HK1087038A - Composition and method for treating diabetes - Google Patents

Description

Compositions and methods for treating diabetes

Background

Type I diabetes is a disease caused by a progressive autoimmune response that selectively destroys insulin-producing beta cells, and islet cells are lost in type I diabetics. Type II diabetes is a so-called adult disease, but the proportion of occurrences in obese young people is also increasing, with beta cells in type II diabetics being reduced by as much as 60% of normal levels. The number of functional beta cells in the islets of langerhans is of crucial importance for the onset, progression and consequences of diabetes. In type I diabetic patients, the number of beta cells drops to less than 2% of normal levels. Even type II diabetes, which has severe rejection of insulin, occurs only if the number of beta cells is not sufficiently increased. Thus, either type of diabetes is due to the inability of β cells to grow adaptively and the resulting lack of insulin secretion. Therefore, the ability to stimulate the production of islets and beta cells by precursor cells, i.e. "islet neogenesis", would be a novel and attractive approach to ameliorating the symptoms of diabetes.

Through a series of experiments, a pancreatic extract called "Ilotropin" was prepared. Experiments have shown that this compound stimulates the already present precursor cells, which are associated with the pancreatic ductal system, to regenerate beta cells. The search and identification of active ingredients in ilotropin was carried out on the assumption that transformation of pancreatic ductal cells to cause islet regeneration is determined by internal growth factors, genes and protein products. It is also this line of research that has led to the discovery of a novel gene and its associated protein (INGAP), which is in turn involved in islet regeneration.

INGAP peptide (INGAP)^104-118) The 15 amino acid sequence contained within INGAP, 175 amino acids, was shown to stimulate proliferation of pancreatic duct cells in the rat cheek. INGAP peptide is amino acids 103 to 117 of sequence identification No. 2 of U.S. patent 5,834,590, which is incorporated herein by reference.

Summary of The Invention

The present invention relates to a dosing regimen and formulation of INGAP peptides. The formulations disclosed herein have acceptable stability as pharmaceutical agents and adequate safety for use in human clinical trials. INGAP peptides prepared as follows were further shown to be able to regenerate functional islet cells that maintain normal feedback control.

It is therefore an object of the present invention to provide a pharmaceutically acceptable and stable INGAP peptide composition which is involved in the regeneration process of Langerhans islets.

It is another object of the present invention to provide a method of treating diabetes in a mammal.

It is yet another object of the present invention to provide a method of treating physiological dysregulation of blood glucose in a mammal.

It is a further object of the present invention to provide a method of increasing the number of pancreatic beta cells or Langerhans islets in a mammal.

It is a further object of the present invention to provide a method of treating a mammal receiving a transplant of pancreatic islet cells.

It is still another object of the present invention to provide a method for inducing differentiation of pancreatic precursor cells.

All cited relevant documents are incorporated in corresponding part of the present invention by reference. The citation of any reference is not to be construed as an admission that it is prior art with respect to the present invention.

Description of the figures

FIG. 1 shows ARIP cells (rat pancreatic ductal cells) treated with INGAP peptide, showing that the number of cells increases with increasing INGAP.

Figure 2 shows the increase in islet cell number following INGAP administration to normal syrian cheek mice.

FIG. 3 shows the time course of blood glucose after in vivo administration of INGAP peptide or physiological saline in C57BL/J6 mice induced by streptozotocin to cause diabetes.

FIG. 4 shows the normal distribution of insulin and glucagon in the pancreas of C57BL/J6 mice that are induced by streptozotocin to cause diabetes after treatment with INGAP peptide.

FIG. 5 shows that INGAP peptide induces PDX-1 expression in cells in the pancreatic ductal wall of C57BL/J6 mice.

FIG. 6 shows the islet tissue comparison between C57BL/J6 mice treated with streptozotocin and mice treated sequentially with streptozotocin and INGAP.

Figure 7 shows the percent insulin increase in the insulin immunoreactive tissue region of normal mice 31 days after treatment with INGAP peptide.

Figure 8 shows the percent insulin increase in the insulin immunoreactive tissue region of normal dogs 34 days after treatment with INGAP peptide.

Detailed Description

Glossary

The following is a list of definitions of terms used in the present invention:

"pharmaceutically acceptable salt" refers to a cationic salt formed from any acidic group (e.g., carboxyl) or an anionic salt formed from any basic group (e.g., amino, alkylamino, dialkylamino, morphinyl, and other similar groups) on the compounds of the present invention. Since the INGAP peptide is amphiphilic, both salts are possible and acceptable. Many such salts are known in the art. Preferred cationic salts include, but are not limited to, alkali metal ion salts (e.g., sodium and potassium), alkaline earth metal ion salts (e.g., magnesium and calcium), and organic salts, such as ammonium salts. Preferred anionic salts include halides, sulfonates, carboxylates, phosphates and other similar salts. It is expressly contemplated in this salt to provide addition salts with an optically active center, since no optically active center is present prior to addition. For example, chiral tartrate salts may be prepared using the compounds of the present invention, and such chiral salts are included in this definition. The salt is considered to be non-toxic in the amount administered to the affected animal, mammal or human. Suitable additional salts include, but are not limited to, hydrochloride, hydrobromide, hydroiodide, sulfate, bisulfate, acetate, trifluoroacetate, nitrate, citrate, fumarate, formate, stearate, succinate, maleate, malonate, adipate, glutarate, lactate, propionate, butyrate, tartrate, methanesulfonate, trifluoromethanesulfonate, p-toluenesulfonate, dodecylsulfonate, cyclohexylsulfamate and the like.

"biohydrolyzable ester" refers to ester compounds of the present invention wherein the ester compound should not substantially interfere, preferably not interfere at all, with the biological activity of the compound, or be selected for easy conversion in the patient to produce an ester of the active compound. Many such esters are known in the art, as described in U.S. patent 4,783,443 to Johnston and Mobashery, published 1988, 11/8. Such esters include lower alkyl esters, alkyl esters of lower acyl groups (e.g., methyl acetate, ethyl acetate, methyl carbamate, methyl isovalerate, and ethyl isovalerate), lactones (e.g., 2-benzo [ c ] furanone and thiophthalide), alkyl lower alkoxycarboxylates (e.g., dimethyl carbonate, diethyl carbonate, isopropyl ethyl carbonate), alkoxyalkyl esters, choline esters, and amidoalkyl esters (e.g., acetamidomethyl ester).

As used herein, "treatment" refers at least to the ability of a compound of the invention to alleviate a disorder resulting from a physiological glucose imbalance, which is suitable for a subject, preferably a mammal, more preferably a human. Thus, the term "treating" includes: preventing a disorder in a subject caused by a physiological glycemic abnormality, particularly when the subject is highly predisposed to, but not yet diagnosed with, the disease; inhibiting disorders caused by physiological blood glucose abnormalities; and/or alleviating or reversing a disorder in a subject caused by a physiological glycemic abnormality. At present, the method of the invention is mainly used for preventing the disorder of the experimental subject caused by physiological blood sugar abnormality. It is to be understood that "prevention" as referred to herein does not require a complete cure of the diseased state (Webster ninth edition university student dictionary), and that prevention as referred to herein refers to the ability of a skilled artisan to identify persons prone to disorders due to physiological blood glucose abnormalities, and thus, the compounds of the present invention may be advantageously applied to prevent such disorders prior to the onset of such disorders due to physiological blood glucose abnormalities. The reference to "prevention" in the present invention does not mean that the occurrence of the disease can be completely avoided. The population with disorders due to physiological blood glucose abnormalities (type I and type II diabetes) is mainly those who have a predisposing gene for diabetes through a family history. Other risk factors include, for example, obesity and improper diet.

Manufacture and stability

INGAP peptide is an amino acid sequence consisting of 15 amino acids, 104 to 118, contained in 175 natural amino acids INGAP. INGAP peptides can be synthesized by a variety of different methods known in the art, although preferably by solid phase synthesis using methyl 9-fluorocarboxylate (Fmoc). A preferred form of INGAP peptide is INGAP peptide in the form of a pharmaceutically acceptable salt, preferably acetate. The preparation of peptide salts is well known in the art. A synthesis using methyl 9-fluorocarboxylate (moc) is described in U.S. Pat. No. 4,108,846. Fmoc cleaved the methoxycarbonyl group with piperidine and cleaved the peptide from the resin with trifluoroacetic acid. The crude INGAP product produced by this process can be purified by preparative high performance chromatography.

The amino acid sequence of INGAP peptide is as follows:

NH₂-Ile-Gly-Leu-His-Asp-Pro-Ser-His-Gly-Thr-Leu-Pro-Asn-Gly-Ser-COOH(SEQ ID NO：3)

INGAP peptidesHas a molecular formula of C₆₄H₁₀₀N₂₀O₂₂Molecular weight is 1501.6 + -1 Dalton, and optical rotation is-103.2 ° in 1% strength acetic acid.

The structure of INGAP peptide can be confirmed by analyzing the constituent amino acids obtained by hydrolysis of INGAP peptide molecules. The correct molar ratio of the various amino acids present can be quantified on the basis of molecular structure, and the molecular weight of the peptide can be confirmed by electrospray mass spectrometry and should be consistent with the calculated, theoretical mass (1501.6. + -. 1 mass unit).

To confirm whether the synthesized molecule has biological activity, the activity can be confirmed by bioassay. A rat pancreatic ductal cell □ ARIP cell obtained from ATCC (Manassas, Va.) was used for the bioassay. The test cells were loaded in a culture plate having 96 wells at 10,000 cells per well, and cultured in DMEM medium containing 10% fetal bovine serum for 24 hours. After 24 hours, DMEM medium without serum was replaced. Then using different doses (0, 10)^-3And 10^-5G/ml) of INGAP peptide to treat the same wells. After 21 hours, the cells were incubated for 3 hours after adding a bromodeoxyuridine (BrdU) labeling solution in a BrDU cell proliferation ELISA kit from Roche Molecular Biochemicals into the culture medium. After 24 hours, the cells were dried at 60 ℃ for 60 minutes, fixed and denatured. According to the kit instructions, these cells were placed in BrdU antibody for 90 minutes and reacted for 15 minutes. Cells with BrdU labeling can be counted using a Wallac Victor model 1420 multiple label counter. The results were compared to a standard curve of cell proliferation performed in the same plate, starting at a cell density of 100 to 20,000 cells per well. As shown in fig. 1, the bioassay results indicated that: cells treated with INGAP peptide at 0.1 μ g/ml proliferated approximately 1.6-fold compared to the control.

Stability of INGAP peptides

Stability is determined by comparing various parameters including, but not limited to, purity of the sample, total percent impurities, individual percent impurities (as determined by high performance liquid chromatography or other suitable quantitative method), appearance, and water content. Methods of high performance liquid chromatography can be used to determine the increase in the content of various degradation products relative to INGAP peptides. INGAP peptide samples (whether solution or lyophilized powder) were stored in various temperature, moist or dry states, and in clear or light-protected vials. Degradation under different storage conditions leads to an increase in impurities and a decrease in the amount of INGAP peptide. It is desirable that the purity of the sample preparation is greater than 80%, preferably greater than 90%, more preferably greater than 95%, and most preferably greater than 97%.

INGAP peptide is stable under any storage conditions when stored as a freeze-dried powder. Under these storage conditions, the purity of INGAP peptide can be well maintained and degradation products are below acceptable levels. Further storage periods of up to six months did not cause any significant degradation of INGAP peptide.

Composition comprising a metal oxide and a metal oxide

Another aspect of the invention is a composition comprising: (a) a safe and effective amount of a peptide of the present invention, (b) a pharmaceutically acceptable carrier. Standard pharmaceutical compounding techniques are employed, for example, those disclosed in the latest version of Remington's pharmaceutical Sciences, edited by Easton, Pa., published by Mack publishing company.

By "safe and effective amount" is meant an amount of a peptide of the present invention that is sufficient to elicit a significant therapeutic effect, but low enough to minimize adverse effects (e.g., toxicity, irritation, or allergic response) to the subject animal, preferably a mammal, more preferably a human, in need of the peptide, and to use the present invention to achieve a reasonable "effect/risk ratio". It is understood that the term "safe and effective amount" used herein may vary depending on the subject, such as the physical condition, the duration of treatment, the nature of concurrent therapy, the dosage used, the carrier used, the solubility of the peptide therein, and the administration of the pharmaceutical agent. One skilled in the art will determine a "safe and effective amount" in accordance with the following methods consistent with the present invention. Reference may be made to the following documents:

spilker B, eds., (Guide to Clinical Studies and development Protocols), New York: raven Press Books, Ltd., 1984, pp.7-13, 54-60;

spilker, Guide to Clinical laboratories, New York: ravenPress Books, Ltd., 1991, pp.93-101;

craig c, and r.stitzel, eds, Modern Pharmacology (Modern Pharmacology) (second edition), Little, Brown and co., Boston: 1986, pp.127-33;

speight, Effury drug therapy: principles, pharmaceutical Clinical practices and therapies (Avery's drug Treatment: Principles and Practice of Clinical Pharmacology and therapeutics (third edition), Williams and Wilkins, Baltimore, 1987, pp.50-56;

r.tallarida, r.raffa and p.mcgonigle, General principles of Pharmacology (Principlesin General Pharmacology), Springer-Verlag, New York: 1988, pp.18-20.

The peptides of the invention are dissolved or suspended in a pharmaceutically acceptable buffer. The buffer solution in which the peptide is dissolved affects the pH, solubility and thus bioavailability of the peptide. The choice of buffer solution depends on the peptide composition, the method of administration, the desired solubility of the peptide, the half-life of the peptide on a physiological basis, and the pH and buffering capacity of physiological fluids. The pH of the ideal buffer solution may be closer to the pK of the peptide_aThe value, or it depends on the physiological environment conducted in vivo with the peptide. Suitable buffer solutions include, but are not limited to, phosphates, acetates, carbonates, bicarbonates, aminoacetates, citrates, imidazolium salts, and other salts. Particularly preferred is acetate buffer solution.

In addition to the host peptide, the compositions of the invention also comprise a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable" carrier as used herein refers to one or more compatible solid or liquid filler diluents or encapsulating substances suitable for use in animals, preferably mammals, more preferably humans. The term "compatible" as used herein refers to an interaction in which the various components of the composition can be mixed with the peptide without substantially reducing the pharmaceutical efficacy of the compound under typical use conditions. The pharmaceutically acceptable carrier must, of course, be of sufficiently high purity and sufficiently low toxicity and suitable for administration to an animal, preferably a mammal, more preferably a human. The choice of a pharmaceutically acceptable carrier for use in combination with the peptide is essentially determined by the mode of administration of the peptide. If the subject peptide is intended for injection, the preferred pharmaceutically acceptable carrier should be sterile and have a colloidal suspension compatible with blood.

More specifically, pharmaceutically acceptable carriers for systemic administration include sugars, starches, cellulose and its derivatives, malt, gelatin, talc, calcium sulfate, vegetable oils, synthetic oils, polyols, alginic acid, phosphate buffered solutions, emulsifiers, physiological saline and pyrogen-free water. Preferred carriers for parenteral use include propylene glycol, ethyl oleate, pyrrolinones, ethanol, and sesame oil. In compositions for parenteral administration, the pharmaceutically preferred acceptable carrier comprises at least 90% by weight of the total composition.

The compositions of the present invention are preferably provided in unit dosage form. As used herein, "unit dosage form" refers to a composition of the invention comprising a single dose of INGAP peptide suitable for use in an animal, preferably a mammal, more preferably a human subject, according to relatively good clinical practice. These compositions preferably contain from about 0.1 mg to about 300 mg, more preferably from about 5 mg to about 150 mg of INGAP peptide. The frequency of treatment with the compositions of the invention can be modified to obtain the desired high dose and to avoid side effects. Thus, non-limiting examples of treatment plans include once a day, twice a day, three times a day, once a week, once every two weeks, once a month, and combinations thereof. Alternatively, administration may be by continuous infusion.

The compositions of the invention may be in a variety of different forms, for example, oral, topical, nasal, or injectable. Depending on the particular mode of administration desired, a variety of carriers well known in the art and pharmaceutically acceptable may be employed. These carriers include fillers, diluents, hydrotropes, surfactants and encapsulating substances in either solid or liquid form. Other optional pharmaceutically active materials that do not substantially interfere with the activity of INGAP peptides may also be included. The carrier used in combination with INGAP peptide is sufficient to provide sufficient peptide per unit dose. Preparation techniques and compositions useful in the methods of the invention are described in the following references: in 1979, modern pharmacology (modern pharmaceuticals), ninth and tenth chapter, edited by Banker & Rhodes; in 1981, Lieberman et al, written "dosage forms of drugs: tablets (Pharmaceutical Dosage Forms: Tablets); in 1976, Ansel's "Introduction to Pharmaceutical Dosage Forms" (second edition).

Preparation of INGAP peptide

Among the various formulations of INGAP peptide, preferred is an injection solution prepared with sterile water and sodium chloride required to regulate muscle tone, prepared in four concentrations: 0, 7.5, 30 and 120 mg/0.5 ml/bottle. Hydrochloric acid and sodium hydroxide may be used to adjust the PH to the desired level, if desired. Other concentrations can be prepared by diluting higher concentration solutions with physiological saline. Dilution did not affect the biological activity of INGAP peptide.

INGAP peptide thus prepared is stable in the range of pH 4 to 6 when stored at 5 ℃ in the dark or light. However, if stored at 25 ℃, the composition may be partially degraded. The composition is more susceptible to degradation at a pH of 6 than at a pH of 4.5. From the above, it is known that INGAP peptide is more stable when stored at temperatures below 8 ℃ and at pH values below 6.

EXAMPLE 1 INGAP peptide solution for injection

A solution of 120 mg INGAP peptide may be formulated according to the following specifications.

TABLE 1

Parameter(s)	Specification of
		Appearance of the product	Colorless transparent solution
Composition of matter	Each bottle contains 90.0% to 110.0% INGAP peptide
		Content of impurities	Each impurity: 1.0% total content of impurities: 3.0 percent
pH value	4.0 to 5.0
		Bacterial endotoxins	NMT 2.92EU/mg
Sterile condition	Meets USP

Example 2 administration of INGAP peptide to Normal cheek mice

This experiment investigated the effect of INGAP peptide on islet production in normal buccal mice. Subjects were injected intraperitoneally with 5 mg/kg (25 mg/m) INGAP peptide solution once a day for four consecutive weeks, and the number of β -cells was assessed on the tenth and thirty days. Animals treated with INGAP peptide showed a significant increase in islet cell number compared to placebo-treated animals (see figure 2). The regenerative effect of islets can be demonstrated by the production of more insulin and an increase in the number of islets in the pancreas. Newly formed beta cells grow and appear on the pancreatic ductal wall. These insulin-containing cells are produced by ductal epidermal cell differentiation and islet cell growth, and their apparent effect of staining is directly proportional to the dose of INGAP peptide and the duration of action. Over time, these cells migrate from the duct and form islets in the soft body of the pancreas. After 10 consecutive days of INGAP peptide, the number of islets increased by 30%, and after 30 consecutive days, the number of islets in the tissue doubled, consistent with previous experimental results using ilotropin, rINGAP and cellophane embedding in animal model experiments.

Example 3 in vivo efficacy Studies

C57BL/6J mice that were diabetic treated for 5 consecutive days with an amount of 35 (mg/kg/day) STZ were divided into two groups of four, one group treated twice daily with an amount of 250 μ g INGAP peptide and the other group treated with physiological saline as a control group. All four mice treated with INGAP peptide returned to normal blood glucose levels, while four mice treated with saline remained hyperglycemic (see fig. 3). Further observations of the cessation of the drug administration after 39 days showed that the drug action was sustained by the time the experiment was stopped for 48 days. Histopathological evaluation of INHAP peptide treated animals showed: both the normal appearance of islets and the recruitment and distribution of new islet-forming regions, including insulin and glucagon secreting cells, have occurred (see fig. 4 and 6). Since glucagon plays a very important role in the prevention of hyperglycemia, the appearance of glucagon-secreting cells is very attractive. This feature of INGAP peptide induction of islet neogenesis is a great help to reverse the already impaired negative regulation of hypoglycemia, which is closely associated with the over-treatment of diabetes. None of the animals treated with INGAP peptide were found to be hyperglycemic, while no islet formation was observed in the saline-treated control animals. The use of INGAP peptide induces differentiation of catheter cells, as evidenced by the expression of the transcription factor PDX-1 by the cells (see fig. 5). Islets of animals that were diabetic by STZ treated with saline showed severe inflammatory cell invasion and were necrotic. Whereas in the animals treated with INGAP peptide, the inflammatory condition was significantly reduced and the islets were healthy (see fig. 6).

FIG. 4 shows immunocytochemical characteristics of pancreas of C57BL/6J mice treated sequentially with streptozotocin and INGAP peptide. The upper left graph shows that islets remain associated with epithelial cells stained with insulin antibodies on a portion of the duct, demonstrating the normal presence and distribution of insulin proteins. The lower left panel shows that islet surface regions still exhibit normal islet cell protein distribution after staining of the same islets with antibodies to glucagon and somatostatin. The upper right panel shows newly formed islets on the delivery catheter stained with H & E. The lower right panel shows insulin-containing cells on the wall of the infusion tube.

Example 4 31-day study (repeat dose) on mice

Repeated dose toxicity studies were also performed on rats with injection of INGAP peptide at 0, 2, 20 and 100 (mg/kg/day) for 31 days. In this study, four treatment groups each consisting of 10 male mice and 10 female mice and two recovery groups each consisting of 5 female mice and 5 male mice were divided. At the end of the experiment, blood samples were also collected and necropsy was performed for gross and microscopic observations. And approximately half of the animals in each group were evaluated for clinical pathology and serum levels. Selected organs (brain, adrenal gland, heart, kidney, liver, lung, pancreas and spleen) were weighed and the relative organ weights were calculated. A portion of the pancreas was isolated and frozen in liquid nitrogen to assess insulin content, and a portion of the pancreas tissue was submitted to independent microscopic examination by relevant researchers. The same experimental treatment was carried out 28 days after the cessation of the drug treatment in the healthy rats. Various parameters for further studies and potentially drug-related abnormal findings were evaluated to determine the reproducibility and potential clinical significance of the experiment.

The administration of INGAP peptide agents intramuscularly to rats over 31 consecutive days did not produce the side effects associated with the present agents in the assessment of discontinuation of the agent and 28 days post-treatment thereafter. With the increase in the number of injections of the highest dose, inflammatory responses were observed at the injection sites in male and female experimental mice, but no stimulation response was observed in the same dose in the healthy mice, indicating the reversibility of inflammation. In this 31-day experimental study, extramedullary hematopoiesis was found to occur in the spleen of one high dose treated male mouse. No signs of inflammatory cell infiltration, edema, necrosis or atrophy were observed under a microscope. The very prominent observation is the increase in the number of islets in the ductal associated tissues and the pancreatic bleb tissue. After two hours after 31 consecutive days of drug action, INGAP levels in serum of the experimental mice were below the limiting values. The increase in insulin-containing tissue area observed in these animals was a measure of pharmacological activity (see FIG. 7). The experimental result shows that: the non-adverse effect level (NOAEL) was greater than 100 mg/kg in CD-1 mice dosed for 31 days.

Example 5A 34-day study (repeat dose) on dogs

Beagle dogs were also subjected to repeated dose toxicity tests over a 34 day period with daily intramuscular injections of INGAP peptide at 0, 0.5, 1.5 and 10 (mg/kg/day), respectively. Pancreatic tissues in which beta cells were quantified by immunohistochemistry were obtained from the mice treated at the end of the treatment period of 34 days and from the mice recovered to be healthy 25 days after the treatment period. Pancreatic β -cells increase with increasing use of INGAP peptide (see fig. 8). These results show that: intramuscular injection of INGAP peptide at the dose studied in normal experimental dogs gave a biologically important response. Furthermore, more intensive studies on pancreatic tissue regions have not revealed symptoms such as edema, inflammatory cell invasion, necrosis or atrophy.

EXAMPLE 6 human clinical study

The dosage of the drug is generally based on drug efficacy and drug safety studies on animals. Patients with type I or type II diabetes are treated with two doses of INGAP peptide for example. These two doses were 7.5 mg (0.125 mg/kg, or 4.625 mg/sq m for a patient of 60 kg body weight) and 120 mg (1.6 mg/kg, or 74 mg/sq m for a patient of 60 kg body weight), respectively. To determine the effect of INGAP peptide treatment, the following parameters were evaluated:

1. maintaining the total insulin dose, fasting glucose levels decreased by greater than 35 mg/dl.

2. Maintaining fasting glucose levels within the limits established by the American Diabetes Association (ADA) standards reduces insulin dosage by 25%.

3. Fasting C-peptide amount is increased to 1 ng/ml, Sustacal is drunk^*Boost^*The amount of C-peptide thereafter increased to 2 ng/ml.

Each patient received a random intramuscular injection of INGAP peptide. After evaluating drug efficacy and safety data, the patient may further undergo INGAP peptide injections to the patient at the discretion of the physician.

A partial list of the assessments that are of patients receiving INGAP or placebo treatment is briefly summarized in the following table.

TABLE 2Evaluation table

Procedure	Screening	Radix	The treatment period is 1-34 days						Tracking days 35 to 63. + -.2 days
													Date of visit	1	7	14	21	28	34	42	49	56	63
Physical examination			X	X						X															X
	Symptoms of significance	X											X	X	X	X	X	X	X	X	X	X	X
Detection of clinical experiments			X	X		X	X	X	X	X															X
	PK values of INGAP peptides in plasma												X	X	X	X	X	X	X	X

C-peptide stimulation experiments

The C-peptide stimulation experiments were performed overnight, the next morning after at least 10 hours of fasting. The experiment can only be performed if the fasting glucose index is between 80 and 250 mg/dl. The patient may take the medication for treating diabetes the evening before the experiment, but not during the morning until the end of the experiment. The blood samples to be collected for determining C-peptide should be at Boost^*The extract was taken immediately before drinking and at 0.5 hour, 2 hour and 4 hour after drinking. Boost^*By oral ingestion. If a patient has fasting levels of C-peptide below 1.0 ng/ml, the patient is considered to be insulin deficient and the patient has a maximum of C-peptide stimulation below 2.0 ng/ml.

The results of the above treatments show: patients treated with INGAP peptide exhibit increased glucose tolerance, decreased fasting glucose levels, decreased insulin demand, increased fasting C-peptide levels, and consumption of Boost^*The latter C-peptide levels increased. However, the condition of patients treated with placebo showed no such improvement.

Unless otherwise indicated, all amounts, including amounts, percentages, fractions, and ratios, are to be understood as having the meaning of "about", which is not to be construed as a numerical limitation on the number itself.

The definite articles "a" and "the" mean "one or more" unless specifically noted otherwise.

While particular embodiments of the present invention have been illustrated and described, it would be obvious to those skilled in the art that various changes and modifications can be made without departing from the spirit and scope of the invention. It is therefore intended to cover in the appended claims all such changes and modifications that are within the scope of this invention.

Sequence listing

<110> A.I. Weinik (Vinik, Aaron I)

L, Rosenberg (Rosenberg, Lawrence)

G. Pittenge (Pittenger, Gary)

D. Taylor-Fishwick (Taylor-Fishwick, David)

M. Salemm (Salem, Michael)

S, Moranded (Mohrland, Scott)

<120> compositions and methods for treating diabetes

<130>9016#L$

<140>Not Yet Assigned

<141>2002-09-24

<150>US 60/329,330

<151>2001-10-16

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Claims (9)

1. A pharmaceutical composition comprising a polypeptide having at least fifteen consecutive amino acids, said polypeptide being associated with a native neogenin of a mammalian pancreatic islet, wherein said amino acid sequence is SEQ id no: 1. SEQ ID NO: 2 or SEQ ID NO: 3 and fragments thereof, and a pH of about 4 to about 6.

2. The pharmaceutical composition of claim 1, wherein the composition is in the form of a lyophilized powder or solution.

3. The pharmaceutical composition of any one of the preceding claims, wherein the composition has a pH of about 4 to about 5.

4. A pharmaceutical composition according to any preceding claim, wherein the polypeptide is in a form selected from the group consisting of pharmaceutically acceptable esters, salts and mixtures thereof.

5. The pharmaceutical composition of any one of the preceding claims, comprising from about 0.1 mg to about 300 mg of the polypeptide.

6. Use of a pharmaceutical composition according to any one of the preceding claims for the manufacture of a medicament for the treatment of diabetes in a human or other animal.

7. Use of a pharmaceutical composition according to any one of the preceding claims for the manufacture of a medicament for regenerating islets of Langerhans, pancreatic beta cells or establishing normal physiological blood glucose regulation in a mammal.

8. The use of a pharmaceutical composition according to any of the preceding claims, wherein the frequency of administration is once daily, twice daily, three times daily, once weekly, once biweekly, once monthly, continuous infusion or a combination thereof.

9. A kit for use according to any preceding claim, the kit comprising:

1) the pharmaceutical composition of claim 1; and

2) and (4) instructions for use.