonist alone. Relating to the pharmacological prescription of

  In one embodiment, a pharmacological formulation is provided consisting essentially of a covalent valence conjugate comprising a KGF agonist covalently linked to a gastrin compound without an intermediate spacer or linker.

(Apply)
The present invention uses the compositions of the invention or the combination treatment of the invention to prevent and / or ameliorate disease severity, disease symptoms and / or the recurrent cycle of the disease or condition described herein. Also intended. The invention also relates to the prevention and treatment of a condition or disease in a mammal using the compositions, combination treatments and conjugates of the invention.

  In one aspect, the present invention administers to a subject a therapeutically effective amount of at least one KGF agonist and at least one gastrin compound to provide a beneficial effect, preferably a sustained beneficial effect. Providing a combination therapy for preventing and / or treating a condition or disease in a subject.

  The invention includes an additive or synergistic amount that provides additive or synergistic activity, or a combination that conveys a therapeutically effective amount of a KGF agonist and gastrin compound, or an amount that provides a conjugate or composition of the invention Treatment is included. Accordingly, pharmaceutical compositions suitable for use in the present invention include compositions wherein the active ingredients are contained in a synergistic or therapeutically effective amount.

  Embodiments of the invention provide improved methods and compositions for the use of KGF and gastrin compounds to treat diabetes. The present invention, in one embodiment, achieves a better effect, efficacy and utility than that achieved with KGF alone, resulting in an improved therapeutic ratio with the combination of gastrin with KGF. Provide a combination. The greater effect resulted in glucose in severe diabetes after treatment was discontinued (Example 6) and also in newly started diabetes (Example 7), with prolonged blood glucose improvement. It can be shown by improving tolerance.

  Treatment with a combination of gastrin and KGF provided a decrease in blood glucose that was greater than that observed after treatment with KGF alone, this decrease began in severe diabetes (Example 5) and recently In diabetes (Example 7), it can be sustained for a long time after the treatment is stopped.

  In combination with gastrin, systemic administration of KGF may have greater efficacy compared to administering KGF alone. An improvement in glucose tolerance is observed with the KGF / gastrin combination using a lower dose of KGF that is not effective when administered alone, ie, a dose of less than 1 mg / kg body weight.

  The greater effect and effectiveness of the combination of gastrin and KGF is that the use of lower doses of KGF reduces undesirable side effects and toxicity due to KGF that induces the growth of other organs such as the liver. Improve the therapeutic ratio of treatment. The combination also enhances the usefulness of improving diabetes over time, even when treatment is begun at a long time after beta cell decay (see Examples 1 and 2). The gastrin / KGF combination may be effective in reducing blood glucose with treatments initiated 3-4 weeks after streptozotocin-induced beta cell disruption.

  The improvement in glucose tolerance after treatment with the gastrin / KGF combination is a result of β cell regeneration and the accompanying increase in β cell weight (Example 4). Histological analysis may indicate that treatment with gastrin / KGF stimulates β-cell regeneration with an increase in β-cell weight as measured morphologically. This can be confirmed biochemically by increasing the pancreatic insulin component. An increase in β-cell weight can also be an increase in insulin secretion in the bloodstream, which can be indicated by an increase in circulating C-peptide in plasma (Example 3).

  The present invention also provides a beneficial effect, preferably a sustained beneficial effect, when administered to a subject with diabetes symptoms manifested as a decrease in blood glucose levels and / or an increase in pancreatic insulin. It also relates to a method of treatment comprising administering a therapeutically effective amount of at least one KGF agonist in combination with administration of at least one gastrin compound.

  In one aspect of the invention, the therapeutically effective amount of the KGF agonist and gastrin compound are mixed prior to administration to the subject. In one embodiment, a therapeutically effective amount of a KGF agonist and a gastrin compound are mixed at a physiologically acceptable pH.

  In a further embodiment, the present invention treats Type I or Type II diabetes comprising administering a therapeutically effective amount of a composition or conjugate of the present invention or a combination of a KGF agonist and a gastrin compound. Methods are provided for preventing and / or treating.

  In a further embodiment, the present invention treats Type I or Type II diabetes comprising administering a therapeutically effective amount of a composition or conjugate of the present invention or a combination of a KGF agonist and a gastrin compound. Methods are provided for improving disease progression or obtaining a lower disease severity stage in an affected individual.

  The invention relates to the progression of impaired glucose tolerance, or non-insulin requirement II, comprising administering a therapeutically effective amount of a composition or conjugate of the invention or a combination of a KGF agonist and a gastrin compound. The present invention relates to a method for delaying progression of type 2 diabetes to insulin-requiring type II diabetes.

  The invention also relates to a method of increasing a subject's ability to synthesize insulin, comprising administering a therapeutically effective amount of a composition or conjugate of the invention, or administering a combination of a KGF agonist and a gastrin compound. Also related.

  An approach to beta cell replacement that avoids cell transplantation is to stimulate beta cell regeneration. Although early studies pointed out that β cells have limited regenerative capacity, it has become increasingly clear that pancreatic insulin secreting β cells contain a dynamic cell population. The weight of β cells can be increased through the proliferation of existing β cells (β cell replication). During pregnancy, luteinizing hormone, growth hormone (Holstad, M. et al. J. Endocrinol. 163: 229-234), and placental lactogen (Nielsen, JH. Et al., J. Mol. Med. 77). : 62-66, 1999) stimulates β-cell proliferation and increases β-cell weight. However, this increased weight depends on continuous hormonal stimulation. After birth, increased β-cell weight decreases to non-pregnant levels with a decrease in luteinizing hormone and placental lactogen ((Logothepopulos, J. (1972), Handbook of Physiology (Am. Physiol. Soc., Washington, DC)). ), Section 7 Chapter 3, pp. 67-76) Thus, in response to growth factor administration, an important aspect in assessing β-cell regeneration is that increased β-cell weight is associated with growth factor treatment. Whether it lasts a significant time after discontinuation.

  In one embodiment, the present invention comprises administering a therapeutically effective amount of a composition or conjugate of the present invention, or administering a combination of a KGF agonist and a gastrin compound in a subject. A method for stimulating proliferation is provided.

  In other embodiments, the present invention comprises administering a therapeutically effective amount of a composition or conjugate of the present invention, or administering a combination of a KGF agonist and a gastrin compound in a beta cell in a subject. A method is provided for increasing the number and / or size of.

  Methods for treating diabetes in an individual in need include the gastrin / CCK receptor ligand and the individual at a dose sufficient to affect the differentiation of islet precursor cells into mature insulin-secreting cells. Administration of a composition that provides both KCG receptor ligands is included. A method for treating insulin-dependent diabetes, especially type I, or juvenile diabetes, comprises administering to a diabetic mammal a differentiated and regenerating amount of both gastrin / CCK receptor ligand and KCG receptor ligand, preferably systemically, Stimulating islet neogenesis is included to increase the number of functional glucose-responsive insulin secreting β cells in the pancreas. The combination of gastrin and KGF receptor ligand can result in a significant enhancement of the islet neogenesis response than that observed only with either gastrin or KGF receptor ligand. A preferred gastrin / CCK receptor ligand is gastrin and a preferred KGF receptor ligand is KGF (FGF-7) or KGF-2 (FGF-10).

  Other embodiments include treating cells or treating a mammalian transplanted pancreatic tissue with a gastrin / CCK receptor ligand and a KGF receptor ligand, and treating the treated cell or pancreatic tissue with a mammal. It is a method including introducing into. In this embodiment, the preferred gastrin / CCK receptor ligand is gastrin and the preferred KGF receptor ligand is KGF (FGF-7) or KGF-2 (FGF-10).

  In another embodiment, the present invention provides a method for gastrin / CCK receptor ligand stimulation comprising providing a pancreatic cell with a chimeric insulin promoter-gastrin fusion gene and expressing the gene. In a further embodiment, a method of KGF receptor ligand stimulation is provided comprising the expression of a KGF receptor ligand transgenically introduced into a mammal, eg, KGF receptor ligand is described in US Pat. No. 5,885,956. KGF (FGF-7), similar to that provided in the issue.

  The present invention provides a gastrin / CCK receptor ligand, eg, gastrin, and a KCG receptor ligand, eg, KGF, KGF-2, at a dose sufficient to affect the differentiation of islet precursor cells into mature insulin-secreting cells. A method for treating diabetes is provided by administering a composition comprising: Both ligands in the composition can be administered systemically. Alternatively, one or both ligands can be expressed in situ by the cell that provided the nucleic acid fusion structure in an expression vector. The fusion structure typically includes a preprogastrin peptide precursor coding sequence and also includes a coding sequence for a KGF receptor ligand.

  Effective islet cell neogenesis is achieved by administration of both a gastrin / CCK receptor ligand, such as gastrin, and a growth factor ligand of the KGF tyrosine kinase receptor (TKR) family, such as KGF.

  Regenerative differentiation of pluripotent pancreatic progenitor cells, such as pancreatic duct cells, into mature insulin-secreting cells is to provide compositions, conjugates and methods for the treatment of diabetes, especially juvenile onset diabetes. And by therapeutic administration of this combination of factors or compositions provided for systemic administration or for in situ expression in the pancreas.

  The present invention provides a method of treating cells, preferably cells in culture, with a KGF agonist and gastrin compound of the present invention, or a composition or conjugate of the present invention. The invention also provides a cell-based treatment method using the KGF agonist and gastrin compound of the invention, or the composition or conjugate of the invention. See PCT / CA03 / 33595 for a description of general culture and cell-based treatment methods.

  In one aspect, the present invention administers a KGF agonist and gastrin compound, composition or conjugate of the present invention, along with a plurality of cells, to a subject in need thereof, thereby providing a beneficial effect, preferably Provides a method of treating a condition or disease comprising producing a sustained beneficial effect.

  A method for treating a subject having a condition or disease described herein comprises contacting a plurality of cells ex vivo with a KGF agonist or gastrin compound, or a composition or conjugate of the present invention, optionally Culturing the cells and administering the cells to a subject in need thereof.

  In an embodiment of the cell-based treatment method described above, the cell is a pancreatic duct cell, and the amount of compound / composition / conjugate used in the method generally refers to the amount of insulin-secreting cells in the subject. It is effective to increase. The cells can be autologous (ie, from the same subject), or from other individuals of the same species, or from different species.

  The invention also provides that islet precursor cells are contacted with a KGF agonist and gastrin compound, composition or conjugate of the invention in an amount sufficient to increase proliferation of the islet precursor cells in the subject. It also relates to inducing islet neogenesis in a subject, including inducing islet neogenesis.

  The invention also proliferates and differentiates stem cells into insulin secreting cells comprising contacting the stem cells with an effective amount of a KGF agonist and gastrin compound, or composition or conjugate of the invention. It also relates to the method. The compound, composition or conjugate may be administered to the subject before, during, or after transplanting the stem cells into the subject. Stem cells can be obtained from islets, umbilical cords, embryos or stem cell lines. The method further includes administering an immunosuppressive agent.

  The present invention contemplates a method of expanding functional beta cell weight of islet transplantation in diabetic patients, said method comprising a therapeutically effective amount of a KGF agonist and gastrin compound of the present invention, or a composition or conjugate. Is administered to the patient.

  The present invention also includes transplanting an islet preparation into a subject and administering a therapeutically effective amount of a KGF agonist and gastrin compound, or composition or conjugate of the present invention to diabetes in a subject. A method for treating is also contemplated.

  The method of the present invention further comprises one or more of the following markers: blood glucose, serum glucose, glycosylated hemoglobin, pancreatic beta cell weight, serum insulin, pancreatic insulin level, morphologically determined beta cell weight, Measuring the amount of insulin secreting cells and the glucose responsiveness of the insulin secreting cells can be included.

  The invention also provides a combination of at least one KGF agonist and at least one gastrin compound for the preparation of a medicament that provides a beneficial effect in treating a condition or disease, preferably a sustained beneficial effect. Contemplated for use of the composition.

  In one embodiment, the present invention provides a therapeutically effective amount of at least one KGF agonist for the preparation of a medicament that provides a beneficial effect, preferably a sustained beneficial effect, in treating a condition or disease. And using at least one gastrin compound.

  In one embodiment, the present invention provides a KGF agonist and gastrin compound for the preparation of a medicament for an increase (preferably a sustained increase) in the number and / or size of beta cells in a subject after treatment. Provide the use of.

  In another embodiment, the use of a KGF agonist and gastrin compound for the preparation of a medicament for stimulation of beta cell proliferation (preferably sustained stimulation) after treatment is provided.

  In yet a further embodiment, the present invention provides the use of a KGF agonist and gastrin compound for the preparation of a medicament for the treatment of type I or type II diabetes.

  The present invention further provides the use of the pharmaceutical compositions and conjugates of the present invention in the preparation of a medicament for beneficial effects, preferably sustained beneficial effects, in the treatment of diseases and conditions.

The therapeutic effects and toxicity of the compositions, conjugates and methods of the invention are ED ₅₀ (dose that is therapeutically effective in 50% of the population), or LD ₅₀ (lethal to 50% of the population). Dose) can be determined by standard pharmacological procedures in cell culture, such as by calculating statistical parameters, or in experimental animals. The therapeutic index is the dose ratio of therapeutic effect to toxic effect and can be expressed as ED ₅₀ / LD ₅₀ . Pharmaceutical compositions that exhibit large therapeutic indices are preferred.

  The present invention also provides immunosuppressive agents (eg, rapamycin, cyclosporine, ISAtx247, and FK506), anti-obesity agents, anti-diabetic agents, appetite regulating agents, hypertensive agents, conditions or diseases, particularly diabetes and obesity results, or these One or more additional therapeutic agents, including, but not limited to, drugs for the treatment and / or prevention of related complications, anti-emetics, anti-headache drugs, and generic drugs that treat or prevent side effects In combination with a method of use of the composition of the invention.

(Administration)
The gastrin compound in combination with a KGF agonist, the conjugates and compositions of the present invention provide for contacting the active agent (s) with a drug active site in the body of a subject or patient by any method. It can be administered. The active ingredients can be administered at the same time or sequentially and in any order at different time points to provide the desired beneficial effect. The compounds, conjugates and compositions can be formulated for sustained release for local or systemic delivery. It is the skill of a physician or veterinarian to select the form and route of administration that optimizes the effectiveness of the compositions, conjugates and treatments of the present invention.

  Parenteral modes of administration include, but are not limited to, transdermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, and oral routes. The compounds can be administered by conventional routes, for example by infusion or by bolus injection, by absorption through the epithelium or mucosal epithelium (eg oral mucosa, rectum and intestinal mucosa, etc.) Can be administered together. The preferred route of administration is systemic, eg, subcutaneous injection. For parenteral administration, the compounds or conjugates described herein can be mixed with saline, PBS, or other suitable buffer at an appropriate pH. Sustained release formulations can also be used for either or both therapeutic agents.

  The composition may be administered in oral dosage forms such as tablets, capsules (including sustained or sustained release formulations, respectively), pills, powders, granules, elixirs, tinctures, suspensions, syrups and emulsions. They may also be administered in intravenous (bolus or infusion), intraperitoneal, subcutaneous, or intramuscular form, all of which are dosage forms well known to those skilled in the pharmacological arts. . The compositions of the present invention may be administered via the intranasal route through topical use of a suitable intranasal vehicle or via a transdermal route, eg, using a conventional transdermal skin patch. Dosing protocols for administration using a transdermal delivery system may be continuous rather than intermittent during the dosage regimen.

  The dosing regimen of the present invention comprises the pharmacokinetic properties of the drug, and the mode and route of administration, patient species, age, gender, health, medical condition and weight, nature and extent of symptoms, type of combination treatment, treatment Depending on known factors such as the frequency of administration, the route of administration, the kidney and liver function of the patient, and the desired effect. The effective amount of drug needed to prevent, counter or prevent the progression of the condition can be readily determined by a physician or veterinarian.

  The therapeutic amount of the present invention, which can be effective in the treatment of a particular condition or disease, can depend on the nature of the condition or disease and can be determined by standard clinical techniques. The exact dose to be used in the formulation will also depend on the route of administration, condition or severity of the disease and should be determined according to the judgment of the physician and the circumstances of each patient. Routine determination of blood levels of insulin or C-peptide, fasting glucose or glucose challenge levels is determined by those skilled in the art. Suitable dosage ranges for administration are from about 0.01 micrograms to about 10,000 micrograms of each active compound per kilogram body weight per day, for example from about 0.01 micrograms to about 1 microgram / kg, about 0.1 microgram / kg to about 10 microgram / kg, about 1 microgram / kg to about 500 microgram / kg, or about 10 microgram / kg to about 10 mg / kg body weight / day. Suitable dosage ranges for administration are therefore generally from about 0.01 microgram / kg body weight / day to about 10 mg / kg body weight / day.

  Effective doses can be extrapolated from dose-response curves derived from in vitro or animal model test systems.

  The composition or treatment of the present invention may comprise a unit dose of at least one KGF agonist and a unit dose of at least one gastrin. A “unit dose” is a single unit, ie, a single dose that can be administered to a patient and is easy to handle and pack, either the active agent itself, or one or more solids or liquids A unit that remains as a physically and chemically stable unit, including either pharmacological excipients, carriers or combinations with excipients.

  In one aspect, the pharmaceutical composition reduces or reduces glucose levels for a sustained period of time, as compared to a dose of either a gastrin compound or a KGF agonist alone, or following treatment, beta cell proliferation or differentiation. It is provided with sub-optimal doses effective in the treatment of KGF agonists and gastrin compounds that are more effective in increasing.

  In another aspect, an improved pharmaceutical composition is provided comprising a suboptimal amount of a KGF agonist and gastrin compound effective for treatment in the form for chronic or acute treatment of a disease or condition, particularly diabetes. .

  In one embodiment, the composition provides a beneficial effect, preferably a sustained beneficial effect, equal to or at least 1.1 doses of each compound required to treat the disease or condition. , 1.5, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times lower doses containing KGF agonist and gastrin compound.

  In one aspect, the present invention relates to 0.5-6000, 100-1500, 100-6000, 1000-6000, 2000-6000 and 3000-6000 microgram KGF agonist per single unit, and per unit , 0.5-6000, 100-3000, 100-6000, 1000-6000, 2000-6000, and 3000-6000 microgram gastrin compound.

  In another aspect, the present invention relates to a KGF agonist between 0.1-20, 0.1-30, 0.1-40, 0.1-50 and 0.1-100 microgram / kg / day. , 0.1-20, 0.1-30, 0.1-40, 0.1-50, and 0.1-60 microgram / kg / day of a gastrin compound.

  A composition or formulation of the invention is administered to a subject for 2 weeks to 12 months, 2 weeks to 6 months, 2 to 16 weeks, 2 weeks to 12 weeks, and / or 2 to 8 weeks, or periodically. Good.

  In one embodiment, the ratio of KGF agonist to gastrin compound in the composition of the invention increases the activity of the KGF agonist and / or gastrin compound and has a beneficial effect, preferably a sustained beneficial effect. Selected to provide.

  KGF agonists and gastrin compounds increase the activity of one or both compounds and produce beneficial effects, preferably additive or synergistic effects, or beneficial effects, preferably sustained beneficial effects, It may be a selected ratio. In certain embodiments, the ratio of KGF agonist to gastrin compound is from 1: 1 to 1: 110, 1: 1 to 1: 100, 1: 1 to 1:75, 1: 1 to 1:50, 1: 1. It may be ˜1: 25, 1: 1 to 1:10, 1: 1 to 1: 5 and 1: 1. In other specific embodiments, the ratio of gastrin compound to KGF agonist is from 1: 1 to 1: 110, 1: 1 to 1: 100, 1: 1 to 1:75, 1: 1 to 1:50, : 1-1: 25, 1: 1-1: 10 and 1: 1-1: 5.

  In one embodiment, the KGF agonist may be used in combination with a gastrin compound in a therapeutically effective weight ratio of about 1: 1 to 1: 150, preferably 1: 1 to 1:50. In other embodiments, the gastrin compound may be used in combination with a KGF agonist at a therapeutically effective weight ratio of about 1: 1 to 1: 150, preferably 1: 1 to 1:50.

  The compositions of the invention or fractions thereof are typically selected based on the intended dosage form and are suitable pharmacological diluents, excipients, excipients consistent with conventional pharmacological practices. Includes vehicles or carriers. The carrier, vehicle, etc. may be adapted to provide an additive, synergistic or therapeutically effective amount of the active compound.

  Suitable pharmacological diluents, excipients, vehicles and carriers are described in the standard text, Remington's Pharmaceutical Sciences, Mack Publishing Company. As an example for oral administration in the form of capsules or tablets, the active ingredient is oral, such as lactose, starch, sucrose, methylcellulose, magnesium stearate, glucose, calcium sulfate, dicalcium phosphate, mannitol, sorbitol, etc. It can be mixed with a non-toxic, pharmaceutically acceptable inert carrier. For oral administration in liquid form, the drug component can be mixed with any oral, non-toxic, pharmaceutically acceptable inert carrier such as ethanol, glycerol, water and the like. Suitable binders (eg gelatin, starch, corn sweeteners, natural sugars including glucose, natural and synthetic gums and waxes), lubricating oils (eg sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, acetic acid) Sodium and sodium chloride), disintegrants (eg starch, methylcellulose, agar, bentonite, and xanthan gum), flavoring and coloring agents may also be incorporated into the composition or components thereof. The compositions described herein can further include wetting or emulsifying agents, or pH buffering agents.

  The composition can be a liquid solution, suspension, emulsion, tablet, pill, capsule, sustained release formulation, or powder. The composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides. Oral formulations can include standard carriers such as pharmacological grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate and the like. Various delivery systems are known and can be used to administer the compositions of the invention, such as, for example, encapsulation in liposomes, microparticles, microcapsules.

  In one aspect of the invention, the pharmaceutical composition has a pH of about 7-10.

  Formulations for parenteral administration of the compositions of this invention may include aqueous or oily suspensions and emulsions with edible oils, such as aqueous solutions, syrups, cottonseed oil, coconut oil or peanut oil. Dispersants or suspensions that can be used for aqueous suspensions include synthetic or natural gums such as tragacanth, alginate, acacia, dextran, sodium carboxymethylcellulose, gelatin, methylcellulose, and polyvinylpyrrolidone. .

  Compositions for parenteral administration include water, isotonic saline, isotonic glucose solutions, buffer solutions, or other solvents conveniently used for parenteral administration of therapeutically active agents. Aseptic aqueous or non-aqueous solvents may be included. Compositions intended for parenteral administration are also conventional additives such as stabilizers, buffers or preservatives, eg, oxidants such as methylhydroxybenzoic acid, or similar additives. May also be included.

  In one embodiment, the compositions herein are formulated according to routine procedures as pharmaceutical compositions adapted for subcutaneous or intravenous administration to humans. Typically, compositions for subcutaneous or intravenous administration are solutions in sterile isotonic aqueous buffer. If necessary, the composition may also include a solubilizing agent and local anesthesia to reduce pain at the site of the injection. In general, the components are administered as a unit, for example, as a dry, lyophilized powder, or a water-free concentrate, in a hermetically sealed container, such as an ampoule or a sachet indicating the amount of active agent Provided in form, either separately or mixed with each other. Where the composition is to be administered by infusion, an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.

  The compositions of the invention can be formulated as natural or salt forms. Pharmaceutically acceptable salts include those formed with free amino groups such as those derived from hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartrate, and the like, and sodium, potassium, ammonium, calcium, hydroxylated And those formed with free carboxyl groups such as those derived from diiron, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine and the like.

  In one embodiment, a solid form pharmaceutical composition is provided comprising a crystalline or amorphous KGF agonist and a gastrin compound (eg, in tablet, capsule, powder or ground form).

  In other embodiments, the solid form pharmaceutical composition is provided comprising a crystalline or amorphous, crystalline or amorphous gastrin compound or KGF agonist (eg, in tablet, capsule, powder or ground form).

  In another embodiment, the present invention provides a liquid drug formulation comprising a pharmaceutically acceptable salt of a KGF agonist and a gastrin compound, and a suspension that is stable and suitable for parenteral administration. It relates to a lyophilized drug formulation that is reconfigurable.

  In certain embodiments, the present invention relates to aqueous compositions comprising KGF agonists and pharmaceutically acceptable salts of gastrin compounds, and solvent systems that affect solubility. The present invention also provides a medicament comprising an aqueous formulation of a pharmaceutically acceptable salt of a KGF agonist and a gastrin compound comprising at least one solubilizer.

  The compositions of the invention can be made sterile by, for example, filtration through a bacteria-retaining filter, adding a sterilant to the composition, irradiating the composition, or heating the composition. Alternatively, the compounds, conjugates and compositions of the invention can be provided as sterile solid preparations, such as lyophilized powders, which are readily dissolved in a sterile solvent immediately prior to use.

  In addition to the formulations described herein, the composition can also be formulated as a depot preparation. Such long acting formulations may be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the fraction may be combined with a suitable polymerized or hydrophobic material (eg, as an acceptable emulsion in oil), or with an ion exchange resin, or as a slightly less soluble derivative, eg, as a slightly less soluble salt. You may prescribe.

  The compositions and components thereof of the present invention may include soluble polymers as targetable drug carriers.

  After the pharmaceutical composition is prepared, it can be placed in a suitable container and labeled for treatment of the indicated condition. For administration of the compositions of the invention, such labels can include the amount, frequency and method of administration.

  Since the present invention relates to a method of treatment comprising a combination of active agents that can be administered separately or as conjugates or as a composition, the present invention also relates to KGF agonists and gastrin compounds, pharmaceutical compositions in kit form. Also provided is a kit comprising the product or conjugate. The present invention also provides a pharmacological kit comprising one bottle containing a KGF agonist and another bottle containing a gastrin compound in one box.

  In an embodiment of the present invention, a pharmacological pack or kit is provided comprising one or more containers filled with one or more components of the pharmaceutical composition of the present invention. Such container (s) shall be manufactured, used or sold for human administration, as directed by the instructions or government agencies regulating the manufacture, use or sale of pharmaceuticals or biological products. Various descriptive documents are included, such as a note in a form that reflects the permission of the institution.

  In accordance with another aspect of the present invention, a kit is provided. The kit is a package that contains a container containing the covalent valence conjugate of the present invention and an instruction manual for administering the covalent valence conjugate to a subject.

  The invention will be described in greater detail by way of specific examples. The following examples are given for illustrative purposes and are not intended to limit the invention in any way. One skilled in the art can readily recognize a variety of non-critical parameters that can be changed or modified to produce essentially similar results.

Example 1. Experimental schedule for preparation of streptozotocin diabetic rats and treatment with KGF / gastrin
Male Wistar rats are injected with 30 mg / kg streptozotocin intravenously (iv) on 3 consecutive days. Blood glucose is controlled by insulin pellets (1 pellet per rat) incorporated subcutaneously prior to treatment. Blood glucose measurements are performed prior to incorporating insulin pellets subcutaneously (sc) and non-fasting blood glucose levels are monitored to determine the degree of glucose control. A combination of KGF and gastrin (50 and 300 μg / kg, respectively) is administered as a subcutaneous bolus injection 3 times a day for 4 weeks, starting 3 weeks after the STZ injection. An intraperitoneal glucose tolerance test (IPGTT) is performed 6 days after cessation of treatment. In addition, C-peptide and pancreatic insulin can be assessed.

Example 2. Assay of non-fasting blood glucose
Non-fasting blood in diabetic rats as compared to rats treated with vehicle alone by treating rats with a composition that is a combination of KGF / gastrin using the rats from Example 1. Glucose levels should be improved. Furthermore, following intraperitoneal glucose challenge, blood glucose should return to normal levels more quickly in KGF and gastrin treated rats.

Example 3. Glucose tolerance and insulin secretion assay
Using streptozotocin rats from Example 1, it can be demonstrated that treatment with the KGF / gastrin combination can improve insulin secretion and C-peptide release. The improvement in glucose tolerance in diabetic rats treated with KGF / gastrin must be the result of increased insulin secretion.

Example 4. KGF gastrin treatment increases pancreatic beta cell weight)
Rats were made diabetic by giving 65 mg / kg streptozotocin (STZ) at the time 10 days prior to treatment, including 250 μg / kg KGF each day and 250 μg / kg daily gastrin by subcutaneous injection for 2 weeks. Administered and treated. A similar diabetic control group receives vehicle by the same subcutaneous injection route as the KGF / gastrin group. One week after the end of the treatment period, the rats are sacrificed and the pancreas is collected for insulin measurement and β cell weight measurement.

  Beta cell weight is determined by total score morphology according to the following protocol. Paraffin sections (5 μm) are immunoperoxidase stained for insulin measurement. Diaminobenzidine is used as a substrate for peroxidase and the slides are counterstained with hematoxylin alone. One section from each pancreas is examined by total score. A rectangular 16 × 12 point grid, using a total of 192 points. Each pancreas section is counted using a 10 × magnification lens. The field of view is projected onto a computer screen and the final magnification is 265x. At this magnification, the beta-cell diameter is 3-5 mm. The total number of points counted per section is generally between 11,000 and 20,000, depending on the size of the section. β-cell content is calculated by dividing the number of points on insulin-stained cells by the total number of points on pancreatic tissue. β-cell content is expressed as a percentage. β-cell weight is calculated in mg, pancreatic weight, multiplied by β-cell content (%). Pancreatic insulin content is determined by immunoassay of pancreatic ethanol water extract.

  STZ causes a severe depletion of β-cell weight compared to that in normal (untreated) rats.

  There must be an increase in β-cell weight after 7 days of treatment with the KGF / gastrin combination. A proportional relationship must also be obtained between β-cell weight and insulin content. Thus, an increase in pancreatic insulin content may be the result of greater β-cell weight.

(Example 5. Combination of gastrin with KGF enhances the effectiveness of KGF)
Rats were made diabetic by intravenously giving streptozotocin (60 mg / kg) and after 14 days (not the 21-day interval in the above example), rats were divided into 4 groups, twice daily via intraperitoneal (ip), KGF. Administer 10 μg / kg, twice daily, 10 μg / kg gastrin administered ip, 10 μg / kg KGF and 10 μg / kg gastrin each in combination twice daily, or excipients Divide twice a day with ip.

  Prior to treatment, blood glucose levels are restored to non-diabetic levels by depot insulin replacement treatment. At the end of the 10-day treatment period, IPGTT was performed and the results should show that glucose tolerance was improved only in rats receiving the KGF / gastrin combination compared to rats receiving vehicle alone. Absent.

(Example 6. Long-term effect of KGF / gastrin treatment)
After 10 days of treatment with the KGF / gastrin combination, treated Stz rats were untreated diabetic rats in the vehicle group (STZ rats that had similar levels of diabetes prior to treatment with vehicle alone. ) Must show a long-term improvement in fasting blood glucose levels. Rats are treated with 10 μg / kg KGF and 10 μg / kg gastrin by ip injection twice daily for 10 days. After IPGTT, rats are measured and followed for both fasting blood glucose and glucose tolerance for up to 4 months. Treatment of rats with the KGF / gastrin combination causes a significant reduction in fasting blood glucose compared to untreated diabetic STZ rats, which is closer to that of non-diabetic control rats. Even at times such as 4 months after treatment, blood glucose in treated STZ rats will be significantly lower than in untreated control rats.

Example 7. Treatment with KGF and gastrin prevents disease progression in recently started diabetic NOD mice
The non-obese diabetic (NOD) mouse species is an excellent model for autoimmune (type I) diabetes because the phenotype reflects many features of the etiology in human type I diabetes. NOD mice are typically 4 weeks old and show destructive autoimmune pancreatic isletitis and β-cell destruction. Diabetes onset occurs in these mice at 10-15 weeks of age, and typical blood glucose levels are usually observed at 7-10 mM (in the range of about 3.0-6.6 mM in normal mice), Pancreatic insulin levels are over 95% lower than that of normal mice. As the disease progresses, NOD mice gradually show signs of severe chronic diabetes, with blood glucose levels reaching between about 25-30 mM and pancreatic insulin levels decreasing to substantially absent. . In severe stages of the disease, over 99% of β-cells are destroyed.

  In this example, the effect of treatment with the combination of KGF + gastrin is tested in a recently started diabetic NOD mouse. Among other things, the aim is to determine whether administration of both low doses of KGF and gastrin prevents severe hyperglycemia and increases pancreatic insulin content in recently-started diabetic NOD mice. Gastrin is a synthetic human gastrin I of 17 amino acid residues with a Leu residue at amino acid position 15 and KGF is recombinant human KGF.

  Non-obese diabetic (NOD) female mice, 10-12 weeks of age, were monitored for the development of diabetes (fasting glucose> 6.6 mmol / l) within 36 hours of onset of symptoms, and each of the three groups Mice are treated as follows. Vehicle, 0.25 μg / kg / day KGF, 1.5 μg / kg / day gastrin, or 0.25 μg / kg / day KGF + 1.5 μg / kg / day gastrin. Each treatment consists of i. p. By route, dosing twice a day. KGF in the range of about 0.1 μg / kg / day to about 100 μg / kg / day and gastrin in the range of about 1 μg / kg / day to about 60 μg / kg / day can be used. Treatment is administered for 14-28 days. At the discontinuation of treatment, the animals are left untreated for an additional 10-14 days and sacrificed to determine if the prevention of severe hyperglycemia persists after the end of treatment. Mice receive neither insulin-replacement treatment nor immunosuppression. The following parameters are assessed: survival rate, status of liver function, pancreatic insulin levels and fasting blood glucose levels.

  In animals in the vehicle-treated subject group, fasting blood glucose (FBG) values progressively increase during the time course of treatment, and the majority of vehicle-treated mice have severe hyperglycemia and keto It is known that it will eventually die from acidosis. In contrast, all mice in the group treated with KGF and gastrin survive and their FGB values are significantly lower compared to vehicle-treated mice. Improved control of blood glucose levels in mice treated with KGF and gastrin may be associated with a significant increase in insulin content in the pancreas of these mice. Treatment with KGF and gastrin will be more effective than treatment with KGF alone with respect to the parameters measured.

  In summary, the examples show that short-term, low-dose KGF and gastrin treatment in newly-started diabetic mice prevents disease progression, improves pancreatic insulin content, and these effects are the end of treatment. Later, it can be shown that it is maintained for a long time. Treatment with a combination of KGF and gastrin is expected to result in a decrease in fasting blood glucose levels and an increase in pancreatic insulin content that is greater than changes in these parameters in mice treated with KGF alone. Instead, lower concentrations of KGF can be used in combination with gastrin, which can reduce known side effects for KGF at higher concentrations.

  Treatment with KGF and gastrin prevents and / or compensates for the autoimmune destruction of islet β-cells, thereby at a level sufficient to prevent severe hyperglycemia, ketoacidosis and death. Maintain weight and pancreatic insulin content. In addition, administration of both KGF and gastrin prevents progression in recent diabetic mice without simultaneous insulin replacement and immunosuppression.

Example 8. Effect of gastrin and keratinocyte growth factor (KGF) in the treatment of acute diabetic non-obese diabetic (NOD) mice
This study aimed to inhibit the progression of diabetes in diabetic NOD mice in vivo via systemic treatment with gastrin and KGF. For 12-14 week old female NOD mice, diabetes developed for 18 days with vehicle (PBS), or KGF 0.25 μg / kg / day + gastrin 1.5 μg / kg / day, and fasted blood glucose ( Within 2 days after the FGB) level was 9-15 mM (normal FGB <6.6 mM), treatment was performed by intraperitoneal injection. There were a total of 4 animals per group.

  18 days after treatment, FBG was 18.3 ± 4.2 mM (mean ± SE) in vehicle-treated mice. On the other hand, the FBG of the treatment group was significantly lower than that of the vehicle-treated mice. In mice treated with a combination of KGF and gastrin, FBG was 10.2 ± 1.0 mM (mean ± SE) (FIG. 1).

  Thus, short-term KGF and gastrin treatment of diabetic NOD mice with recently-onset diabetes reduces hyperglycemia and inhibits the progression of diabetes.

  Such embodiments are merely intended to illustrate one aspect of the invention, and any functionally equivalent embodiment is within the scope of the invention, and thus, according to the particular embodiment described in the present invention. The object of the present invention is not limited. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and accompanying drawings. Such modifications are intended to fall within the scope of the appended claims.

  All publications, patents, and patent specifications cited herein are intended to indicate that each individual publication, patent or patent specification is specifically and individually incorporated by reference in its entirety. As is done, to the same extent, all of which is incorporated in the reference. The citation of any references herein is not an understanding that such references are available as prior art to the present invention.

FIG. 1 is a graph showing fasted blood glucose levels in vehicle-treated mice and mice treated with a combination of KGF and gastrin.

Claims (68)

A pharmaceutical composition comprising a KGF agonist and gastrin compound, and optionally a pharmaceutically acceptable carrier, excipient or vehicle, which provides a beneficial effect compared to each compound alone. 2. A pharmaceutical composition according to claim 1 which provides a lasting beneficial effect. 3. The pharmaceutical composition of claim 2, in a form that provides normal blood glucose levels in a subject that persist for a long period of time after administration. 4. The pharmaceutical composition according to any of claims 1 to 3, comprising a therapeutically effective amount of a KGF agonist and a gastrin compound in a form for chronic or acute treatment of a subject in need of treatment. 5. The pharmaceutical composition according to claim 4, wherein the therapeutically effective amount is suboptimal compared to the amount of each compound administered alone for the treatment of diabetes. 6. The pharmaceutical composition according to any of claims 1 to 5, wherein the ratio of KGF agonist to gastrin compound is selected to increase the activity of the KGF agonist or gastrin compound. The ratio of the KGF agonist to gastrin compound is about 1: 1 to 1: 110, 1: 1 to 1: 100, 1: 1 to 1:75, 1: 1 to 1:50, 1: 1 to 1:25. The pharmaceutical composition of claim 6, derived from 1: 1 to 1:10, 1: 1 to 1: 5 and 1: 1. The gastrin compound to KGF agonist ratio is about 1: 1 to 1: 110, 1: 1 to 1: 100, 1: 1 to 1:75, 1: 1 to 1:50, 1: 1 to 1:25. The pharmaceutical composition of claim 6, derived from 1: 1 to 1:10, and 1: 1 to 1: 5. The KGF agonist is used in combination with a gastrin compound in a therapeutically effective weight ratio between about 1: 1.5 and 1: 150, preferably between 1: 2 and 1:50. A pharmaceutical composition according to any of -8. The KGF agonist and the gastrin compound are at least about 1.1 to 1.4 times, 1.5 times, 2 times, 3 times greater than the dose of each compound alone required to treat the disease or condition, 10. The pharmaceutical composition according to any of claims 1-9, present at a dose that is 4, 5, 6, 7, 8, 9, or 10 times lower. 2. The pharmaceutical composition of claim 1, comprising an additive or synergistic amount of a KGF agonist and gastrin compound in a pharmaceutically acceptable excipient, carrier or vehicle. 0.1-20 microgram / kg / day, 0.1-30 microgram / kg / day, 0.1-40 microgram / kg / day, 0.1-50 microgram / kg / day and 1-60 microgram / kg / day KGF agonist, and 0.1-20 microgram / kg / day, 0.1-30 microgram / kg / day, 0.1-40 microgram / kg / day, The pharmaceutical composition according to claim 1, comprising 0.1-50 microgram / kg / day and 0.1-60 microgram / kg / day of a gastrin compound. The beneficial effect is one or more of: reducing or eliminating islet inflammation, reducing disease progression, increasing survival, or reducing symptoms of a disease or condition The pharmaceutical composition according to claim 2, which is an effect. 14. The pharmaceutical composition according to any of claims 1 to 13, wherein the beneficial effect is a sustained beneficial effect that is maintained for a long time after the end of treatment. The beneficial effect lasts for at least about 2-4 weeks, 2-8 weeks, 2-12 weeks, 2-24 weeks, 2 weeks to 12 months, and 2 weeks to 18 months after treatment. Item 14. A pharmaceutical composition according to Item 13. 14. The sustained beneficial effect can manifest as increased C-peptide production, increased pancreatic insulin production, and near normal or low blood glucose levels for a long period after treatment. Pharmaceutical composition. The pharmacological agent according to any of claims 1 to 16, wherein the sustained beneficial effect is statistically significant by statistical analysis of the effect of the KGF agonist and gastrin compound compared to the effect of each compound. Composition. The beneficial effect is at least about 0.05%, about 0.1%, about 0.5%, about 1%, about 2%, about 5%, about 10%, about 15% of pancreatic insulin level, 18. A pharmaceutical composition according to any of claims 1 to 17, which is an increase of about 20%, about 30%, about 33%, about 35%, about 40%, about 45% or about 50%. The beneficial effect is at least about 2%, about 5%, about 10%, about 15%, about 20%, about 30%, about 40%, about 50%, about 60%, about 60% of blood glucose level. 19. The pharmaceutical composition according to any of claims 1-18, which is a reduction of 70%, about 80%, or about 90%. The beneficial effect is at least about 2-4 weeks, about 2-8 weeks, about 2-12 weeks, about 2-24 weeks, about 2-12 months, and about 2-18 months after treatment. 20. The pharmaceutical composition according to any of claims 1 to 19, which is a decrease in blood glucose level over a period of. 21. The pharmaceutical composition according to any of claims 1 to 20, wherein the KGF agonist is KGF, KGF-2, fragments, analogs and derivatives thereof, and active metabolites and prodrugs of KGF. The pharmaceutical composition according to any one of claims 1 to 21, wherein the KGF agonist is an analog or derivative of KGF listed in Table 1. Preventing and / or preventing a condition or disease in a subject comprising administering to the subject a therapeutically effective amount of at least one KGF agonist and a gastrin compound to produce a sustained beneficial effect. How to treat. A therapeutically effective amount of at least one KGF agonist in combination with administration of at least one gastrin compound that provides a sustained beneficial effect upon administration to a subject having diabetes symptoms. A method of treatment comprising the step of: 25. The method of claim 24, wherein administration of at least one KGF agonist in combination with administration of at least one gastrin compound provides a sustained beneficial effect of at least one diabetic condition. 25. The method of claim 24, wherein a therapeutically effective amount of the KGF agonist and gastrin compound are mixed prior to administration to the subject. 25. The method of claim 24, wherein a therapeutically effective amount of the KGF agonist and gastrin compound is administered to the subject sequentially. 28. The method of any of claims 23-27, wherein the therapeutically effective amount of the KGF agonist and gastrin compound are administered at a physiologically acceptable pH. A conjugate comprising a KGF agonist linked to a gastrin compound to provide a beneficial effect, particularly a sustained beneficial effect. KGF agonist, gastrin compound and pharmaceutically acceptable effective to physically stabilize the KGF agonist and adapted to provide beneficial effects, preferably sustained beneficial effects A method of preparing a stable pharmaceutical composition of a KGF agonist comprising the step of mixing possible carriers, excipients or vehicles. Administering to a subject in need of treatment a therapeutically effective amount of a KGF agonist and a gastrin compound, or a composition according to any of claims 1-22, or a conjugate according to claim 29, A method of treating a condition or disease thereby comprising the step of producing a beneficial effect, preferably a sustained beneficial effect. Administering a KGF agonist and a gastrin compound, or a composition according to any of claims 1-22 or a conjugate according to claim 29, together with a plurality of cells, to a subject in need of treatment. , Thereby treating a condition or disease comprising the step of producing a beneficial effect, preferably a sustained beneficial effect. Contacting a plurality of cells ex vivo with a KGF agonist and a gastrin compound, or a composition according to any of claims 1-22, or a conjugate according to claim 29, optionally culturing the cells And a method for treating a subject having a condition or disease comprising administering the cells to a subject in need of treatment. The condition or disease is dyslipidemia, hyperglycemia, severe hypoglycemic event, stroke, left ventricular hypertrophy, arrhythmia, bacteremia, sepsis, irritable bowel syndrome, functional dyspepsia, diabetes, post-operative catabolic changes Stress induced hyperglycemia, gastric ulcer, myocardial infarction, impaired glucose tolerance, hypertension, Alzheimer's disease and other central and peripheral neurodegenerative conditions, chronic heart failure, fluid retention, metabolic syndrome and related diseases, and disorders and obesity 34. A method according to any one of claims 23 to 28 and 30 to 33. 23. A KGF agonist and gastrin compound, or a composition or claim according to any of claims 1-22, in an amount sufficient to increase proliferation of islet precursor cells in a subject and thereby induce islet neogenesis. Item 30. A method for inducing islet neogenesis in a subject comprising the step of contacting the conjugate of Item 29 with islet precursor cells. A stem cell to an insulin-secreting cell comprising the step of contacting the stem cell with an effective amount of a KGF agonist and a gastrin compound, or a composition according to any one of claims 1 to 22 or a conjugate according to claim 29. Methods for proliferation and differentiation. Use of a composition comprising a combination of at least one KGF agonist and at least one gastrin compound for the preparation of a medicament for the treatment of a condition or disease. The condition or disease is dyslipidemia, hyperglycemia, severe hypoglycemic event, stroke, left ventricular hypertrophy, arrhythmia, bacteremia, sepsis, irritable bowel syndrome, functional dyspepsia, diabetes, post-operative catabolic changes Stress induced hyperglycemia, gastric ulcer, myocardial infarction, impaired glucose tolerance, hypertension, Alzheimer's disease and other central and peripheral neurodegenerative conditions, chronic heart failure, fluid retention, metabolic syndrome and related diseases, and disorders and obesity 38. The use according to claim 37, wherein A kit form of the composition according to any one of claims 1 to 22 or the conjugate according to claim 29. A method for preventing and / or treating diabetes, wherein the mammal in need thereof is in an amount sufficient to increase the number of pancreatic insulin secreting beta cells in the mammal. Administering a composition containing a combination of a ligand and a gastrin / CCK receptor ligand, thereby preventing and / or treating diabetes. A method for preventing and / or treating diabetes, in a mammal in need thereof, in an amount sufficient to increase proliferation of islet precursor cells in pancreatic tissue and Administering a composition containing a gastrin / CCK receptor ligand combination, thereby preventing and / or treating diabetes. A method for preventing and / or treating diabetes, wherein ex vivo, a plurality of cells is added in an amount sufficient to increase the proliferation of islet precursor cells and the amount of insulin secreting islet cells. Contacting with a composition comprising a receptor ligand and a gastrin / CCK receptor ligand; and administering the contacted cells to a mammal in need thereof, thereby preventing and / or treating diabetes. Including. The amount of KGF in the composition is substantially less than the minimum effective dose of KGF required to reduce blood glucose in a diabetic mammal in the absence of gastrin / CCK receptor ligand. 43. The method according to 42. A method for preventing and / or treating diabetes, wherein a KGF receptor is provided to a mammal in need thereof in an amount sufficient to increase the number of pancreatic insulin secreting beta cells in the mammal. Administering a composition comprising a ligand and a gastrin / CCK receptor ligand combination, and determining the amount of islet neogenesis, thereby preventing and / or treating diabetes. Determining the amount of islet neogenesis from a group of blood glucose, serum glucose, blood glycosylated hemoglobin, pancreatic beta cell weight, serum insulin, pancreatic insulin content, and morphologically determined beta cell weight; 45. The method of claim 44, wherein the method is a step of measuring a selected parameter. 46. The method of claim 45, wherein administering the composition reduces blood glucose as compared to blood glucose assayed prior to administering the composition. 46. The method of claim 45, wherein administering the composition reduces blood glucose by 50% compared to blood glucose assayed prior to administering the composition. 46. The method of claim 45, wherein the glycosylated hemoglobin concentration is reduced compared to a glycosylated hemoglobin concentration in a control mammal that is not administered the composition. 46. The method of claim 45, wherein the serum C peptide insulin concentration is increased compared to the serum insulin concentration in the mammal assayed prior to administering the composition. 46. The method of claim 45, wherein the pancreatic insulin concentration is increased compared to the pancreatic insulin concentration in the mammal assayed prior to administering the composition. 46. The method of claim 45, wherein the cell is a pancreatic duct cell. 46. The method of claim 45, wherein the KGF receptor ligand and the gastrin / CCK receptor ligand are provided in an amount sufficient to induce differentiation of islet precursor cells into glucose-responsive insulin secreting islet cells. 46. The method of claim 45, wherein the KGF receptor ligand and the gastrin / CCK receptor ligand are provided in an amount sufficient to induce differentiation of islet precursor cells into glucose-responsive insulin secreting islet cells. 46. The method of claim 45, wherein the composition is provided in an amount sufficient to achieve differentiation of islet precursor cells in pancreatic tissue into mature insulin secreting islet cells. 46. The method of claim 45, wherein the composition is provided in an amount sufficient to increase islet precursor cell proliferation. A method for inducing islet neogenesis in a mammal, the KGF receptor ligand and gastrin / CCK receptor in an amount sufficient to increase proliferation of islet precursor cells in the pancreatic tissue. Administering a composition containing a combination with a ligand, thereby inducing islet neogenesis. 57. The method of claim 56, wherein the plurality of cells are multicellular. 57. The method of claim 56, wherein the plurality of cells are transmitted systemically to the mammal. A method for inducing islet neogenesis in a mammal comprising an amount of a KGF receptor ligand and a gastrin / CCK receptor ligand in an amount sufficient to increase the number of pancreatic insulin secreting β cells in the mammal. Administering a composition containing the combination. A method for inducing islet neogenesis in an animal cell, comprising a nucleic acid sequence encoding a gastrin / CCK receptor ligand operably linked to an insulin promoter receptor ligand, and a KGF receptor operably linked to a metallothionein promoter Contacting the cell with a nucleic acid sequence encoding a ligand. 61. The method of claim 60, wherein the cell is a germ cell. 61. The method of claim 60, wherein the cell is an autologous cell cultured ex vivo. A nucleic acid construct comprising a nucleic acid sequence encoding a mammalian KGF receptor ligand operably linked to a heterologous promoter and a nucleic acid sequence encoding a mammalian gastrin / CCK receptor ligand operably linked to a heterologous promoter. A composition comprising a gastrin / CCK receptor ligand and a KGF receptor ligand. 65. The composition of claim 64, at a dosage effective to induce proliferation of islet precursor cells into large amounts of mature insulin secreting cells. 65. The composition of claim 64, at a dosage effective to induce differentiation of islet precursor cells into mature insulin secreting cells. A nucleic acid sequence encoding a mammalian KGF receptor ligand, wherein the germ cells of the transgenic animal are operably linked to a heterologous promoter, and a mammalian gastrin / CCK receptor operably linked to a heterologous promoter A transgenic animal comprising a nucleic acid sequence encoding a ligand. A kit for preventing and / or treating diabetes comprising a composition containing gastrin / CCK receptor ligand and KGF receptor ligand, a container and instructions for use.

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