Exendin-3 and Exendin-4 Polypeptides, and Pharmaceutical Compositions Comprising Same

1. Field of the Invention

This invention is in the field of the prevention and treatment of diabetes mellitus.

2. Description of the Prior Art

Diabetes mellitus (DM) is a major chronic illness found in humans with many consequences. Some complications arising from long-standing diabetes are blindness, kidney failure, and limb amputations. Insulin-dependent diabetes mellitus (IDDM) accounts for 10 to 15% of all cases of diabetes mellitus. The action of IDDM is to cause hyperglycemia (elevated blood glucose concentration) and a tendency towards diabetic ketoacidosis (DKA). Currently treatment requires chronic administration of insulin. Non-insulin dependent diabetes mellitus (NIDDM) is marked by hyperglycemia that is not linked with DKA. Sporadic or persistent incidence of hyperglycemia can be controlled by administering insulin. Uncontrolled hyperglycemia can damage the cells of the pancreas which produce insulin (the .beta.-islet cells) and in the long term create greater insulin deficiencies. Currently, oral sulfonylureas and insulin are the only two therapeutic agents available in the United States. for treatment of Diabetes mellitus. Both agents have the potential for producing hypoglycemia as a side effect, reducing the blood glucose concentration to dangerous levels. There is no generally applicable and consistently effective means of maintaining an essentially normal fluctuation in glucose levels in DM. The resultant treatment attempts to minimize the risks of hypoglycemia while keeping the glucose levels below a target value. The drug regimen is combined with control of dietary intake of carbohydrates to keep glucose levels in control.

A fragment of human peptide molecule called, glucagon-like peptide-1 (GLP-1) has been found to be a glucose-dependent insulinotropic agent (Gutniak, M., et al. N. Engl. J. Bled. 1992; 326:1316-1322). GLP-1 is itself a fragment of the human proglucagon molecule. Another active fragment, glucagon-like insulinotropic peptide (GLIP), corresponds to GLP-1(7-36). It was reasoned that since GLIP is the naturally active form found in humans after a meal, this peptide may aid in glucose regulation in IDDM and NIDDM.

In normal subjects, the infusion of GLIP significantly lowered the meal-related increases in blood glucose concentration, and the plasma concentrations of insulin and glucagon. In patients with NIDDM, the treatment reduced the requirement for insulin by 8 fold. In patients with IDDM, the GLIP treatment lowered the insulin required by one half. This glucose-dependent activity is a very desirable characteristic for a therapeutic agent that can be used to treat DM avoiding tile complications of hypoglycemic side effects.

In 1981, it was discovered that Gila monster (Heloderma suspectum) venom stimulated pancreatic enzyme secretion in vitro (Raufman, J. P., et al., Gastroenterology 80:1257 abst. (1981); Raufman, J. P., et al., Am. J. Physiol. 242: G470-G474 (1982)). Several peptides have been isolated from the venom that can stimulate increased cAMP and amylase release from dispersed pancreatic acinar cells. These structural analogs to the mammalian peptides VIP (vasoactive intestinal peptide) and secretin include helospectin-I, helospectin-II (Parker, D. S. et al., J. Biol. Chem. 259:11751-11755 (1984)),and helodermin (Hoshino, M. et al., FEBS Lett. 178:233-239 (1984)). Recently, we discovered another peptide that increases cAMP and stimulated the release of amylase in dispersed acinar cells. This peptide was found in Heloderma horridum venom and was termed exendin-3 (Eng, J. et al., J. BIol. Chem. 265: 20259-20262 (1990). Exendin-3 shares homology with VIP, secretin, helospectin-I and -II, and helodermin. The venom of Heloderma suspectum was examined and another peptide was purified from it. This peptide called exendin-4 is an analogue of exendin-3 with an identical sequence except for substitutions in residues 2 and 3 from the amino terminus (Eng, J. et al.,J. Biol. Chem. 267:742-7405 (1992)). Experiments were done to establish that the exendins could stimulate cAMP activity in dispersed pancreatic acinar cells, and a specific antagonist, exendin(9-39) amide, which can inhibit the effects of the exendins, was identified. (Raufman, J. P. et al., J. BIol. Chem. 266: 2897-2902 (1991 )) Experiments were performed to establish that GLP-1 could interact with possible exendin receptors in dispersed pancreatic acinar cells in vitro (Raufman, J. P. et al., J. BIol. Chem. 267:21432-21437 (1992)).

This invention encompasses pharmaceutical compositions containing exendin-3 or exendin-4, or any combination thereof, and methods for the treatment of diabetes mellitus and the prevention of hyperglycemia.

The compositions of the invention will normalize hyperglycemia through glucose-dependent, insulin-dependent and insulin-independent mechanisms. Therefore they will be useful as primary agents for the treatment of type II diabetes mellitus and as adjunctive agents for the treatment of type I diabetes mellitus. The invention specifically provides for exendin-4(1-39) as an insulinotropic agent.

The use of an effective amount of exendins as a treatment for diabetes mellitus has the advantage of being more potent than other insulinotropic peptides. The present invention is especially suited for the treatment of patients with diabetes, both type I and type II, in that the action of the peptide is dependent on the glucose concentration of the blood, and thus the risk of hypoglycemic side effects are greatly reduced over the risks in using current methods of treatment. Thus the use of insulinotropic peptides such as exendin-3 and exendin-4, has many advantages in the treatment of diabetes mellitus over current methods.

The present invention also provides for inhibitory agents derived from the exendins. In particular, exendin-4(9-39) as an inhibitor of exendin-4 and GLP-1 insulinotropic activity.

The present invention also provides for a method for treating diabetes mellitus in an individual, wherein said method comprises providing an amount of an insulinotropic composition sufficient to treat said diabetes; said composition containing an insulinotropic molecule; wherein said molecule is selected from the group consisting of:

(a) a peptide having the amino acid sequence substantially identicle to the sequence of exendin-3 or exendin-4 or fragments thereof; and

(b) a derivative of said peptide (a), wherein said derivative is selected from the group consisting of:

(1) a pharmaceutically acceptable acid addition salt of said peptide;

(2) a pharmaceutically acceptable carboxylate salt of said peptide;

(3) a pharmaceutically acceptable lower alkyl ester of said peptide; and,

(4) a pharmaceutically acceptable amide of said peptide wherein said pharmaceutically acceptable amide is selected from the group consisting of amide, lower alkyl amide and lower dialkyl amide; wherein said molecule has an insulinotropic activity which exceeds the insulinotropic activity of exendin-3 or exendin-4 or fragments thereof.

Thus the invention provides for the peptides or peptide fragments, made synthetically or purified from natural sources, which embody the biological activity of the exendins, or fragments thereof, as described by the present specification.

FIG. 1 is a graph showing exendin stimulated insulin secretion in a dog. Endogenous insulin secretion stimulated by exendin-3 (200 nmol) in a conscious dog. Exendin-3 was given as a bolus injection into a leg vain at time 0. Plasma was measured by radioimmunoassay.

FIG. 2a & 2b & 2c are graphs showing the serial injection of GLP-1 and Exendin-4. FIG. 2a, Dog #1. FIG. 2b, Dog #2. FIG. 2c, Dog #3. Serial injections of GLP-1(7-36) amide alternating with exendin-4 into the left atrium via a chronically indwelling catheter. GLP-1(7-36) amide was given at time 0 (0.1 nmol) and at 40 min (1 nmol). Exendin-4 was given at 20 min (0.1 nmol) and at 60 min (1 nmol). In 2c, the rise and fall in the baseline insulin between time 0 and 60 min is unexplained.

FIG. 3 is a graph illustrating the effect of exendin with and without antagonist. Insulin response in a normal dog to exendin-4 with or without exendin(9-39) amide. Glucose was infused at 100 mg/min. Exendin-4 (1 nmol) was given as an intravenous bolus at 60, 120 and 180 min. Exendin(9-39) amide, 42 nmol, was given together with exendin-4 at 120 min. The first phase of insulin release is greatly reduced and the second phase is abolished by this ratio of antagonist to agonist.

FIG. 4 is a graph illustrating the effect of exendin on cultured beta cells. .beta.TC-3 cell insulin response to exendin-4, insulin mg/well vs. exendin-4 logM.

FIG. 5 is a graph demonstrating the effect of exendin antagonist on glucose -stimulated increase in insulin. Conscious dog infused with glucose at 200 mg/min beginning at time 0. A bolus injection of exendin(9-39) amide was made at 60 minutes.

FIG. 6 is a graph illustrating the effect of Y31 exendin-4(1-31) amide. Conscious, fasted dog injected with a bolus of Y31 exendin-4(1-31) amide at time 0.

FIG. 7 is a graph illustrating the effect of GLP-1 with and without antagonist. Insulin responses in a fasted dog to GLP-1 (1 nmol) injected either alone at time 0 or together with exendin(9-39) amide (180 nmol) at time 60 min. GLP-1's insulinotropic activity is inhibited by exendin (9-39) amide.

The present invention provides for novel polypeptides which are unexpectedly useful as insulinotropic agents. Insulinotropic agents being agents which can stimulate, or cause the stimulation of, the synthesis or expression of the hormone insulin. The polypeptides of the present invention are termed exendin-3 and exendin-4. These peptides were originally isolated from the venom of Heloderma horridum and Heloderma suspectum respectively. In one embodiment of the invention, polypeptides corresponding to the amino acid sequence of exendin-3 and exendin-4 are synthesized by the solid phase method as previously described (Merrifield, J. M., Chem. Soc. 85: 2149 (1962); Stewart and Young, Solid Phase Peptide Synthesis, Freeman, San Francisco, 1969, pp. 27-66). In addition, it is also possible to isolate naturally occuring polypeptides from venom samples in a fashion similar to the original isolation of exendins 3 and 4. It is further possible to obtain the desired polypeptides by using recombinant DNA techniques (Maniatis, T. et al., Molecular Biology: A Laboratory Manual, Cold Spring Harbor, N.Y., 1982). The invention encompasses polypeptides which are insulinotropic and can be derived from naturally-occuring amino acid sequences. These proteins consist of the following amino acid sequences:

Exendin-3 [SEQ ID No:1] HSDGTFTSDL SKQMEEEAVR LFIEWLKNGG PSSGAPPPS

Exendin-4 [SEQ ID No:2] HGEGTFTSDL SKQMEEEAVR LFIEWLKNGG PSSGAPPPS

The invention also encompasses the insulinotropic fragments of exendin-4 comprising the amino acid sequentes:

Exendin-4(1-31) [SEQ ID No:3] HGEGTFTSDL SKQMEEAVR LFIEWLKNGG P

y.sup.31 Exendin-4(1-31 ) [SEQ ID No:4] HGEGTFTSDL SKQMEEEAVR LFIEWLKNGG Y

The invention also encompasses the inhibitory fragment of exendin-4 comprising the amino acid sequence:

Exendin-4(9-39 ) [SEQ ID No:5] DL SKQMEEEAVR LFIEWLKNGG PSSGAPPPS

The invention further encompasses a method for the enhancement of insulin production or expression which comprises the steps of providing to a mammalian beta type pancreatic islet cell an effective amount of the insulinotropic peptides disclosed above.

Also provided for by the present invention are those amino acid sequences in the above peptides which are capable of functioning as insulinotropic hormones. In addition, the invention also provides for the addition of amino acids to enhance attachment to carrier molecules, or added to enhance the insulinotropic effect.

A material is said to be "substantially free of natural contaminants" if it has been substantially purified from materials with which it is normally and naturally found. Examples of natural contaminants of exendin-3 or exendin-4 are: other peptides, carbohydrates, glycosylated peptides, lipids, membrane, other venom components etc. A material is also said to be substantially free of natural contaminants if these contaminants are substantially absent of from a sample of the material.

The compounds of the present invention can be formulated according to known methods to prepare pharmaceutically useful compositions. In these compositions, exendin-3 and or exendin-4, or their functional derivatives are combined in admixture with a pharmaceutically acceptable carrier vehicle. Suitable vehicles and their formulations, inclusive of other human proteins, e.g. human serum albumin, are well known. In order to form an effective pharmaceutical composition, the composition will contain an effective amount of the exendin-3 or exendin-4, or functional derivatives together with a suitable amount of carrier vehicle. Other compositions may combine exendin-3 and exendin-4, or their functional derivatives with other effective drugs that may treat other symptoms, or the same symptoms.

The use of exendin-3 and 4 in compositions that may be injected intravenously, intramuscularly, subcutaneously, or intraperitoneally, would call for dosages of about 0. 1 pg/kg to 1,000 mg/kg body weight depending on many individual factors such as age, severity of disease, total body weight, sex and other mitigating factors.

The insulinotropic properties of a compound may be determined by in vitro or in vivo assay. The compound in question may be administered to animals and monitoring the release of insulin. It is possible to monitor the increase in insulin production in cell culture as well.

The sequences of the invention also provide a means for identifying any specific mamalian analogs to the exendins. This can be accomplished by direct comparison of amino acid sequences, or by the translation of RNA or DNA sequences which may encode for the amino acid sequences of the invention, or by inhibition of activity by the specific exendin inhibitor, exendin (9-39) amide.

The sequences of the invention also provides a means for generating antibodies specific for the exendins, and further for the production of monoclonal antibodies for the exendins and fragments thereof. Thus the invention provides a means for purifying mammalian or other analogs to the exendins by the method of affinity chromatography.

Testing was done to establish if exendin-3 or exendin-4 could stimulate pancreatic insulin secretion in mammals. Since both exendin-3 and exendin-4 peptides have about 50% homology with glucagon and GLP-1(7-36) (glucagon-like peptide-1), and GLP-1(7-36) was found to bind to exendin receptors, it was thought possible that exendins could act in similar fashion as GLP-1 on other receptors.

Polypeptides corresponding to the amino acid sequence of exendin-3 and exendin-4 were synthesised by the solid phase method as previously described (Merrifield, J. M., Chem. Soc. 85:2149 (19625; Stewart and Young, Solid Phase Peptide Synthesis, Freeman, San Francisco, 1969, pp. 27-66). It is also possible to isolate naturally occuring polypeptides from venom samples in a fashion similar to the original isolation of exendins 3 and 4. It is further possible to obtain the desired polypeptides by using recombinant DNA techniques (Maniatis,T. et al., Molecular Biology: A Laboratory Manual, Cold Spring Harbor, N.Y., 19825).

The examples which follow are illustrative of specific embodiments of the invention, and various uses thereof. They are set forth for explanatory purposes only, and are not to be taken as limiting the invention.

The exendins are insulinotropins

Naural or synthetic exendin-3 and exendin-4 were tested in several biological systems, including conscious dog, anesthetized dog with chronic indwelling left atrial catheters, and beta TC-3 insulinoma cell line (described in D'Ambra et al., Endocrinology 126:2815-2822 (1990)) in cell culture. FIG. 1 shows an insulin secretory response to bolus injection of exendin-3 in a conscious dog with a seven-fold increase in insulin concentration above basal levels. Similar results are obtained using exendin-4. Since exendin-4 does not interact with VIP receptors and acts solely on exendin receptors, it has been used for subsequent studies.

Exendin-4 insulin secretagogue activity is glucose dependent

Dogs with glucose concentrations clamped at graded levels show a glucose-dependent insulinotropic response to exendin-4. Dosages of exendin-4 which do not stimulate insulin release at fasting glucose concentrations of 50-75 mg/dL (such as 0.1 nmol exendin-4 given as a bolus) are able to produce a peak insulin response of one-fold above basal when given to dogs in a clamped, hyperglycemic state.

Exendin-4 stimulates a greater insulin secretory response than GLP-1

Synthetic exendin-4 was compared with GLP-1 (purchased from Peninsula Labs, Belmont, Calif.) by alternating injections of bolus doses into dogs with chronic indwelling left atrial catheters. Since GLP-1 and exendin-4 are glucose dependent in their insulinotropic response, paired equimolar doses of GLP-1 and exendin-4 were given with GLP-1 administered first to avoid the possibility that falling glucose levels in the animals cause a diminished insulinotropic response to GLP-1 relative to exendin-4. Dogs #1 and #2 in FIGS. 2a and 2b maintained constant fasting glucose concentrations throughout the experiments in a range between 60 and 80 mg/dL. FIGS. 2a, 2b and 2c show a comparison of insulinotropic responses to alternating bolus injections of GLP-1 and exendin-4 at 20 minute intervals and at increasing doses ranging from 0.1 nmol to 10 nmol administered through chronic indwelling left atrial catheters into anesthetized dogs.

In contrast to the euglycemia present in the first two dogs, the third dog in FIG. 3c was exceptionally hyperglycemic, probably as a result of an infected catheter. Several points are illustrated by this experiment. First, euglycemic dogs normally do not respond to 0.1 nmol of either GLP-1 or exendin-4 with an insulin secretory resonse as illustrated by the first two dogs, whereas the hyperglycemic dog had clear insulinotropic responses to this lower dose of peptide. Second, the rapid normalization of hyperglycemia to euglycemic levels following modest doses of the two peptides reflects the great potential for use of these peptides in treatment of diabetic states. Third, despite the rapid normalization of the hyperglycemia, hypoglycemia does not occur. This class of therapeutic agents might be termed "euglycemic" agents. The potential for hypoglycemia caused by overdosages of these agents is minimized. Hypoglycemia is avoided even when the agents are given in the euglycemic state. Fourth, despite the administration of exendin-4 following an equivilent dose of GLP-1 in the setting of decreasing glucose levels, the insulin response as defined by area under the curve, is consistently 2-3 fold greater for exendin-4 compared to GLP-1. The greater response to exendin-4 holds true for the two euglycemic animals as well. A summary of the insulin responses is shown in Table 2.

Table 2 shows the relative ratio of insulin secretion stimulated by serial injections of GLP-1 (7-36) amide and exendin-4 expressed as area under the curve (AUC). AUC=T-B where T=total insulin secreted (sum of concentrations at times 2,4,6, 10 mad 14 min. and B=baseline insulin=average of insulin concentrations at times 0 and 20 min. Multiplied by a factor of 5.

Exendin(9-39) amide inhibits endogenous, exendin-4, and GLP-1 insulinotropic activity.

Antagonistic peptides can arise by a number of mechanisms. The gene encoding the exendins may also encode for related peptides which have antagonistic activity. The production of antagonistic peptides may then be either initiated or suppressed by differential cleavage of the pre-propeptide. The antagonistic peptides may also arise through post-translational modification of the agonist peptide, specifically through differential cleavage to produce extended or truncated forms of the agonist peptide. In our studies of the structure-function relationship of exendin peptide sequences, the NH.sub.2 -terminally truncated exendin analog, exendin(9-39) amide, was shown to have potent antagonistic activity against exendin-3 and exendin-4 in a pancreatic acinar cell system measuring cAMP activity. (Raufman et al., J. Biol. Chem. 266:2897-2902 (1991); Eng et al., J. Biol. Chem. 267:7402-7505 (1992)). FIG. 5 shows the effect of exendin(9-39) amide when administered alone on circulating insulin levels while glucose levels are clamped at approximately 100% above fasting levels. Following injection of the antagonist there is a rapid decrease in circulating insulin levels to 60% of the maximum concentration. This result indicates that the antagonist inhibited an endogenous insulinotropin that accounted for a substantial portion of the insulin secretory response to hyperglycemia.

When exendin(9-39) amide is given together with exendin-4 at a molar ratio of 40:1, there is substantial inhibition of the insulin secretory response, as shown in FIG. 3. The second phase of insulin release is completely inhibited while the first phase is more resistant to complete inhibition. This finding suggests a differential sensitivity to inhibition between the first and second phases of insulin release. A pathological condition which may correlate to this phenomenon is a loss of first phase insulin secretion in type 2 diabetes.

When exendin(9-39) amide is given together with GLP-1 at molar ratio of 180:1 there is substantial inhibition of the insulin secretory response, as shown in FIG. 7.

Exendin-4 acts directly on the beta cell

Beta TC-3 cells were obtained through Norman Fleischer (Diabetes Research and Training Center, Albert Einstein College of Medicine, N.Y.) and cultured in serum-containing media in 48-well culture dishes to confluency. Fresh media was added 24 hours before the cells were tested. The cells were tested in Earle's balanced salt solution containing IBMX, BSA and 16.7 mM glucose with graded concentrations of exendin-4 for 1 hour at 37.degree. C. before collection of media supernate and assay for insulin concentrations. FIG. 4 shows a dose response curve to exendin-4 indicating that exendin-4 acts directly on beta cells to stimulate insulin secretion.

Exendin-4 reduces the hyperglycemic state in a diabetic animal model

The db/db mouse is a genetically obese and diabetic strain of mouse. The db/db mouse develops hyperglycemia and hyperinsulinemia concomitant with its development of obesity and thus serves as a model of obese type 2 diabetes (NIDDM). Five 11-week old db/db mice purchased from The Jackson Laboratories (Bar Harbor, Me.) had sub-orbital sinus blood samples taken before and 60 minutes after intraperitoneal injection of exendin-4 at 10 nmol each animal (1 microgram/gram body weight). Blood glucose measurements were made with a glucose meter (YSI 1500 glucose analyzer, Yellow Springs, Ohio). The blood glucose levels in the animals were (average.+-.standard error, in mg/dL glucose) 310.+-.37 before and 181.+-.37 one hour after administration of exendin-4. Thus, exendin-4 was able to reduce the diabetic levels of blood glucose by 40% in these animals.

We have compared the effect of COOH-terminal truncations on the insulinotropic activity of exendin-4. The Y.sup.31 mutation of exendin-4( 1-31) amide has a TYR for PRO substitution at position 31 from the amino terminus. This mutant was shown to have insulinotropic activity when infused into dog. FIG. 6 shows this result. This result indicates that the amino acids in the exendin-4 sequence located between residues 1 and 31 are important for the insulinotropic activity.

This invention thus provides for compounds that are an unexpectedly efficient means of stimulating insulin production in vitro and in vivo that will be useful for the treatment of diabetes mellitus, as well as specific inhibitors therof.