# Patent

## Title
Treatment of gestational diabetes

## Abstract

Methods for treating gestational diabetes which comprise administration of an effective amount of an exendin or an exendin agonist, alone or in conjunction with other compounds or compositions that lower blood glucose levels.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a continuation of U.S. patent application Ser. No. 10/342,014 filed Jan. 13, 2003, which is a continuation of U.S. patent application Ser. No. 09/323,867 filed Jun. 1, 1999, now U.S. Pat. No. 6,506,724, each of which is incorporated by reference in their entirety.


INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED ON COMPACT DISCS
The sequence listing in the present application is being submitted on two compact discs labeled “Sequence Listing Copy 1” and “Sequence Listing Copy 2”; each containing a file of 106 KB in size named “0204-CON1-0_seq_lst.txt” created on Nov. 14, 2005, the contents of which are hereby incorporated by reference.
BACKGROUND
The following description summarizes information relevant to the present invention. It is not an admission that any of the information provided herein is prior art to the presently claimed invention, not that any of the publications specifically or implicitly referenced are prior art to that invention
Gestational Diabetes Mellitus
Gestational diabetes mellitus (“GDM”) is a disorder associated with elevated circulating plasma glucose. Although the diagnostic criteria for GDM have been the subject of controversy of decades, it was defined by the Third Workshop Conference on Gestational Diabetes Mellitus as carbohydrate intolerance of varying severity with onset or first recognition during pregnancy, irrespective of the glycemic status after delivery. Metzger (ed.) Proceedings of the Third International Workshop Conference on Gestational Diabetes Mellitus, Diabetes 40 (Suppl. 2), 1991. Despite advances in clinical management of GDM, there are problems associated with GDM which persist, including elevated rate of perinatal morbidity and elevated rate of malformations in newborns. Persson et al., Diabetes and Pregnancy, In International Textbook of Diabetes Mellitus, Second Edition, John Wiley & Sons 1997 (Alberti et al. Eds.). For example, it has been reported that, when the mean blood glucose level is greater than 105 mg/dl, there is a greater risk for the development of large-for-gestational age (“LGA”) infants when compared with a control population. Id. Additional reported consequences of untreated GDM include an increased incidence of macrosomia, respiratory distress syndrome, and other abnormalities of fetal metabolism. Langer, Am. J. Obstet Gynecol. 176:S186, 1997; American Diabetes Association: Self-Monitoring of Blood Glucose Consensus Statement, Diabetes Care 17:81-82, 1994 (“ABA Consensus Statement”); Coetzee & Jackson, S. Afr. Med. J. 56:467-475, 1979. It has been clearly established by those in the field that tight glycemic control can serve as the primary prevention of fetal disease relating to GDM. Drexel et al., Diabetes Care 11:761-768, 1988; Roversi et al., Diabetes Care 3:489-494, 1980; Langer & Mazze, Am. J. Obstet Gynecol. 159:1478-1483, 1988; Langer et al., Am. J. Obstet Gynecol. 161:646-653, 1989). GDM results in a greater incidence of intrauterine death or neonatal mortality. Position Statement American Diabetes Association: Gestational Diabetes Mellitus, Diabetes Care 21 (Suppl. 1):S60-61, 1998. GDM pregnancies are at an increased risk for fetal macrosomia and neonatal morbidities including neural tube defects, hypoglycemia, hypocalcemiea, hypomagnsemia, polycythemia and hyperbilirubinemia and subsequent childhood and adolescent obesity. Siccardi, Gestational Diabetes. Other complications to the woman include increased rates of cesarean delivery, hypertensive disorders including preeclamsia and urinary tract infections.
It has been reported that approximately 4% of all pregnancies (135,000 cases annually) are complicated by GDM, however, it has been estimated that the incidence may range from 1% to 14% of all pregnancies, depending on the population and diagnostic tests employed. ADA Consensus Statement, supra.
Normally during pregnancy, fasting plasma levels of insulin gradually increase to reach concentrations that are approximately twice as high in the third trimester as they were outside of pregnancy. Women with gestational diabetes mellitus (“GDM”) have fasting insulin levels comparable to or higher than those of normal pregnant women with the highest levels seen in women with GDM who are obese. Insulin secretion also increases gradually in pregnancy and also reaches a maximum during the third trimester. However, the relative increase in secretion is significantly smaller in women with GDM than in normal glucose tolerant (“NGT”) women. The first-phase insulin response in NGT women is significantly higher than in GDM women; second phase insulin response was similarly increased during pregnancy in both groups. This finding is consistent with the finding that GDM women have a later time of peak insulin concentration during an oral glucose tolerance test than do NGT women. Consistent with this observation, the insulin response per unit of glycemic stimulus is significantly higher in NGT women than in GDM women (90% and 40%, respectively). The fact that glucose tolerance deteriorates in both normal and GDM pregnancies while at the same time, insulin secretion increases indicates a decrease in insulin sensitivity. Comparative results from an intravenous glucose tolerance test and a hyperinsulinemic, euglycemic clamp showed a sensitivity decrease during pregnancy in both groups of 50-60%, but GDM women had a slightly lower sensitivity. In another study using radioactive glucose, turnover of glucose and amino acids in GDM women was comparable to NGT women only when insulin concentrations 3-5 fold higher in the GDM group were used. Thus, it appears that GDM is due to a combination of diminished insulin sensitivity and an impaired ability to increase insulin secretion and has, in fact, many features in common with type 2 diabetes. Normal or near normal glycemic control returns upon parturition.
Clinical Diagnosis:
it is common clinical practice to screen women for elevated glucose and glucose intolerance between weeks 24 and 28 of gestation, especially women with any one the following four characteristics: age ≧25; race/ethnicity of Hispanic, Native American, Asian, African-American or Pacific Islander origin; obese or a family history of diabetes. In addition, women with previous pregnancies with complications due to a large weight fetus/neonate are usually tested. In some medical centers all pregnant women are tested. Indeed, certain investigators have found that historical risk factors account for only roughly half of the women known to have GDM. Carr, Diabetes Care 21 (Suppl. 2):B14-B18, 1998. Additionally, there is some reported evidence that advancing maternal age is associated with increased incidence of GDM. Id.
The clinical diagnosis is generally based on a multi-step process. The evaluation is most typically performed by measuring plasma glucose 1 hour after a 50-gram oral glucose challenge test in either the fasted or the unfasted state. If the value in the glucose challenge test is ≧140 mg/dl, a 3-hr 100 g oral glucose tolerance test is done. If two or more of the following criteria are met, the patient is considered in need of glycemic control: fasted venous plasma≧105 mg/dl; venous plasma≧190 ma/dl at 1 hr, venous plasma≧165 mg/dl at 2 hr or venous plasma≧145 mg/dl at 3 hr. Williams et al., Diabetes Care 22: 418-421, 1999. Variations of this test are also used by some. See, e.g., Coustan, Gestational Diabetes In Diabetes in America, 2d ed. National Institutes of Health Publication No. 95-1468, 1995.
Current Clinical Therapy:
The current therapeutic approach for GDM is to control plasma glucose for the remainder of the gestation (i.e., the third trimester through parturition). GDM has many features in common with type 2 diabetes. The endocrine (impaired insulin secretion) and metabolic (insulin resistance) abnormalities that characterize both forms of diabetes are similar. In general, pregnancy is characterized by increases in both insulin resistance and insulin secretion. Women with GDM fail to respond with increased insulin to the decrease in insulin sensitivity.
A significant correlation has been shown to exist between late-stage gestational maternal glucose levels and preeclamsia, macrosomia, Cesarean section delivery and phototherapy for hyperbilirubinemia. Sermer et al., Diabetic Care 21 (Suppl. 2):B33-B42, 1998. It has also been determined that the length of hospitalization of the new mother and the length of time the neonate spent in the nursery could be correlated to the degree of elevation of plasma glucose in the pregnant woman. Id. Tallarigo, et al. reported a striking rise in the risk of fetal macrosomia (9.9 vs. 27.5%) and preeclamsia/Cesarean sections (19.9 vs. 40.0%) in women with abnormal glucose tolerance when compared to NGT women. Tallarigo et al., N. Engl. J. Med. 315:989-992, 1986.
Thus, the goals for therapy of GDM are to achieve and maintain as near normal glycemia as feasible with a special emphasis to keep postprandial glucose concentrations within the normal range. Optimal therapeutic strategies are safe and efficacious in achieving a metabolic balancing without creating complications, which may include ketosis and/or hypoglycemia. Jovanovic, Diabetes Care 21(Suppl. 2):B131-B137, 1998. The initial therapeutic approach is through diet. Jovanovic-Peterson & Peterson, J. Am. Coll. Nutr. 9:320-325, 1990.
If diet or diet and exercise are not effective (i.e., failure is fasting glucose≧105 mg/dl and/or a 2-hr postprandial plasma glucose of ≧120 mg/dl on 2 or more occasions within a 1-to 2-week period), then insulin therapy (preferably, human insulin) is considered appropriate. ADA Position Statement, supra.
Oral glucose-lowering agents are not recommended during pregnancy. Kuhl et al., Diabetic Care 21 (Suppl. 2): B19-B26, 1998. Although sulfonylureas are used in the treatment of type 2 diabetes due to their activity in increasing insulin sensitivity, these agents are contraindicated for use in GDM. Jovanovic, Diabetes Care 21 (Suppl. 2):B131-B137, 1998. See also Kahn & Shechter, Insulin, Oral Hypoglycemic Agents, and the Pharmacology of the Endocrine Pancreas, In Goodman & Gilman's The Pharmacological Basis of Therapeutics (8th ed. 1993 Goodman Gilman et al. eds.). Oral hypoglycemic drugs traverse the placenta, and may cause prolonged severe hypoglycemia in the newborn. Persson et al., supra.
The difficulties with, and the highly variable approaches to insulin therapy in GDM have been reviewed, for example, by Langer, et al. Langer, Diabetes Care 21 (Suppl.2):B91-B98, 1998. The problems commonly associated with insulin therapy in a non-pregnant population remain when used in the treatment of GDM. They are determination of the proper dose, maintenance of good glucose control through each 24-hr period, possible hypoglycemia and weight gain. Hypoglycemia can result when insulin is administered to control postprandial plasma glucose, but the fetus demands for energy in the presence of excess insulin later causes the glucose level to drop to a hypoglycemic level. This physiological state can be dangerous to both the mother and the fetus. Excess weight gain is undesirable in any pregnancy. Another problem with insulin therapy is the day-to-day and week-to-week variability in glucose control vs.insulin dose.
Thus, it can be appreciated that an effective means to treat gestational diabetes remains a major challenge and a superior method of treatment would be of great utility. Such a method, and compounds and compositions which are useful therefor, have been invented and are described and claimed herein.
Exendins and Exendin Agonists
Exendins are peptides that were first isolated from the salivary secretions of the Gila-monster, a lizard found in Arizona, and the Mexican Beaded Lizard. Exendin-3 is present in the salivary secretions of Heloderma horridum, and exendin-4 is present in the salivary secretions of Heloderma suspectum (Eng, J., et al., J. Biol. Chem., 265:20259-62, 1990; Eng, J., et al., J. Biol. Chem., 267:7402-05, 1992). The exendins have some sequence similarity to several members of the glucagon-like peptide family, the highest homology, 53%, being to GLP-1 [7-36]NH2 (Goke, et al., J. Biol. Chem., 268:19650-55, 1993). GLP-1 [7-36]NH2, also known as proglucagon [78-107] and most commonly as “GLP-1,” has an insulinotropic effect, stimulating insulin secretion from pancreatic β-cells; GLP-1 also inhibits glucagon secretion from pancreatic α-cells (Orskov, et al., Diabetes, 42:658-61, 1993: D'Alessio, et al., J. Clin. Invest., 97:133-38, 1996). GLP-1 is reported to inhibit gastric emptying (Williams, B., et al., J. Clin. Edocrinol Metab., 81 (1):327-32, 1996; Wettergren, A., et al., Dig. Dis. Sci., 38 (4):665-73, 1993), and gastric acid secretion. (Schjoldager, B T., et al., Dig. Dis. Sci., 34 (5):703-8, 1989; O'Halloran, D J., et al., J. Endrocrinol, 126 (1):169-73, 1990; Wettergren, A., et al., Dig. Dis. Sci., 38 (4):665-73, 1993). GLP-1 [7-37], which has an additional glycine residue at its carboxy terminus, also stimulates insulin secretion in humans (Orskov, et al., Diabetes, 42:658-61, 1993). A transmembrane G-protein adenylate-cyclase-coupled receptor believed to be responsible for the insulinotropic effect of GLP-1 is reported to have been cloned from a β-cell line (Thorens, Proc. Natl. Acad. Sci. USA 89:8641-45 (1992)).
Exendin-4 potently binds at GLP-1 receptors on insulin-secreting βTC1 cells, at dispersed acinar cells from guinea pig pancreas, and at parietal cells from stomach; the peptide is also said to stimulate somatostatin release and inhibit gastrin release in isolated stomachs (Goke, et al., J. Biol. Chem. 268:19650-55, 1993; Schepp, et al., Eur. J. Pharmacol., 69:183-91, 1994; Eissele, et al., Life Sci., 55:629-34, 1994). Exendin-3 and exendin-4 were reported to stimulate cAMP production in, and amylase release from, pancreatic acinar cells (Malhotra, R., et al., Regulatory Peptides, 41:149-56, 1992; Raufman, et al., J. Biol. Chem. 267:21432-37, 1992; Singh, et al., Regul. Pept. 53:47-59, 1994). The use of exendin-3 and exendin-4 as insulinotrophic agents for the treatment of diabetes mellitus and the prevention of hyperglycemia has been proposed (Eng, U.S. Pat. No. 5,424,286).
C-terminally truncated exendin peptides such as exendin-4[9-39], a carboxyamidated molecule, and fragments 3-39 through 9-39 have been reported to be potent and selective antagonists of GLP-1 (Goke, et al., J. Biol. Chem., 268:19650-55, 1993; Raufman, J. P., et al., J. Biol. Chem. 266:2897-902, 1991; Schepp, W., et al., Eur. J. Pharm. 269:183-91, 1994; Montrose-Rafizadeh, et al., Diabetes, 45(Suppl. 2):152A, 1996). Exendin-4[9-39] is said to block endogenous GLP-1 in vivo, resulting in reduced insulin secretion. Wang, et al., J. Clin. Invest., 95:417-21, 1995; D'Alessio, et al., J. Clin. Invest., 97:133-38, 1996). The receptor apparently responsible for the insulinotropic effect of GLP-1 has reportedly been cloned from rat pancreatic islet cell (Thorens, B., Proc. Natl. Acad. Sci. USA 89:8641-8645, 1992). Exendins and exendin-4[9-39] are said to bind to the cloned GLP-1 receptor (rat pancreatic β-cell GLP-1 receptor (Fehmann H C, et al., Peptides 15 (3): 453-6, 1994) and human GLP-1 receptor (Thorens B, et al., Diabetes 42 (11): 1678-82, 1993). In cells transfected with the cloned GLP-1 receptor, exendin-4 is reportedly an agonist, i.e., it increases cAMP, while exendin[9-39] is identified as an antagonist, i.e., it blocks the stimulatory actions of exendin-4 and GLP-1. Id.
Exendin-4[9-39] is also reported to act as an antagonist of the full length exendins, inhibiting stimulation of pancreatic acinar cells by exendin-3 and exendin-4 (Raufman, et al., J. Biol. Chem. 266:2897-902, 1991; Raufman, et al., J. Biol. Chem., 266:21432-37, 1992). It is also reported thaw exendin[9-39] inhibits the stimulation of plasma insulin levels by exendin-4, and inhibits the somatostatin release-stimulating and gastrin release-inhibiting activities of exendin-4 and GLP-1 (Kolligs, F., et al., Diabetes, 44:16-19, 1995; Eissele, et al., Life Sciences, 55:629-34, 1994).
Methods for regulating gastrointestinal motility using exendin agonists are described and claimed in U.S. application Ser. No. 08/908,867, filed Aug. 8, 1997, entitled, “Methods for Regulating Gastrointestinal Motility,” which application is a continuation-in-part of U.S. application Ser. No. 08/694,954, filed Aug. 8, 1996, which enjoys common ownership with the present invention and is hereby incorporated by reference.
Methods of reducing food intake using exendin agonists are described and claimed in U.S. application Ser. No. 09/003,869, filed Jan. 7, 1998, entitled, “Use of Exendin and Agonists Thereof for the Reduction of Food Intake,” claiming the benefit of Provisional Application Nos. 60/034,905, filed Jan. 7, 1997, 60/055,404, filed Aug. 7, 1997, 60/065,442 filed Nov. 14, 1997, and 60/066,029 filed Nov. 14, 1997. These applications also enjoy common ownership with the present invention and are hereby incorporated by reference.
Exendins have also been found to have inotropic and diuretic effects. International Application No. PCT/US99/02554, filed Feb. 5, 1999, 1998, claiming the benefit of Provisional Application No. 60/075,122, filed Feb. 13, 1998. These applications also enjoy common ownership with the present invention and are hereby incorporated by reference.
Additionally, exendins have been found to suppress glucagon secretion (U.S. Provisional Application No. 60/132,017, entitled, “Methods for Glucagon Suppression,” filed Apr. 30, 1999, which enjoys common ownership with the present invention and is hereby incorporated by reference).
Exendin [9-39] has been used to investigate the physiological relevance of central GLP-1 in control of food intake (Turton, M. D., et al., Nature, 379:69-72, 1996). GLP-1 administered by intracerebroventricular injection inhibits food intake in rats. This satiety-inducing effect of GLP-1 delivered ICV is reported to be inhibited by ICV injection of exendin [9-39] (Turton, supra). However, it has been reported that GLP-1 does not inhibit food intake in mice when administered by peripheral injection (Turton, M. D., Nature, 379:69-72, 1996; Bhavsar, S. P., Soc. Neurosci. Abstr., 21:460 (188.8), 1995).
SUMMARY OF THE INVENTION
The present invention concerns the surprising discovery that exendins and exendin agonists do not cross the placenta, and yet have a profound and prolonged effect on blood glucose, rendering them ideal agents for the treatment of gestational diabetes mellitus.
The present invention is directed to novel methods for treating gestational diabetes mellitus comprising the administration of an exendin, for example, exendin-3 [SEQ ID No. 1: His Ser Asp Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser Ser Gly Ala Pro Pro Pro Ser], or exendin-4 [SEQ ID No. 2: His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser Ser Gly Ala Pro Pro Pro Ser], or other compounds which effectively bind to the receptor at which exendin exerts its actions which are beneficial in the treatment of gestational diabetes mellitus.
In a first aspect, the invention features a method of treating gestational diabetes mellitus in a subject comprising administering to the subject a therapeutically effective amount of an exendin or an exendin agonist. By an “exendin agonist” is meant a compound that mimics the effects of exendin in the treatment of gestational diabetes mellitus by binding to the receptor or receptors where exendin causes one or more of these effects. Exendins and exendin agonists should be especially beneficial in the treatment of GDM because, due to their actions to inhibit gastric emptying, administration of such compounds should not result in increased weight gain. Additionally, in animal and human studies to date, administration of exendins and exendin agonists have not resulted in an increased incidence of hypoglycemia.
Exendin agonist compounds include exendin acids, for example exendin-3 acid and exendin-4 acid. Preferred exendin agonist compounds include those described in International Application No. PCT/US98/16387, entitled, “Novel Exendin Agonist Compounds,” filed Aug. 6, 1998, claiming the benefit of U.S. Provisional Patent Application Ser. No. 60/055,404, entitled, “Novel Exendin Agonist Compounds,” filed Aug. 8, 1997; International Application No. PCT/US98/24210, entitled, “Novel Exendin Agonist Compounds,” filed Nov. 13, 1998, claiming priority to U.S. States Provisional Patent Application Ser. No. 60/065,442, filed Nov. 14, 1997; and International Application No. PCT/US98/24273, entitled, “Novel Exendin Agonist Compounds,” filed Nov. 13, 1998, claiming priority to U.S. Provisional Patent Application Ser. No. 60/066,029, filed Nov. 14, 1997; all of which enjoy common ownership with the present application and all of which are incorporated by this reference into the present application as though fully set forth herein. Additional preferred exendin agonist compounds are those described and claimed in U.S. Provisional Application Ser. No. 60/132,018, entitled, “Modified Exendins and Exendin Agonists,” filed Apr. 30, 1999, which enjoys common ownership with the present application and which is incorporated by this reference into the present application as though fully set forth herein.
By “gestational diabetes mellitus” or “GDM is meant any degree of glucose intolerance with onset or first recognition during pregnancy.
Thus, in a first embodiment, the present invention provides a method for treating gestational diabetes in a subject comprising administering to said subject a therapeutically effective amount of an exendin or an exendin agonist. Preferred exendin agonist compounds include those described in International Application Nos., PCT/US98/16387, PCT/US98/24210, and PCT/US98/24273, which have been incorporated by reference in the present application. Preferably, the subject is a vertebrate, more preferably a mammal, and most preferably a human woman. In preferred aspects, the exendin or exendin agonist is administered parenterally, more preferably by injection. In a most preferred aspect, the injection is a peripheral injection. Preferably, about 1 μg-30 μg to about 1 mg of the exendin or exendin agonist is administered per day. More preferably, about 1-30 μg to about 500 μg, or about 1-30 μg to about 50 μg of the exendin or exendin agonist is administered per day. Most preferably, about 3 μg to about 50 μg of the exendin or exendin agonist is administered per day.
In one preferred aspect, the exendin or exendin agonist used in the methods of the present invention is exendin-3. In another preferred aspect, said exendin is exendin-4. Other preferred exendin agonists include exendin-4 (1-30) [SEQ ID NO 6: His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly], exendin-4 (1-30) amide [SEQ ID NO 7: His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly-NH2], exendin-4 (1-28) amide [SEQ ID NO 40: His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn-NH2], 14Leu, 25Phe exendin-4 amide [SEQ ID NO 9: His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Leu Glu Glu Glu Ala Val Arg Leu Phe Ile Glu Phe Leu Lys Asn Gly Gly Pro Ser Ser Gly Ala Pro Pro Pro Ser-NH2], 14Leu, 25Phe exendin-4 (1-28) amide {SEQ ID NO 41: His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Leu Glu Glu Glu Ala Val Arg Leu Phe Ile Glu Phe Leu Lys Asn-NH2], and 14Leu, 22Ala, 25Phe exendin-4 (1-28) amide [SEQ ID NO 8: His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Leu Glu Glu Glu Ala Val Arg Leu Ala Ile Glu Phe Leu Lys Asn-NH2].
In the methods of the present invention, the exendins and exendin agonists may be administered separately or together with one or more other compounds and compositions that exhibit a long-term or short-term blood glucose control action, including, but not limited to other compounds and composition that comprise an insulin or an amylin agonist. Suitable amylin agonists include, for example, [25,28,29Pro-]-human amylin (also known as “Pramlintide,” previously referred to as “AC-137,” and, referred to in its acetate salt form by its trademark SYMLIN® (Pramlintide acetate), as described in “Amylin Agonist Peptides and Uses Therefor,” U.S. Pat. No. 5,686,411, issued Nov. 11, 1997, and salmon calcitonin.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 depicts the generic exendin agonist peptide having the amino acid sequence of SEQ ID NO: 3, and exemplary exendin agonist peptides having the amino acid sequences of SEQ ID NOs: 9-39 that are useful in the present invention.
FIG. 2 depicts concentrations of exendin-4 (SEQ ID NO: 2) in plasma and amniotic fluid of rats after 21 μg subcutaneous injection.
FIG. 3 depicts concentrations of (SEQ ID NO: 2) in plasma and amniotic fluid of rats after 210 μg subcutaneous injection.



DETAILED DESCRIPTION OF THE INVENTION
Exendins and exendin agonists are useful as described herein in view of their pharmacological properties. Activity as exendin agonists can be indicated by activity in the assays described below. Effects of exendins or exendin agonists in treating gestational diabetes can be identified, evaluated, or screened for, using the methods described in the Examples below, or other methods known in the art for determining effects on blood glucose control.
Exendin Agonist Compounds
Exendin agonist compounds are those described in International Application No. PCT/US98/16387, filed Aug. 6, 1998, entitled, “Novel Exendin Agonist Compounds,” which claims the benefit of United States Provisional Application No. 60/055,404, filed Aug. 8, 1997, including compounds of the formula (I) [SEQ ID NO. 3]:

    Xaa1 Xaa2 Xaa3 Gly Thr Xaa4 Xaa5 Xaa6 Xaa7 Xaa8 Ser Lys Gln Xaa9 Glu Glu Glu Ala Val Arg Leu Xaa10 Xaa11 Xaa12 Xaa13 Leu Lys Asn Gly Gly Xaa14 Ser Ser Gly Ala Xaa15 Xaa16 Xaa17 Xaa18-Z

wherein Xaa1 is His, Arg or Tyr; Xaa2 is Ser, Gly, Ala or Thr; Xaa3 is Asp or Glu; Xaa4 is Phe, Tyr or naphthylalanine; Xaa5 is Thr or Ser; Xaa6 is Ser or Thr; Xaa7 is Asp or Glu; Xaa8 is Leu, Ile, Val, pentylglycine or Met; Xaa9 is Leu, Ile, pentylglycine, Val or Met; Xaa10 is Phe, Tyr or naphthylalanine; Xaa11 is Ile, Val, Leu, pentylglycine, tert-butylglycine or Met; Xaa12 is Glu or Asp; Xaa13 is Trp, Phe, Tyr, or naphthylalanine; Xaa14, Xaa15, Xaa16 and Xaa17 are independently Pro, homoproline, 3Hyp, 4Hyp, thioproline, N-alkylglycine, N-alkylpentylglycine or N-alkylalanine; Xaa18 is Ser, Thr or Tyr; and Z is —OH or —NH2; with the proviso that the compound is not exendin-3 or exendin-4.



Preferred N-alkyl groups for N-alkylglycine, N-alkylpentylglycine and N-alkylalanine include lower alkyl groups preferably of 1 to about 6 carbon atoms, more preferably of 1 to 4 carbon atoms. Suitable compounds include those listed in FIG. 10 having amino acid sequences of SEQ. ID. NOS. 9 to 39.
Preferred exendin agonist compounds include those wherein Xaa1 is His or Tyr. More preferably Xaa1 is His.
Preferred are those compounds wherein Xaa2 is Gly.
Preferred are those compounds wherein Xaa9 is Leu, pentylglycine or Met.
Preferred compounds include those wherein Xaa11 is Trp or Phe.
Also preferred are compounds where Xaa4 is Phe or naphthylalanine; Xaa11 is Ile or Val and Xaa14, Xaa15, Xaa16 and Xaa17 are independently selected from Pro, homoproline, thioproline or N-alkylalanine. Preferably N-alkylalanine has a N-alkyl group of 1 to about 6 carbon atoms.
According to an especially preferred aspect, Xaa15, Xaa16 and Xaa17 are the same amino acid reside.
Preferred are compounds wherein Xaa18 is Ser or Tyr, more preferably Ser.
Preferably Z is —NH2.
According to one aspect, preferred are compounds of formula (I) wherein Xaa1 is His or Tyr, more preferably His; Xaa2 is Gly; Xaa4 is Phe or naphthylalanine; Xaa9 is Leu, pentylglycine or Met; Xaa10 is Phe or naphthylalanine; Xaa11 is Ile or Val; Xaa14, Xaa15, Xaa16 and Xaa17 are independently selected from Pro, homoproline, thioproline or N-alkylalanine; and Xaa18 is Ser or Tyr, more preferably Ser. More preferably Z is —NH2.
According to an especially preferred aspect, especially preferred compounds include those of formula (I) wherein: Xaa1 is His or Arg; Xaa2 is Gly; Xaa3 is Asp or Glu; Xaa4 is Phe or napthylalanine; Xaa5 is Thr or Ser; Xaa6 is Ser or Thr; Xaa7 is Asp or Glu; Xaa8 is Leu or pentylglycine; Xaa9 is Leu or pentylglycine; Xaa10 is Phe or naphthylalanine; Xaa11 is Ile, Val or t-butyltylglycine; Xaa12 is Glu or Asp; Xaa13 is Trp or Phe; Xaa14, Xaa15, Xaa16, and Xaa17 are independently Pro, homoproline, thioproline, or N-methylalanine; Xaa18 is Ser or Tyr: and Z is —OH or —NH2; with the proviso that the compound does not have the formula of either SEQ. ID. NOS. 1 or 2. More preferably Z is —NH2. Especially preferred compounds include those having the amino acid sequence of SEQ. ID. NOS. 9, 10, 21, 22, 23, 26, 28, 34, 35 and 39.
According to an especially preferred aspect, provided are compounds where Xaa9 is Leu, Ile, Val or pentylglycine, more preferably Leu or pentylglycine, and Xaa13 is Phe, Tyr or naphthylalanine, more preferably Phe or naphthylalanine. These compounds will exhibit advantageous duration of action and be less subject to oxidative degradation, both in vitro and in vivo, as well as during synthesis of the compound.
Exendin agonist compounds also include those described in International Application No. PCT/US98/24210, filed Nov. 13, 1998, entitled, “Novel Exendin Agonist compounds,” which claims the benefit of U.S. Provisional Application No. 60/065,442, filed Nov. 14, 1997, including compounds of the formula (II) [SEQ ID NO. 4]:

    Xaa1 Xaa2 Xaa3 Gly Xaa5 Xaa6 Xaa7 Xaa8 Xaa9 Xaa10 Xaa11 Xaa12 Xaa13 Xaa14 Xaa15 Xaa16 Xaa17 Ala Xaa19 Xaa20 Xaa21 Xaa22 Xaa23 Xaa24 Xaa25 Xaa26 Xaa27 Xaa28-Z1; wherein
    Xaa1 is His, Arg or Tyr;
    Xaa2 is Ser, Gly, Ala or Thr;
    Xaa3 is Asp or Glu;
    Xaa5 is Ala or Thr;
    Xaa6 is Ala, Phe, Tyr or naphthylalanine;
    Xaa7 is Thr or Ser;
    Xaa8 is Ala, Ser or Thr;
    Xaa9 is Asp or Glu;
    Xaa10 is Ala, Leu, Ile, Val, pentylglycine or Met;
    Xaa11 is Ala or Ser;
    Xaa12 is Ala or Lys;
    Xaa13 is Ala or Gln;
    Xaa14 is Ala, Leu Ile; pentylglycine, Val or Met;
    Xaa15 is Ala or Glu;
    Xaa16 is Ala or Glu;
    Xaa17 is Ala or Glu;
    Xaa19 is Ala or Val;
    Xaa20 is Ala or Arg;
    Xaa21 is Ala or Leu;
    Xaa22 is Ala, Phe, Tyr or naphthylalanine;
    Xaa23 is Ile, Val, Leu, pentylglycine, tert-butylglycine or Met;
    Xaa24 is Ala, Glu or Asp;
    Xaa25 is Ala, Trp, Phe, Tyr or naphthylalanine;
    Xaa26 is Ala or Leu;
    Xaa27 is Ala or Lys;
    Xaa28 is Ala or Asn;
    Z1 is —OH,
     —NH2,
     Gly-Z2,
     Gly Gly-Z2,
     Gly Gly Xaa31-Z2,
     Gly Gly Xaa31Ser-Z2,
     Gly Gly Xaa31Ser Ser-Z2,
     Gly Gly Xaa31Ser Ser Gly-Z2,
     Gly Gly Xaa31Ser Ser Gly Ala-Z2
     Gly Gly Xaa31Ser Ser Gly Ala Xaa36-Z2,
     Gly Gly Xaa31Ser Ser Gly Ala Xaa36Xaa37-Z2, or
     Gly Gly Xaa31Ser Ser Gly Ala Xaa36Xaa37Xaa38-Z2(SEQ ID NO. 189);
     Xaa31, Xaa36, Xaa37 and Xaa38 are independently Pro, homoproline, 3Hyp, 4Hyp, thioproline, N-alkylglycine, N-alkylpentylglycine or N-alkylalanine; and
     Z2 is —OH or —NH2;

provided that no more than three of Xaa3, Xaa5, Xaa6, Xaa8, Xaa10, Xaa11, Xaa12, Xaa13, Xaa14, Xaa15, Xaa16, Xaa17, Xaa19, Xaa20, Xaa21, Xaa24, Xaa25, Xaa26, Xaa27 and Xaa28 are Ala. Preferred N-alkyl groups for N-aklylglycine, N-alkylpentylglycine and N-alkylalanine include lower alkyl groups preferably of 1 to about 6 carbon atoms, more preferably of 1 to 4 carbon atoms.



Preferred exendin agonist compounds include those wherein Xaa1 is His or Tyr. More preferably Xaa1 is His.
Preferred are those compounds wherein Xaa2 is Gly.
Preferred are those compounds wherein Xaa14 is Leu, pentylglycine or Met.
Preferred compounds are those wherein Xaa25 is Trp or Phe.
Preferred compounds are those where Xaa6 is Phe or naphthylalanine; Xaa22 is Phe or naphthylalanine and Xaa23 is Ile or Val.
Preferred are compounds wherein Xaa31, Xaa36, Xaa37 and Xaa38 are independently selected from Pro, homoproline, thioproline and N-alkylalanine.
Preferably Z1 is —NH2.
Preferable Z2 is —NH2.
According to one aspect, preferred are compounds of formula (II) wherein Xaa1 is His or Tyr, more preferably His; Xaa2 is Gly; Xaa6 is Phe or naphthylalanine; Xaa14 is Leu, pentylglycine or Met; Xaa22 is Phe or naphthylalanine; Xaa23 is Ile or Val; Xaa31 Xaa36, Xaa37 and Xaa38 are independently selected from Pro, homoproline, thioproline or N-alkylalanine. More preferably Z1 is —NH2.
According to an especially preferred aspect, especially preferred compounds include those of formula (II) wherein: Xaa1 is His or Arg; Xaa2 is Gly or Ala; Xaa3 is Asp or Glu; Xaa5 is Ala or Thr; Xaa6 is Ala, Phe or nephthylalaine; Xaa7 is Thr or Ser; Xaa8 is Ala, Ser or Thr; Xaa9 is Asp or Glu; Xaa10 is Ala, Leu or pentylglycine; Xaa11 is Ala or Ser; Xaa12 is Ala or Lys; Xaa13 is Ala or Gln; Xaa14 is Ala, Leu or pentylglycine; Xaa15 is Ala or Glu; Xaa16 is Ala or Glu; Xaa17 is Ala or Glu; Xaa19 is Ala or Val; Xaa20 is Ala or Arg; Xaa21 is Ala or Leu; Xaa22 is Phe or naphthylalanine; Xaa23 is Ile, Val or tert-butylglycine; Xaa24 is Ala, Glu or Asp; Xaa25 is Ala, Trp or Phe; Xaa26 is Ala or Leu; Xaa27 is Ala or Lys; Xaa28 is Ala or Asn; Z1 is —OH, —NH2, Gly-Z2, Gly Gly-Z2, Gly Gly Xaa31-Z2, Gly Gly Xaa313 Ser-Z2, Gly Gly Xaa31 Ser Ser-Z2, Gly Gly Xaa31 Ser Ser Gly-Z2, Gly Gly Xaa31 Ser Ser Gly Ala-Z2, Gly Gly Xaa31 Ser Ser Gly Ala Xaa36-Z2, Gly Gly Xaa31 Ser Ser Gly Ala Xaa36 Xaa37-Z2, Gly Gly Xaa31 Ser Ser Gly Ala Xaa36 Xaa37 Xaa38-Z2; Xaa31, Xaa36 Xaa37 and Xaa38 being independently Pro homoproline, thioproline or N-methylalanine; and Z2 being —OH or —NH2; provided that no more than three of Xaa3, Xaa5, Xaa6, Xaa8, Xaa10, Xaa11, Xaa12, Xaa13, Xaa14, Xaa15, Xaa16, Xaa17, Xaa19, Xaa20, Xaa21, Xaa24, Xaa25, Xaa26, Xaa27 and Xaa28 are Ala. Especially preferred compounds include those having the amino acid sequence of SEQ. ID. NOS. 40-61.
According to an especially preferred aspect, provided are compounds where Xaa14 is Leu, Ile, Val or pentylglycine, more preferably Leu or pentylglycine, and Xaa25 is Phe, Tyr or naphthylalanine, more preferably Phe or naphthylalanine. These compounds will be less susceptive to oxidative degration, both in vitro and in vivo, as well as during
Synthesis of the Compound.
Exendin agonist compounds also include those described in International Patent Application No. PCT/US98/24273, filed Nov. 13, 1998, entitled, “Novel Exendin Agonist Compounds,” which claims the benefit of U.S. Provisional Application No. 60/066,029, filed Nov. 14,1997, including compounds of the formula (III) [SEQ ID NO. 5]:

    Xaa1 Xaa2 Xaa3 Xaa4 Xaa5 Xaa6 Xaa7 Xaa8 Xaa9 Xaa10 Xaa11 Xaa12 Xaa13 Xaa14 Xaa15 Xaa16 Xaa17 Ala Xaa19 Xaa20 Xaa21 Xaa22 Xaa23 Xaa24 Xaa25 Xaa26 Xaa27 Xaa28-Z1; wherein
    Xaa1 is His, Arg, Tyr, Ala, Norval, Val or Norleu;
    Xaa2 is Ser, Gly, Ala or Thr;
    Xaa3 is Ala, Asp or Glu;
    Xaa4 is Ala, N or Val, Val, Norleu or Gly;
    Xaa5 is Ala or Thr;
    Xaa6 is Phe, Tyr or naphthylalanine;
    Xaa7 is Thr or Ser;
    Xaa8 is Ala, Ser or Thr;
    Xaa9 is Ala, Norval, Val, Norleu, Asp or Glu;
    Xaa10 is Ala, Leu, Ile, Val, pentylglycine or Met;
    Xaa11 is Ala or Ser;
    Xaa12 is Ala or Lys;
    Xaa13 is Ala or Gln;
    Xaa14 is Ala or Glu;
    Xaa15 is Ala or Glu;
    Xaa16 is Ala or Glu;
    Xaa17 is Ala or Glu;
    Xaa19 is Ala or Val;
    Xaa20 is Ala or Arg;
    Xaa21 is Ala or Leu;
    Xaa22 is Phe, Tyr or naphthylalanine;
    Xaa23 is Ile, Val, Leu, pentylglycine, tert-butylglycine or Met;
    Xaa24 is Ala, Glu or Asp;
    Xaa25 is Ala, Trp, Phe, Tyr or naphthylalanine;
    Xaa26 is Ala or Leu;
    Xaa27 is Ala or Lys;
    Xaa28 is Ala or Asn;
    Z1 is —OH,
     —NH2,
     Gly-Z2,
     Gly Gly-Z2,
     Gly Gly Xaa31-Z2,
     Gly Gly Xaa31 Ser-Z2,
     Gly Gly Xaa31 Ser Ser-Z2,
     Gly Gly Xaa31 Ser Ser Gly-Z2,
     Gly Gly Xaa31 Ser Ser Gly Ala-Z2,
     Gly Gly Xaa31 Ser Ser Gly Ala Xaa36-Z2,
     Gly Gly Xaa31 Ser Ser Gly Ala Xaa36 Xaa37-Z2,
     Gly Gly Xaa31 Ser Ser Gly Ala Xaa36 Xaa37 Xaa38-Z2 or Gly
     Gly Gly Xaa31 Ser Ser Gly Ala Xaa36 Xaa37 Xaa38 Xaa39-Z2 (SEQ ID NO. 190);
     wherein Xaa31, Xaa36, Xaa37 and Xaa38 are independently Pro, homoproline, 3Hyp, 4Hyp, thioproline, N-alkylglycine, N-alkylpentylglycine or N-alkylalanine; and
     Z2 is —OH or —NH2;

provided that no more than three of Xaa3, Xaa4, Xaa5, Xaa6, Xaa8, Xaa9, Xaa10, Xaa11, Xaa12, Xaa13, Xaa14, Xaa15, Xaa16, Xaa17, Xaa19, Xaa20, Xaa21, Xaa24, Xaa25, Xaa26, Xaa27 and Xaa28 are Ala; and provided also that, if Xaa1 is His, Arg or Tyr, then at least one of Xaa3, Xaa4 and Xaa9 is Ala.



DEFINITIONS
In accordance with the present invention and as used herein, the following terms are defined to have the following meanings, unless explicitly stated otherwise.
The term “amino acid” refers to natural amino acids, unnatural amino acids, and amino acid analogs, all in their D and L stereoisomers if their structure allow such stereoisomeric forms. Natural amino acids include alanine (Ala), arginine (Arg), asparagine (Asn), aspartic acid (Asp), cysteine (Cys), glutamine (Gln), glutamic acid (Glu), glycine (Gly), histidine (His), isoleucine (Ile), leucine (Leu), lysine (Lys), methionine (Met), phenylalanine (Phe), proline (Pro), serine (Ser), threonine (thr), tryptophan (Trp), tyrosine (Tyr) and valine (Val). Unnatural amino acids include, but are not limited to azetidinecarboxylic acid, 2-aminoadipic acid, 3-aminoadipic acid, beta-alanine, aminopropionic acid, 2-aminobutyric acid, 4-aminobutyric acid, 6-aminocaproic acid, 2-aminoheptanoic acid, 2-aminoisobutyric acid, 3-aminoisbutyric acid, 2-aminopimelic acid, tertiary-butylglycine, 2,4-diaminoisobutyric acid, desmosine, 2,2′-diaminopimelic acid, 2,3-diaminopropionic acid, N-ethylglycine, N-ethylasparagine, homoproline, hydroxylysine, allo-hydroxylysine, 3-hydroxyproline, 4-hydroxyproline, isodesmosine, allo-isoleucine, N-methylalanine, N-methylglycine, N-methylisoleucine, N-methylpentylglycine, N-methylvaline, naphthalanine, norvaline, norleucine, ornithine, pentylglycine, pipecolic acid and thioproline. Amino acid analogs include the natural and unnatural amino acids which are chemically blocked, reversibly or irreversibly, or modified on their N-terminal amino group or their side-chain groups, as for example, methionine sulfoxide, methionine sulfone, S-(carboxymethyl)-cysteine, S-(carboxymethyl)-cysteine sulfoxide and S-(carboxymethyl)-cysteine sulfone.
The term “amino acid analog” refers to an amino acid wherein either the C-terminal carboxy group, the N-terminal amino group or side-chain functional group has been chemically codified to another functional group. For example, aspartic acid-(beta-methyl ester) is an amino acid analog of aspartic acid; N-ethylglycine is an amino acid analog of glycine; or alanine carboxamide is an amino acid analog of alanine.
The term “amino acid residue” refers to radicals having the structure: (1) —C(O)—R—NH—, wherein R typically is —CH(R′)—, wherein R′ is an amino acid side chain, typically H or a carbon containing substitutent; or (2)




wherein p is 1, 2 or 3 representing the azetidinecarboxylic acid, proline or pipecolic acid residues, respectively.

The term “lower” referred to herein in connection with organic radicals such as alkyl groups defines such groups with up to and including about 6, preferably up to and including 4 and advantageously one or two carbon atoms. Such groups may be straight chain or branched chain.
“Pharmaceutically acceptable salt” includes salts of the compounds described herein derived from the combination of such compounds and an organic or inorganic acid. In practice the use of the salt form amounts to use of the base form. The compounds are useful in both free base and salt form.
In addition, the following abbreviations stand for the following:
“ACN” or “CH3CN” refers to acetonitrile.

    “Boc”, “tBoc” or “Tboc” refers to t-butoxy carbonyl.
    “DCC” refers to N,N′-dicyclohexylcarbodiimide.
    “Fmoc” refers to fluorenylmethoxycarbonyl.
    “HBTU” refers to 2-(1H-benzotriazol-1-yl)-1,1,3,3,3-tetramethyluronium hexaflurophosphate.
    “HOBt” refers to 1-hydroxybenzotriazole monohydrate.
    “homoP” or hPro” refers to homoproline.
    “MeAla” or “Nme” refers to N-methylalanine.
    “naph” refers to naphthylalanine.
    “pG” or pGly” refers to pentylglycine.
    “tBuG” refers to tertiary-butylglycine.
    “ThioP” or tPro” refers to thioproline.
    3Hyp” refers to 3-hydroxyproline
    4Hyp” refers to 4-hydroxyproline
    NAG” refers to N-alkylglycine
    NAPG” refers to N-alkylpentylglycine
    “Norval” refers to norvaline


“Norleu” refers to norleucine
Preparation of Compounds
The exendins and exendin agonists described herein may be prepared using standard solid-phase peptide synthesis techniques and preferably an automated or semiautomated peptide synthesizer. Typically, using such techniques, an α-N-carbamoyl protected amino acid and an amino acid attached to the growing peptide chain on a resin are coupled at room temperature in an inert solvent such as dimethylformamide, N-methylpyrrolidinone or methylene chloride in the presence of coupling agents such as dicyclohexylcarbodiimide and 1-hydroxybenzotriazole in the presence of a base such as diisopropylethylamine. The α-N-carbamoyl protecting group is removed from the resulting peptide-resin using a reagent such as trifluoroacetic acid or piperidine, and the coupling reaction repeated with the next desired N-protected amino acid to be added to the peptide chain. Suitable N-protecting groups are well known in the art, with t-butyloxycarbonyl (tBoc) and fluorenylmethoxycarbonyl (Fmoc) being preferred herein.
The solvents, amino acid derivatives and 4-methylbenzhydryl-amine resin used in the peptide synthesizer may be purchased from Applied Biosystems Inc. (Foster City, Calif.). The following side-chain protected amino acids may be purchased from Applied Biosystems, Inc.: Boc-Arg(Mts), Fmoc-Arg(Pmc), Boc-Thr(Bzl), Fmoc-Thr(t-Bu), Boc-Ser(Bzl), Fmoc-Ser(t-Bu), Boc-Tyr(BrZ), Fmoc-Tyr(t-Bu), Boc-Lys(Cl-Z), Fmoc-Lys(Boc), Boc-Glu(Bzl), Fmoc-Glu(t-Bu), Fmoc-His(Trt), Fmoc-Asn(Trt), and Fmoc-Gln(Trt). Boc-His(BOM) may be purchased from Applied Biosystems, Inc. or Bachem Inc. (Torrance, Calif.). Anisole, dimethylsulfide, phenol, ethanedithiol, and thioanisole may be obtained from Aldrich Chemical Company (Milwaukee, Wis.). Air Products and Chemicals (Allentown, Pa.) supplies HF. Ethyl ether, acetic acid and methanol may be purchased from Fisher Scientific (Pittsburgh, Pa.).
Solid phase peptide synthesis may be carried out with an automatic peptide synthesizer (Model 430A, Applied Biosystems Inc., Foster City, Calif.) using the NMP/HOBt (Option 1) system and tBoc or Fmoc chemistry (see, Applied Biosystems User's Manual for the ABI 430A Peptide Synthesizer, Version 1.3B Jul. 1, 1988, section 6, pp. 49-70, Applied Biosystems, Inc., Foster City, Calif.) with capping. Boc-peptide-resins may be cleaved with HF (−5° C. to 0° C., 1 hour). The peptide may be extracted from the resin with alternating water and acetic acid, and the filtrates lyophilized. The Fmoc-peptide resins may be cleaved according to standard methods (Introduction to Cleavage Techniques, Applied Biosystems, Inc., 1990, pp. 6-12). Peptides may be also be assembled using an Advanced Chem Tech Synthesizer (Model MPS 350, Louisville, Ky.).
Peptides may be purified by RP-HPLC (preparative and analytical) using a Waters Delta Prep 3000 system. A C4, C8 or C18 preparative column (10μ, 2.2×25 cm; Vydac, Hesperia, Calif.) may be used to isolate peptides, and purity may be determined using a C4, C8 or C18 analytical column (5μ, 0.46×25 cm; Vydac). Solvents (A=0.1% TFA/water and B=0.1% TFA/CH3CN) may be delivered to the analytical column at a flowrate of 1.0 ml/min and to the preparative column at 15 ml/min. Amino acid analyses may be performed on the Waters Pico Tag system and processed using the Maxima program. Peptides may be hydrolyzed by vapor-phase acid hydrolysis (115° C., 20-24 h). Hydrolysates may be derivatized and analyzed by standard methods (Cohen, et al., The Pico Tag Method: A Manual of Advanced Techniques for Amino Acid Analysis, pp. 11-52, Millipore Corporation, Milford, Mass. (1989)). Fast atom bombardment analysis may be carried out by M-Scan, Incorporated (West Chester, Pa.). Mass calibration may be performed using cesium iodide or cesium iodide/glycerol. Plasma desorption ionization analysis using time of flight detection may be carried out on an Applied Biosystems Bio-Ion 20-mass spectrometer. Electrospray mass spectroscopy may be carried out on a VG-Trio machine.
Peptide compounds useful in the invention may also be prepared using recombinant DNA techniques, using methods now known in the art. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, 2d Ed., Cold Spring Harbor (1989). Non-peptide compounds useful in the present invention may be prepared by art-known methods. For example, phosphate-containing amino acids and peptides containing such amino acids, may be prepared using methods known in the art. See, e.g., Bartlett and Landen, Biorg. Chem. 14:356-377 (1986).
Compositions useful in the invention may conveniently be provided in the form of formulations suitable for parenteral (including intravenous, intramuscular and subcutaneous) or nasal or oral administration. In some cases, it will be convenient to provide an exendin or exendin agonist and another blood glucose-controlling, plasma glucose-lowering agent, such as an insulin, an amylin, an amylin agonist, in a single composition or solution for administration together. In other cases, it may be more advantageous to administer the additional agent separately from said exendin or exendin agonist. A suitable administration format may best be determined by a medical practitioner for each patient individually. Suitable pharmaceutically acceptable carriers and their formulation are described in standard formulation treatises, e.g., Remington's Pharmaceutical Sciences by E. W. Martin. See also Wang, Y. J. and Hanson, M. A. “Parenteral Formulations of Proteins and Peptides: Stability and Stabilizers,” Journal of Parenteral Science and Technology, Technical Report No. 10, Supp. 42:2S (1988).
Compounds useful in the invention can be provided as parenteral compositions for injection or infusion. Preferred formulations are those described and claimed in U.S. application Ser. No. 60/116,380, entitled, “Novel Exendin Agonist Formulations and Methods of Administration Thereof,” filed Jan. 14, 1999, which enjoys common ownership with the present application and which is incorporated by this reference into the present application as though fully set forth herein. They can, for example, be suspended in an inert oil, suitably a vegetable oil such as sesame, peanut, olive oil, or other acceptable carrier. Preferably, they are suspended in an aqueous Carrier, for example, in an isotonic buffer solution at a pH of about 3.0 to 8.0, preferably at a pH of about 3.5 to 5.0. These compositions may be sterilized by conventional sterilization techniques, or may be sterile filtered. The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH buffering agents. Useful buffers include for example, sodium acetate/acetic acid buffers. A form of repository or “depot” slow release preparation may be used so that therapeutically effective amounts of the preparation are delivered into the bloodstream over many hours or days following transdermal injection or delivery.
The desired isotonicity may be accomplished using sodium chloride or other pharmaceutically acceptable agents such as dextrose, boric acid, sodium tartrate, propylene glycol, polyols (such as mannitol and sorbitol), or other inorganic or organic solutes. Sodium chloride is preferred particularly for buffers containing sodium ions.
The claimed compositions can also be formulated as pharmaceutically acceptable salts (e.g., acid addition salts) and/or complexes thereof. Pharmaceutically acceptable salts are non-toxic salts at the concentration at which they are administered. The preparation of such salts can facilitate the pharmacological use by altering the physical-chemical characteristics of the composition without preventing the composition from exerting its physiological effect. Examples of useful alterations in physical properties include lowering the melting point to facilitate tranamucosal administration and increasing the solubility to facilitate the administration of higher concentrations of the drug.
Pharmaceutically acceptable salts include acid addition salts such as those containing sulfate, hydrochloride, phosphate, sulfamate, acetate, citrate, lactate, tartrate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, cyclohexylsulfamate and quinate. Pharmaceutically acceptable salts can be obtained from acids such as hydrochloric acid, sulfuric acid, phosphoric acid, sulfamic acid, acetic acid, citric acid, lactic acid, tartaric acid, malonic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, cyclohexylsulfamic acid, and quinic acid. Such salts may be prepared by, for example, reacting the free acid or base forms of the product with one or more equivalents of the appropriate base or acid in a solvent or medium in which the salt is insoluble, or in a solvent such as water which is then removed in vacuo or by freeze-drying or by exchanging the ions of an existing salt for another ion on a suitable ion exchange resin.
Carriers or excipients can also be used to facilitate administration of the compound. Examples of carriers and excipients include calcium carbonate, calcium phosphate, various sugars such as lactose, glucose, or sucrose, or types of starch, cellulose derivatives, gelatin, vegetable oils, polyethylene -glycols and physiologically compatible solvents. The compositions or pharmaceutical composition can be administered by different routes including intravenously, intraperitoneal, subcutaneous, and intramuscular, orally, topically, transmucosally, or by pulmonary inhalation.
If desired, solutions of the above compositions may be thickened with a thickening agent such as methyl cellulose. They may be prepared in emulsified form, either water in oil or oil in water. Any of a wide variety of pharmaceutically acceptable emulsifying agents may be employed including, for example, acacia powder, a non-ionic surfactant (such as a Tween), or an ionic surfactant (such as alkali polyether alcohol sulfates or sulfonates, e.g., a Triton).
Compositions useful in the invention are prepared by mixing the ingredients following generally accepted procedures. For example, the selected components may be simply mixed in a blender or other standard device to produce a concentrated mixture which may then be adjusted to the final concentration and viscosity by the addition of water or thickening agent and possibly a buffer to control pH or an additional solute to control tonicity.
For use by the physician, the compositions will be provided in dosage unit form containing an amount of an exendin or exendin agonist, for example, exendin-3, and/or exendin-4, with or without another glucosed-lowering agent. Therapeutically effective amounts of an exendin or exendin agonist for use creating a subject with gestational diabetes mellitus are those that lower blood glucose to a desired level. As will be recognized by those in the field, an effective amount of therapeutic agent will vary with many factors including the age and weight of the patient, the patient's physical condition, the blood glucose level and other factors.
The effective daily blood glucose controlling dose of the compounds will typically be in the range of about 3 to 30 μg to about 1 mg/day, preferably about 1 to 30 μg to about 500 μg/day and more preferably about 1 to 30 μg to about 100 μg/day, most preferably about 3 μg to about 50 μg/day, for a 70 kg patient, administered in a single or divided doses. Preferred dosages are described in U.S. application Ser. No. 60/116,380, entitled, “Novel Exendin Agonist Formulations and Methods of Administration Thereof,” filed Jan. 14, 1999, which has been incorporated by reference into the present application. A preferred dose for twice daily administration is about 0.05 to about 0.3 μg per kilogram. The exact dose to be administered is determined by the attending clinician and is dependent upon where the particular compound lies within the above quoted range., as well as upon the age, weight and condition of the individual, and the mode of administration. Administration should begin shortly after diagnosis of GDM and continue for the remainder of the gestation (i.e., the third trimester through parturition). Administration may be by injection, preferably subcutaneous or intramuscular. Administration may also be by non-injectable routes, for example, via the respiratory tract, the mouth and the gut. Orally active compounds may be taken orally, however dosages should be increased 5-10 fold. Preferred methods of administration are described in U.S. application Ser. No. 60/116,380, entitled, “Novel Exendin Agonist Formulations and Methods of Administration Thereof,” filed Jan. 14, 1999, which has been incorporated by reference into the present application. Solid dosage forms, such as those useful for oral, buccal, sublingual, intra-tracheal, nasal or pulmonary delivery may be used. Additionally, preserved or unpreserved liquid formulations or dry powder may be used
The optimal formulation and mode of administration of compounds of the present application to a patient depend on factors known in the art such as the particular disease or disorder, the desired effect, and the type of patient. While the compounds will typically be used to treat human subjects they may also be used to treat similar or identical diseases in other vertebrates such as ocher primates, farm animals such as swine, cattle and poultry, and sports animals and pets such as horses, dogs and cats.
To assist in understanding the present invention, the following Examples are included. The experiments relating to this invention should not, of course, be construed as specifically limiting the invention and such variations of the invention, now known or later developed, which would be within the purview of one skilled in the art are considered to fall within the scope of the invention as described herein and hereinafter claimed.
EXAMPLE 1
Preparation of Amidated Peptide Having SEQ. ID. NO. 9
The above-identified peptide was assembled on 4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.). In general, single-coupling cycles were used throughout the synthesis and Fast Moc (HBTU activation) chemistry was employed. However, at some positions coupling was less efficient than expected and double couplings were required. In particular, residues Asp9, Thr7 and Phe6 all required double coupling. Deprotection (Fmoc group removal)of the growing peptide chain using piperidine was not always efficient. Double deprotection was required at positions Arg20, Val19 and Leu14. Final deprotection of the completed peptide resin was achieved using a mixture of triethylsilane (0.2 mL), ethanedithiol (0.2 mL), anisole (0.2 mL), water (0.2 mL) and trifluoroacetic acid (15 mL) according to standard methods (Introduction to Cleavage Techniques, Applied Biosystems, Inc.) The peptide was precipitated in ether/water (50 mL) and centrifuged. The precipitate was reconstituted in glacial acetic acid and lyophilized. The lyophilized peptide was dissolved in water). Crude purity was about 55%.
Used in purification steps and analysis were Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA in ACN).
The solution containing peptide was applied to a preparative C-18 column and purified (10% to 40% Solvent B in Solvent A over 40 minutes). Purity of fractions was determined isocratically using a C-18 analytical column. Pure fractions were pooled furnishing the above-identified peptide. Analytical RP-HPLC (gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide gave product peptide having an observed retention time of 14.5 minutes. Electrospray Mass Spectrometry (M): calculated 4131.7; found 4129.3.
EXAMPLE 2
Preparation of Peptide Having SEQ. ID. NO. 10
The above-identified peptide was assembled on 4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Example 5. Used in analysis were Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC (gradient 25% to 75% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide gave product peptide having an observed retention time of 21.5 minutes. Electrospray Mass Spectrometry (M): calculated 4168.6; found 4171.2.
EXAMPLE 3
Preparation of Peptide Having SEQ. ID. NO. 11
The above-identified peptide was assembled on 4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Example 1. Used in analysis were Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide cave product peptide having an observed retention time of 17.9 minutes. Electrospray Mass Spectrometry (M): calculated 4147.6; found 4150.2.
EXAMPLE 3
Preparation of Peptide Having SEQ. ID. NO. 12
The above-identified peptide was assembled on 4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Example 1. Used in analysis were Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC (gradient 35% to 65% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide gave product peptide having an observed retention time of 19.7 minutes. Electrospray Mass Spectrometry (M): calculated 4212.6; found 4213.2.
EXAMPLE 4
Preparation of Peptide Having SEQ. ID. NO. 13
The above-identified peptide was assembled on 4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Example 1. Used in analysis were Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 50% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide gave product peptide having an observed retention time of 16.3 minutes. Electrospray Mass Spectrometry (M): calculated 4262.7; found 4262.4.
EXAMPLE 5
Preparation of Peptide Having SEQ. ID. NO. 14
The above-identified peptide is assembled on 4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Example 1. Used in analysis are Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide is then carried out to determine the retention time of the product peptide. Electrospray Mass Spectrometry (M): calculated 4172.6
EXAMPLE 6
Preparation of Peptide Having SEQ. ID. NO. 15
The above-identified peptide is assembled on 4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Example 1. Used in analysis are Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide is then carried out to determine the retention rime of the product peptide. Electrospray Mass Spectrometry (M): calculated 4224.7.
EXAMPLE 7
Preparation of Peptide Having SEQ. ID. NO. 16
The above-identified peptide is assembled on 4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Example 1. Used in analysis are Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide is then carried out to determine the retention time of the product peptide. Electrospray Mass Spectrometry (M): calculated 4172.6
EXAMPLE 8
Preparation of Peptide Having SEQ. ID. NO. 17
The above-identified peptide is assembled on 4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Example 1. Used in analysis are Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide is then carried out to determine the retention time of the product peptide. Electrospray Mass Spectrometry (M): calculated 4186.6
EXAMPLE 9
Preparation of Peptide Having SEQ. ID. NO. 18
The above-identified peptide is assembled on 4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Example 1. Used in analysis are Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide is then carried out to determine the retention time of the product peptide. Electrospray Mass Spectrometry (M): calculated 4200.7
EXAMPLE 10
Preparation of Peptide Having SEQ. ID. NO. 19
The above-identified peptide is assembled on 4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-proctected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Example 1. Used in analysis are Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide is then carried out to determine the retention time of the product peptide. Electrospray Mass Spectrometry (M): calculated 4200.7
EXAMPLE 11
Preparation of Peptide Having SEQ. ID. NO. 20
The above-identified peptide is assembled on 4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Example 1. Used in analysis are Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide is then carried out to determine the retention time of the product peptide. Electrospray Mass Spectrometry (M): calculated 4202.7.
EXAMPLE 12
Preparation of Peptide Having SEQ. ID. NO. 21
The above-identified peptide is assembled on 4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Example 1. Used in analysis are Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide is then carried out to determine the retention time of the product peptide. Electrospray Mass Spectrometry (M): calculated 4145.6.
EXAMPLE 13
Preparation of Peptide Having SEQ. ID. NO. 22
The above-identified peptide is assembled on 4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Example 1. Used in analysis are Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide is then carried out to determine the retention time of the product peptide. Electrospray Mass Spectrometry (M): calculated 4184.6.
EXAMPLE 14
Preparation of Peptide Having SEQ. ID. NO. 23
The above-identified peptide is assembled on 4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Example 1. Used in analysis are Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide is then carried out to determine the retention time of the product peptide. Electrospray Mass Spectrometry (M): calculated 4145.6.
EXAMPLE 15
Preparation of Peptide Having SEQ. ID. NO. 24
The above-identified peptide is assembled on 4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Example 1. Used in analysis are Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide is then carried out to determine the retention time of the product peptide. Electrospray Mass Spectrometry (M): calculated 4224.7.
EXAMPLE 16
Preparation of Peptide Having SEQ. ID. NO. 25
The above-identified peptide is assembled on 4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Example 1. Used in analysis are Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide is then carried out to determine the retention time of the product peptide. Electrospray Mass Spectrometry (M): calculated 4172.6.
EXAMPLE 17
Preparation of Peptide Having SEQ. ID. NO. 26
The above-identified peptide is assembled on 4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Example 1. Used in analysis are Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide is then carried out to determine the retention time of the product peptide. Electrospray Mass Spectrometry (M): calculated 4115.5.
EXAMPLE 18
Preparation of Peptide Having SEQ. ID. NO. 27
The above-identified peptide is assembled on 4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Example 1. Used in analysis are Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent D in Solvent A over 30 minutes) of the lyophilized peptide is then carried out to determine the retention time of the product peptide. Electrospray Mass Spectrometry (M): calculated 4188.6.
EXAMPLE 19
Preparation of Peptide Having SEQ. ID. NO. 28
The above-identified peptide is assembled on 4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Example 1. Used in analysis are Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide is then carried out to determine the retention time of the product peptide. Electrospray Mass Spectrometry (M): calculated 4131.6.
EXAMPLE 20
Preparation of Peptide Having SEQ. ID. NO. 29
The above-identified peptide is assembled on 4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Example 1. Used in analysis are Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide is then carried out to determine the retention time of the product peptide. Electrospray Mass Spectrometry (M): calculated 4172.6.
EXAMPLE 21
Preparation of Peptide Having SEQ. ID. NO. 30
The above-identified peptide is assembled on 4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Example 1. Used in analysis are Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide is then carried out to determine the retention time of the product peptide. Electrospray Mass Spectrometry (M): calculated 4145.6.
EXAMPLE 22
Preparation of Peptide Having SEQ. ID. NO. 31
The above-identified peptide is assembled on 4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Example 1. Additional double couplings are required at the thioproline positions 38, 37, 36 and 31. Used in analysis are Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide is then carried out to determine the retention time of the product peptide. Electrospray Mass Spectrometry (M): calculated 4266.8.
EXAMPLE 23
Preparation of Peptide Having SEQ. ID. NO. 32
The above-identified peptide is assembled on 4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Example 1. Additional double couplings are required at the thioproline positions 38, 37 and 36. Used in analysis are Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide is then carried out to determine the retention time of the product peptide. Electrospray Mass Spectrometry (M): calculated 4246.8.
EXAMPLE 24
Preparation of Peptide Having SEQ. ID. NO. 33
The above-identified peptide is assembled on 4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Example 1. Additional double couplings are required at the homoproline positions 38, 37, 36 and 31. Used in analysis are Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide is then carried out to determine the retention time of the product peptide. Electrospray Mass Spectrometry (M): calculated 4250.8.
EXAMPLE 25
Preparation of Peptide Having SEQ. ID. NO. 34
The above-identified peptide is assembled on 4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Example 1. Additional double couplings are required at the homoproline positions 38, 37, and 36. Used in analysis are Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide is then carried out to determine the retention time of the product peptide. Electrospray Mass Spectrometry (M): calculated 4234.8.
EXAMPLE 26
Preparation of Peptide Having SEQ. ID. NO. 35
The above-identified peptide is assembled on 4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Example 1. Additional double couplings are required at the thioproline positions 38, 37, 36 and 31. Used in analysis are Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide is then carried out to determine the retention time of the product peptide. Electrospray Mass Spectrometry (M): calculated 4209.8.
EXAMPLE 27
Preparation of Peptide Having SEQ. ID. NO. 36
The above-identified peptide is assembled on 4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Example 1. Additional double couplings are required at the homoproline positions 38, 37, 36 and 31. Used in analysis are Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide is then carried out to determine the retention time of the product peptide. Electrospray Mass Spectrometry (M): calculated 4193.7.
EXAMPLE 28
Preparation of Peptide Having SEQ. ID. NO. 37
The above-identified peptide is assembled on 4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Example 1. Additional double couplings are required at the N-methylalanine positions 38, 37, 36 and 31. Used in analysis are Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide is then carried out to determine the retention time of the product peptide. Electrospray Mass Spectrometry (M): calculated 3858.2.
EXAMPLE 29
Preparation of Peptide Having SEQ. ID. NO. 38
The above-identified peptide is assembled on 4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Example 1. Additional double couplings are required at the N-methylalanine positions 38, 37 and 36. Used in analysis are Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide is then carried out to determine the retention time of the product peptide. Electrospray Mass Spectrometry (M): calculated 3940.3.
EXAMPLE 30
Preparation of Peptide Having SEQ. ID. NO. 39
The above-identified peptide is assembled on 4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Example 1. Additional double couplings are required at the N-methylalanine positions 38, 37, 36 and 31. Used in analysis are Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide is then carried out to determine the retention time of the product peptide. Electrospray Mass Spectrometry (M): calculated 3801.1.
EXAMPLE 31
Preparation of C-Terminal Carboxylic Acid Peptides Corresponding to the Above C-Terminal Amide Sequences
The above peptides of Examples 1-5 to 30 are assembled on the so called Wang resin (p-alkoxybenzylalacohol resin (Bachem, 0.54 mmole/g)) using Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Example 1. Used in analysis are Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide is then carried out to determine the retention time of the product peptide. Electrospray Mass Spectrometry provides an experimentally determined (M).
EXAMPLE 32
Preparation of Peptide Having SEQ ID NO. 7









His Gly Glu Gly Thr Phe Thr Ser
[SEQ. ID. NO. 7]



Asp Leu Ser Lys Gln Met Glu Glu


Glu Ala Val Arg Leu Phe Ile Glu


Trp Leu Lys Asn Gly Gly-NH2






The above amidated peptide was assembled on 4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.). In general, single-coupling cycles were used throughout the synthesis and Fast Moc (HBTU activation) chemistry was employed. Deprotection (Fmoc group removal)of the growing peptide chain was achieved using piperidine. Final deprotection of the completed peptide resin was achieved using a mixture of triethylsilane (0.2 mL), ethanedithiol (0.2 mL), anisole (0.2 mL), water (0.2 mL) and trifluoroacetic acid (15 mL) according to standard methods (Introduction to Cleavage Techniques, Applied Biosystems, Inc.) The peptide was precipitated in ether/water (50 mL) and centrifuged. The precipitate was reconstituted in glacial acetic acid and lyophilized. The lyophilized peptide was dissolved in water). Crude purity was about 75%.
Used in purification steps and analysis were Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA in ACN). The solution containing peptide was applied to a preparative C-18 column and purified (10% to 40% Solvent B in Solvent A over 40 minutes). Purity of fractions was determined isocratically using a C-18 analytical column. Pure fractions were pooled furnishing the above-identified peptide. Analytical RP-HPLC (gradient 30% to 50% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide gave product peptide having an observed retention time of 18.9 minutes. Electrospray Mass Spectrometry (M): calculated 3408.0; found 3408.9.
EXAMPLE 33
Preparation of Peptide Having SEQ ID NO. 40









His Gly Glu Gly Thr Phe Thr Ser
[SEQ. ID. NO. 40]



Asp Leu Ser Lys Gln Met Glu Glu


Glu Ala Val Arg Leu Phe Ile Glu


Trp Leu Lys Asn-NH2






The above amidated peptide was assembled on 4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Example 32. Used in analysis were Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 40% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide gave product peptide having an observed retention time of 17.9 minutes. Electrospray Mass Spectrometry (M): calculated 3294.7; found 3294.8.
EXAMPLE 34
Preparation of Peptide Having SEQ ID NO. 41









His Gly Glu Gly Thr Phe Thr Ser
[SEQ. ID. NO. 41]



Asp Leu Ser Lys Gln Leu Glu Glu


Glu Ala Val Arg Leu Phe Ile Glu


Phe Leu Lys Asn-NH2






The above-identified amidated peptide was assembled on 4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Example 32. Used in analysis were Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC (gradient 29% to 36% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide gave product peptide having an observed retention time of 20.7 minutes. Electrospray Mass Spectrometry (M): calculated 3237.6; found 3240.
EXAMPLE 35
Preparation of Peptide Having SEQ ID NO. 42









His Ala Glu Gly Thr Phe Thr Ser
[SEQ. ID. NO. 42]



Asp Leu Ser Lys Gln Leu Glu Glu


Glu Ala Val Arg Leu Phe Ile Glu


Phe Leu Lys Asn-NH2






The above amidated peptide was assembled on 4-(2′-4′dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Example 32. Used in analysis were Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC (gradient 36% to 46% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide gave product peptide having an observed retention time of 15.2 minutes. Electrospray Mass Spectrometry (M): calculated 3251.6; found 3251.5.
EXAMPLE 36
Preparation of Peptide Having SEQ ID NO. 43









His Gly Glu Gly Ala Phe Thr Ser
[SEQ. ID. NO. 43]



Asp Leu Ser Lys Gln Leu Glu Glu


Glu Ala Val Arg Leu Phe Ile Glu


Phe Leu Lys Asn-NH2






The above amidated peptide was assembled on 4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Example 32. Used in analysis were Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC (gradient 36% to 46% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide gave product peptide having an observed retention time of 13.1 minutes. Electrospray Mass Spectrometry (M): calculated 3207.6; found 3208.3.
EXAMPLE 37
Preparation of Peptide Having SEQ ID NO. 44









His Gly Glu Gly Thr Ala Thr Ser
[SEQ. ID. NO. 44]



Asp Leu Ser Lys Gln Leu Glu Glu


Glu Ala Val Arg Leu Phe Ile Glu


Phe Leu Lys Asn-NH2






The above amidated peptide was assembled on 4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Example 32. Used in analysis were Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-MPLC (gradient 35% to 45% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide gave product peptide having an observed retention time of 12.8 minutes. Electrospray Mass Spectrometry (M): calculated 3161.5; found 3163.
EXAMPLE 38
Preparation of Peptide Having SEQ ID NO. 45









His Gly Glu Gly Thr Phe Thr Ala
[SEQ. ID. NO. 45]



Asp Leu Ser Lys Gln Leu Glu Glu


Glu Ala Val Arg Leu Phe Ile Glu


Phe Leu Lys Asn-NH2






The above-identified amidated peptide was assembled on 4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Example 32. Used in analysis were Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC (gradient 36% to 46% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide gave product peptide having an observed retention time of 15.2 minutes. Electrospray Mass Spectrometry (M): calculated 3221.6; found 3222.7.
EXAMPLE 39
Preparation of Peptide Having SEQ ID NO. 46









His Gly Glu Gly Thr Phe Thr Ser
[SEQ. ID. NO. 46]



Asp Ala Ser Lys Gln Leu Glu Glu


Glu Ala Val Arg Leu Phe Ile Glu


Phe Leu Lys Asn-NH2






The above-identified amidated peptide was assembled on 4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Example 32. Used in analysis were Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC (gradient 34% to 44% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide gave product peptide having an observed retention time of 14.3 minutes. Electrospray Mass Spectrometry (M): calculated 3195.5; found 3199.4.
EXAMPLE 40
Preparation of Peptide Having SEQ ID NO. 47









His Gly Glu Gly Thr Phe Thr Ser
[SEQ. ID. NO. 47]



Asp Leu Ala Lys Gln Leu Glu Glu


Glu Ala Val Arg Leu Phe Ile Glu


Phe Leu Lys Asn-NH2






The above-identified amidated peptide was assembled on 4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Example 32. Used in analysis were Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC (gradient 38% to 48% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide gave product peptide having an observed retention time of 15.7 minutes. Electrospray Mass Spectrometry (M): calculated 3221.6; found 3221.6.
EXAMPLE 41
Preparation of Peptide Having SEQ ID NO. 48









His Gly Glu Gly Thr Phe Thr Ser
[SEQ. ID. NO. 48]



Asp Leu Ser Ala Gln Leu Glu Glu


Glu Ala Val Arg Leu Phe Ile Glu


Phe Leu Lys Asn-NH2






The above-identified amidated peptide was assembled on 4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Example 32. Used in analysis were Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC (gradient 38% to 48% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide gave product peptide having an observed retention time of 18.1 minutes. Electrospray Mass Spectrometry (M): calculated 3180.5; found 3180.9.
EXAMPLE 42
Preparation of Peptide Having SEQ ID NO. 49









His Gly Glu Gly Thr Phe Thr Ser
[SEQ. ID. NO. 49]



Asp Leu Ser Lys Ala Leu Glu Glu


Glu Ala Val Arg Leu Phe Ile Glu


Phe Leu Lys Asn-NH2






The above-identified amidated peptide was assembled on 4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Example 32. Used in analysis were Solvent A (0 1% TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-MPLC (gradient 36% to 46% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide gave product peptide having an observed retention time of 17.0 minutes. Electrospray Mass Spectrometry (M): calculated 3180.6; found 3182.8.
EXAMPLE 43
Preparation of Peptide Having SEQ ID NO. 50









His Gly Glu Gly Thr Phe Thr Ser
[SEQ. ID. NO. 50]



Asp Leu Ser Lys Gln Ala Glu Glu


Glu Ala Val Arg Leu Phe Ile Glu


Phe Leu Lys Asn-NH2






The above-identified amidated peptide was assembled on 4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Example 32. Used in analysis were Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC (gradient 32% to 42% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide gave product peptide having an observed retention time of 14.9 minutes. Electrospray Mass Spectrometry (M): calculated 3195.5; found 3195.9.
EXAMPLE 44
Preparation of Peptide Having SEQ ID NO. 51









His Gly Glu Gly Thr Phe Thr Ser
[SEQ. ID. NO. 51]



Asp Leu Ser Lys Gln Leu Ala Glu


Glu Ala Val Arg Leu Phe Ile Glu


Phe Leu Lys Asn-NH2






The above-identified amidated peptide was assembled on 4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Example 32. Used in analysis were Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC (gradient 37% to 47% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide gave product peptide having an observed retention time of 17.9 minutes. Electrospray Mass Spectrometry (M): calculated 3179.6; found 3179.0.
EXAMPLE 45
Preparation of Peptide Having SEQ ID NO. 52









His Gly Glu Gly Thr Phe Thr Ser
[SEQ. ID. NO. 52]



Asp Leu Ser Lys Gln Leu Glu Ala


Glu Ala Val Arg Leu Phe Ile Glu


Phe Leu Lys Asn-NH2






The above-identified amidated peptide was assembled on 4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Example 32. Used in analysis were Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC (gradient 37% to 47% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide gave product peptide having an observed retention time of 14.3 minutes. Electrospray Mass Spectrometry (M): calculated 3179.6; found 3180.0.
EXAMPLE 46
Preparation of Peptide Having SEQ ID NO. 53









His Gly Glu Gly Thr Phe Thr Ser
[SEQ. ID. NO. 53]



Asp Leu Ser Lys Gln Leu Glu Glu


Ala Ala Val Arg Leu Phe Ile Glu


Phe Leu Lys Asn-NH2






The above-identified peptide was assembled on 4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Example 32. Used in analysis were Solvent A (0.1% TFA in water) and Solvent B (0.1% TPA in ACN). Analytical RP-HPLC (gradient 37% to 47% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide gave product peptide having an observed retention time of 13.7 minutes. Electrospray Mass Spectrometry (M): calculated 3179.6; found 3179.0.
EXAMPLE 47
Preparation of Peptide Having SEQ ID NO. S4









His Gly Glu Gly Thr Phe Thr Ser
[SEQ. ID. NO. 54]



Asp Leu Ser Lys Gln Leu Glu Glu


Glu Ala Ala Arg Leu Phe Ile Glu


Phe Leu Lys Asn-NH2






The above-identified amidated peptide was assembled on 4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Example 32. Used in analysis were Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC (gradient 35% to 45% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide gave product peptide having an observed retention time of 14.0 minutes. Electrospray Mass Spectrometry (M): calculated 3209.6; found 3212.8.
EXAMPLE 48
Preparation of Peptide Having SEQ ID NO. 55









His Gly Glu Gly Thr Phe Thr Ser
[SEQ. ID. NO. 55]



Asp Leu Ser Lys Gln Leu Glu Glu


Glu Ala Val Ala Leu Phe Ile Glu


Phe Leu Lys Asn-NH2






The above-identified amidated peptide was assembled on 4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Example 32. Used in analysis were Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC (gradient 38% to 48% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide gave product peptide having an observed retention time of 14.3 minutes. Electrospray Mass Spectrometry (M): calculated 3152.5; found 3153.5.
EXAMPLE 49
Preparation of Peptide Having SEQ ID NO. 56









His Gly Glu Gly Thr Phe Thr Ser
[SEQ. ID. NO. 56]



Asp Leu Ser Lys Gln Leu Glu Glu


Glu Ala Val Arg Ala Phe Ile Glu


Phe Leu Lys Asn-NH2






The above-identified amidated peptide was assembled on 4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Example 32. Used in analysis were Solvent A (0.1% TPA in water) and Solvent B (0.1% TPA in ACN). Analytical RP-HPLC (gradient 35% to 45% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide gave product peptide having an observed retention time of 12.1 minutes. Electrospray Mass Spectrometry (M): calculated 3195.5; found 3197.1.
EXAMPLE 50
Preparation of Peptide Having SEQ ID NO. 57









His Gly Glu Gly Thr Phe Thr Ser
[SEQ. ID. NO. 57]



Asp Leu Ser Lys Gln Leu Glu Glu


Glu Ala Val Arg Leu Phe Ile Ala


Phe Leu Lys Asn-NH2






The above-identified amidated peptide was assembled on 4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Example 32. Used in analysis were Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC (gradient 38% to 48% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide gave product peptide having an observed retention time of 10.9 minutes. Electrospray Mass Spectrometry (M): calculated 3179.6; found 3180.5.
EXAMPLE 51
Preparation of Peptide Having SEQ ID NO. 58









His Gly Glu Gly Thr Phe Thr Ser
[SEQ. ID. NO. 58]



Asp Leu Ser Lys Gln Leu Glu Glu


Glu Ala Val Arg Leu Phe Ile Glu


Ala Leu Lys Asn-NH2






The above-identified amidated peptide was assembled on 4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Example 32. Used in analysis were Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC (gradient 32% to 42% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide gave product peptide having an observed retention time of 17.5 minutes. Electrospray Mass Spectrometry (M): calculated 3161.5; found 3163.0.
EXAMPLE 52
Preparation of Peptide Having SEQ ID NO. 59









His Gly Glu Gly Thr Phe Thr Ser
[SEQ. ID. NO. 59]



Asp Leu Ser Lys Gln Leu Glu Glu


Glu Ala Val Arg Leu Phe Ile Glu


Phe Ala Lys Asn-NH2






The above-identified amidated peptide was assembled on 4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected-and purified in a similar way to Example 32. Used in analysis were Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC (gradient 32% to 42% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide gave product peptide having an observed retention time of 19.5 minutes. Electrospray Mass Spectrometry (M): calculated 3195.5; found 3199.
EXAMPLE 53
Preparation of Peptide Having SEQ ID NO. 60









His Gly Glu Gly Thr Phe Thr Ser
[SEQ. ID. NO. 60]



Asp Leu Ser Lys Gln Leu Glu Glu


Glu Ala Val Arg Leu Phe Ile Glu


Phe Leu Ala Asn-NH2






The above-identified amidated peptide was assembled on 4-(2′-4′dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Example 32. Used in analysis were Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC (gradient 38% to 48% Solvent B in Solvent A over 30 minutes) of lyophilized peptide gave product peptide having an observed retention time of 14.5 minutes. Electrospray Mass Spectrometry (M): calculated 3180.5; found 3183.7.
EXAMPLE 54
Preparation of Peptide Having SEQ ID NO. 61









His Gly Glu Gly Thr Phe Thr Ser
[SEQ ID NO. 61]



Asp Leu Ser Lys Gln Leu Glu Glu


Glu Ala Val Arg Leu Phe Ile Glu


Phe Leu Lys Ala-NH2






The above-identified amidated peptide was assembled on 4-(2′-4′dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Example 32. Used in analysis were Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC (gradient 34% to 44% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide gave product peptide having an observed retention time of 22.8 minutes. Electrospray Mass Spectrometry (M): calculated 3194.6; found 3197.6.
EXAMPLE 55
Preparation of Peptide Having SEQ ID NO. 62









His Gly Glu Gly Thr Phe Thr Ser
[SEQ. ID. NO. 62]



Asp Leu Ser Lys Gln Met Glu Glu


Glu Ala Val Arg Leu Phe Ile Glu


Trp Leu Lys Asn Gly Gly Pro Ser


Ser Gly Ala Pro Pro Pro-NH2






The above-identified amidated peptide is assembled on 4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Example 32. Used in analysis are Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide is then carried out to determine the retention time of the product peptide. Electrospray Mass Spectrometry (M): calculated 4099.6.
EXAMPLE 56
Preparation of Peptide Having SEQ ID NO. 63









His Gly Glu Gly Thr Phe Thr Ser
[SEQ. ID. NO. 63]



Asp Leu Ser Lys Gln Leu Glu Glu


Glu Ala Val Arg Leu Phe Ile Glu


Phe Leu Lys Asn Gly Gly Pro Ser


Ser Gly Ala Pro Pro Pro-NH2






The above-identified amidated peptide is assembled on 4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Example 32. Used in analysis are Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA in ACN) Analytical RP-HPLC (gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide is then carried out to determine the-retention time of the product peptide. Electrospray Mass Spectrometry (M): calculated 4042.5.
EXAMPLE 57
Preparation of Peptide Having SEQ ID NO. 64









His Gly Glu Gly Thr Phe Thr Ser
[SEQ. ID. NO. 64]



Asp Leu Ser Lys Gln Met Glu Glu


Glu Ala Val Arg Leu Phe Ile Glu


Trp Leu Lys Asn Gly Gly Pro Ser


Ser Gly Ala Pro Pro-NH2






The above-identified peptide is assembled on 4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Example 32. Used in analysis are Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide is then carried out to determine the retention time of the product peptide. Electrospray Mass Spectrometry (M): calculated 4002.4
EXAMPLE 58
Preparation of Peptide Having SEQ ID NO. 65









His Gly Glu Gly Thr Phe Thr Ser
[SEQ. ID. NO. 65]



Asp Leu Ser Lys Gln Leu Glu Glu


Glu Ala Val Arg Leu Phe Ile Glu


Phe Leu Lys Asn Gly Gly Pro Ser


Ser Gly Ala Pro Pro-NH2






The above-identified amidated peptide is assembled on 4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Example 32. Used in analysis are Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide is then carried out to determine the retention time of the product peptide. Electrospray Mass Spectrometry (M): calculated 3945.4.
EXAMPLE 59
Preparation of Peptide Having SEQ ID NO. 66









His Gly Glu Gly Thr Phe Thr Ser
[SEQ. ID. NO. 66]



Asp Leu Ser Lys Gln Met Glu Glu


Glu Ala Val Arg Leu Phe Ile Glu


Trp Leu Lys Asn Gly Gly Pro Ser


Ser Gly Ala Pro-NH2






The above-identified amidated peptide is assembled on 4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Example 32. Used in analysis are Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide is then carried out to determine the retention time of the product peptide. Electrospray Mass Spectrometry (M): calculated 3905.3.
EXAMPLE 60
Preparation of Peptide Having SEQ ID NO. 67









His Gly Glu Gly Thr Phe Thr Ser
[SEQ. ID. NO. 67]



Asp Leu Ser Lys Gln Leu Glu Glu


Glu Ala Val Arg Leu Phe Ile Glu


Phe Leu Lys Asn Gly Gly Pro Ser


Ser Gly Ala Pro-NH2






The above-identified amidated peptide is assembled on 4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Example 32. Used in analysis are Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide is then carried out to determine the retention time of the product peptide. Electrospray Mass Spectrometry (M): calculated 3848.2.
EXAMPLE 61
Preparation of Peptide Having SEQ ID NO. 68









His Gly Glu Gly Thr Phe Thr Ser
[SEQ. ID. NO. 68]



Asp Leu Ser Lys Gln Met Glu Glu


Glu Ala Val Arg Leu Phe Ile Glu


Trp Leu Lys Asn Gly Gly Pro Ser


Ser Gly Ala-NH2






The above-identified amidated peptide is assembled on 4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Example 32. Used in analysis are Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide is then carried out to determine the retention time of the product peptide. Electrospray Mass Spectrometry (M): calculated 3808.2.
EXAMPLE 62
Preparation of Peptide Having SEQ ID NO. 69









His Gly Glu Gly Thr Phe Thr Ser
[SEQ. ID. NO. 69]



Asp Leu Ser Lys Gln Leu Glu Glu


Glu Ala Val Arg Leu Phe Ile Glu


Phe Leu Lys Asn Gly Gly Pro Ser


Ser Gly Ala-NH2






The above-identified amidated peptide is assembled on 4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Example 32. Used in analysis are Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA in ACN) Analytical RP-HPLC (gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide is then carried out to determine the retention time of the product peptide. Electrospray Mass Spectrometry (M): calculated 3751.1.
EXAMPLE 63
Preparation of Peptide Having SEQ ID NO. 70









His Gly Glu Gly Thr Phe Thr Ser
[SEQ. ID. NO. 70]



Asp Leu Ser Lys Gln Met Glu Glu


Glu Ala Val Arg Leu Phe Ile Glu


Trp Leu Lys Asn Gly Gly Pro Ser


Ser Gly-NH2






The above-identified amidated peptide is assembled on 4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Example 32. Used in analysis are Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide is then carried out to determine the retention time of the product peptide. Electrospray Mass Spectrometry (M): calculated 3737.1.
EXAMPLE 64
Preparation of Peptide Having SEQ ID NO. 71









His Gly Glu Gly Thr Phe Thr Ser
[SEQ. ID. NO. 71]



Asp Leu Ser Lys Gln Leu Glu Glu


Glu Ala Val Arg Leu Phe Ile Glu


Phe Leu Lys Asn Gly Gly Pro Ser


Ser Gly-NH2






The above-identified amidated peptide is assembled on 4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Example 32. Used in analysis are Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide is then carried out to determine the retention time of the product peptide. Electrospray Mass Spectrometry (M): calculated 3680.1.
EXAMPLE 65
Preparation of Peptide Having SEQ ID NO. 72









His Gly Glu Gly Thr Phe Thr Ser
[SEQ. ID. NO. 72]



Asp Leu Ser Lys Gln Met Glu Glu


Glu Ala Val Arg Leu Phe Ile Glu


Trp Leu Lys Asn Gly Gly Pro Ser


Ser-NH2






The above-identified amidated peptide is assembled on 4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Example 32. Used in analysis are Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide is then carried out to determine the retention time of the product peptide. Electrospray Mass Spectrometry (M): calculated 3680.1
EXAMPLE 66
Preparation of Peptide Having SEQ ID NO. 73









His Gly Glu Gly Thr Phe Thr Ser
[SEQ. ID. NO. 73]



Asp Leu Ser Lys Gln Leu Glu Glu


Glu Ala Val Arg Leu Phe Ile Glu


Phe Leu Lys Asn Gly Gly Pro Ser


Ser-NH2






The above-identified amidated peptide is assembled on 4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Example 32. Used in analysis are Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide is then carried out to determine the retention time of the product peptide. Electrospray Mass Spectrometry (M): calculated 3623.0.
EXAMPLE 67
Preparation of Peptide Having SEQ ID NO. 74









His Gly Glu Gly Thr Phe Thr Ser
[SEQ. ID. NO. 74]



Asp Leu Ser Lys Gln Met Glu Glu


Glu Ala Val Arg Leu Phe Ile Glu


Trp Leu Lys Asn Gly Gly Pro


Ser-NH2






The above-identified amidated peptide is assembled on 4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Example 32. Used in analysis are Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide is then carried out to determine the retention time of the product peptide. Electrospray Mass Spectrometry (M): calculated 3593.0
EXAMPLE 68
Preparation of Peptide Having SEQ ID NO. 75









His Gly Glu Gly Thr Phe Thr Ser
[SEQ. ID. NO. 75]



Asp Leu Ser Lys Gln Leu Glu Glu


Glu Ala Val Arg Leu Phe Ile Glu


Phe Leu Lys Asn Gly Gly Pro


Ser-NH2






The above-identified amidated peptide is assembled on 4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Example 32. Used in analysis are Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide is then carried out to determine the retention time of the product peptide. Electrospray Mass Spectrometry (M): calculated 3535.9
EXAMPLE 69
Preparation of Peptide Having SEQ ID NO. 76









His Gly Glu Gly Thr Phe Thr Ser
[SEQ. ID. NO. 76]



Asp Leu Ser Lys Gln Met Glu Glu


Glu Ala Val Arg Leu Phe Ile Glu


Trp Leu Lys Asn Gly Gly


Pro-NH2






EXAMPLE 70
Preparation of Peptide Having SEQ ID NO. 77









His Gly Glu Gly Thr Phe Thr Ser
[SEQ. ID. NO. 77]



Asp Leu Ser Lys Gln Leu Glu Glu


Glu Ala Val Arg Leu Phe Ile Glu


Phe Leu Lys Asn Gly Gly


Pro-NH2






The above-identified amidated peptide is assembled on 4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Example 32. Used in analysis are Solvent A (0.1% TFA in water) and Solvent A (0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide is then carried out to determine the retention time of the product peptide. Electrospray Mass Spectrometry (M): calculated 3448.8.
EXAMPLE 71
Preparation of Peptide Having SEQ ID NO. 78









His Gly Glu Gly Thr Phe Thr Ser
[SEQ. ID. NO. 78]



Asp Leu Ser Lys Gln Leu Glu Glu


Glu Ala Val Arg Leu Phe Ile Glu


Phe Leu Lys Asn Gly Gly-NH2






The above-identified peptide is assembled on 4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Example 32. Used in analysis are Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide is then carried out to determine the retention time of the product peptide. Electrospray Mass Spectrometry (M): calculated 3351.7.
EXAMPLE 72
Preparation of Peptide Having SEQ ID NO. 79









His Gly Glu Gly Thr Phe Thr Ser
[SEQ. ID. NO. 79]



Asp Leu Ser Lys Gln Met Glu Glu


Glu Ala Val Arg Leu Phe Ile Glu


Trp Leu Lys Asn Gly-NH2






The above-identified peptide is assembled on 4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Example 32. Used in analysis are Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide is then carried out to determine the retention time of the product peptide. Electrospray Mass Spectrometry (M): calculated 3351.8.
EXAMPLE 73
Preparation of Peptide Having SEQ ID NO. 80









His Gly Glu Gly Thr Phe Thr Ser
[SEQ. ID. NO. 80]



Asp Leu Ser Lys Gln Leu Glu Glu


Glu Ala Val Arg Leu Phe Ile Glu


Phe Leu Lys Asn Gly-NH2






The above-identified amidated peptide is assembled on 4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Example 32. Used in analysis are Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide is then carried out to determine the retention time of the product peptide. Electrospray Mass Spectrometry (M); calculated 3294.7.
EXAMPLE 74
Preparation of Peptide Having SEQ ID NO. 81









His Gly Glu Gly Thr Phe Thr Ser
[SEQ. ID. NO. 81]



Asp Leu Ser Lys Gln Met Glu Glu


Glu Ala Val Arg Leu Phe Ile Glu


Trp Leu Lys Asn Gly Gly tPro Ser


Ser Gly Ala tPro tPro tPro-NH2






The above-identified amidated peptide is assembled on 4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Example 32. Double couplings are required at residues 37, 36 and 31. Used in analysis are Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide is then carried out to determine the retention time of the product peptide. Electrospray Mass Spectrometry (M): calculated 4197.1.
EXAMPLE 75
Preparation of Peptide Having SEQ ID NO. 82









His Gly Glu Gly Thr Phe Thr Ser
[SEQ. ID. NO. 82]



Asp Leu Ser Lys Gln Met Glu Glu


Glu Ala Val Arg Leu Phe Ile Glu


Trp Leu Lys Asn Gly Gly Pro Ser


Ser Gly Ala tPro tPro tPro-NH2






The above-identified amidated peptide is assembled on 4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Example 32. Double couplings are required at residues 37, 36 and 31. Used in analysis are Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide is then.carried out to determine the retention time of the product peptide. Electrospray Mass Spectrometry (M): calculated 4179.1.
EXAMPLE 76
Preparation of Peptide Having SEQ ID NO. 83









His Gly Glu Gly Thr Phe Thr Ser
[SEQ. ID. NO. 83]



Asp Leu Ser Lys Gln Met Glu Glu


Glu Ala Val Arg Leu Phe Ile Glu


Trp Leu Lys Asn Gly Gly NMeala


Ser Ser Gly Ala Pro Pro-NH2






The above-identified amidated peptide is assembled on 4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Example 32. Double couplings are required at residues 36 and 31. Used in analysis are Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide is then carried out to determine the retention time of the product peptide. Electrospray Mass Spectrometry (M): calculated 3948.3.
EXAMPLE 77
Preparation of Peptide Having SEQ ID NO. 84









His Gly Glu Gly Thr Phe Thr Ser
[SEQ. ID. NO. 84]



Asp Leu Ser Lys Gln Met Glu Glu


Glu Ala Val Arg Leu Phe Ile Glu


Trp Leu Lys Asn Gly Gly


NMeala Ser Ser Gly Ala NMeala


Nmeala-NH2






The above-identified amidated peptide is assembled on 4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Example 32. Double couplings are required at residues 36 and 31. Used in analysis are Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide is then carried out to determine the retention time of the product peptide. Electrospray Mass Spectrometry (M): calculated 3840.1.
EXAMPLE 78
Preparation of Peptide Having SEQ ID NO. 85









His Gly Glu Gly Thr Phe Thr Ser
[SEQ. ID. NO. 85]



Asp Leu Ser Lys Gln Met Glu Glu


Glu Ala Val Arg Leu Phe Ile Glu


Trp Leu Lys Asn Gly Gly hPro Ser


Ser Gly Ala hPro hPro-NH2






The above-identified amidated peptide is assembled on 4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Example 32. Double couplings are required at residues 36 and 31. Used in analysis are Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide is then carried out to determine the retention time of the product peptide. Electrospray Mass Spectrometry (M): calculated 4050.1.
EXAMPLE 79
Preparation of Peptide Having SEQ ID NO. 86









His Gly Glu Gly Thr Phe Thr Ser
[SEQ. ID. NO. 86]



Asp Leu Ser Lys Gln Met Glu Glu


Glu Ala Val Arg Leu Phe Ile Glu


Trp Leu Lys Asn Gly Gly hPro Ser


Ser Gly Ala hPro-NH2






The above-identified amidated peptide is assembled on 4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Example 32. A double coupling is required at residue 31. Used in analysis are Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide is then carried out to determine the retention time of the product peptide. Electrospray Mass Spectrometry (M): calculated 3937.1
EXAMPLE 80
Preparation of Peptide Having SEQ ID NO. 87









Arg Gly Glu Gly Thr Phe Thr Ser
[SEQ. ID. NO. 87]



Asp Leu Ser Lys Gln Met Glu Glu


Glu Ala Val Arg Leu Phe Ile Glu


Trp Leu Lys Asn Gly Gly Pro Ser


Ser Gly Ala-NH2






The above-identified amidated peptide is assembled on 4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Example 32. Used in analysis are Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide is then carried out to determine the retention time of the product peptide. Electrospray Mass Spectrometry (M); calculated 3827.2.
EXAMPLE 81
Preparation of Peptide Having SEQ ID NO. 88









His Gly Asp Gly Thr Phe Thr Ser
[SEQ. ID. NO. 88]



Asp Leu Ser Lys Gln Met Glu Glu


Glu Ala Val Arg Leu Phe Ile Glu


Trp Leu Lys Asn Gly Gly-NH2






The above-identified amidated peptide is assembled on 4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Example 32. Used in analysis are Solvent A (0.1% TFA in water) and Solvent B (0.1% TPA in ACN). Analytical RP-MPLC (gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide is then carried out to determine the retention time of the product peptide. Electrospray Mass Spectrometry (M): calculated 3394.8.
EXAMPLE 82
Preparation of Peptide Having SEQ ID NO. 89









His Gly Glu Gly Thr Naphthylala
[SEQ. ID. NO. 89]



Thr Ser Asp Leu Ser Lys Gln Leu


Glu Glu Glu Ala Val Arg Leu Phe


Ile Glu Phe Leu Lys Asn-NH2






The above-identified amidated peptide is assembled on 4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Example 32. Used in analysis are Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide is then carried out to determine the retention time of the product peptide. Electrospray Mass Spectrometry (M): calculated 3289.5.
EXAMPLE 83
Preparation of Peptide Having SEQ ID NO. 90









His Gly Glu Gly Thr Phe Ser Ser
[SEQ. ID. NO. 90]



Asp Leu Ser Lys Gln Met Glu Glu


Glu Ala Val Arg Leu Phe Ile Glu


Trp Leu Lys Asn-NH2






The above-identified amidated peptide is assembled on 4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Example 32. Used in analysis are Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide is then carried out to determine the retention time of the product peptide. Electrospray Mass Spectrometry (M): calculated 3280.7.
EXAMPLE 84
Preparation of Peptide Having SEQ ID No. 91









His Gly Glu Gly Thr Phe Ser Thr
[SEQ. ID. NO. 91]



Asp Leu Ser Lys Gln Met Glu Glu


Glu Ala Val Arg Leu Phe Ile Glu


Trp Leu Lys Asn-NH2






The above-identified amidated peptide is assembled on 4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Example 32. Used in analysis are Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide is then carried out to determine the retention time of the product peptide. Electrospray Mass Spectrometry (M): calculated 3294.7.
EXAMPLE 85
Preparation of Peptide Having SEQ ID NO. 92









His Gly Glu Gly Thr Phe Thr Ser
[SEQ. ID. NO. 92]



Glu Leu Ser Lys Gln Met Ala Glu


Glu Ala Val Arg Leu Phe Ile Glu


Trp Leu Lys Asn-NH2






The above-identified amidated peptide is assembled on 4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mole/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Example 32. Used in analysis are Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide is then carried out to determine the retention time of the product peptide. Electrospray Mass Spectrometry (M): calculated 3250.7.
EXAMPLE 86
Preparation of Peptide Having SEQ ID NO. 93









His Gly Glu Gly Thr Phe Thr Ser
[SEQ. ID. NO. 93]



Asp pentylgly Ser Lys Gln Leu


Glu Glu Glu Ala Val Arg Leu Phe


Ile Glu Phe Leu Lys Asn-NH2






The above-identified amidated peptide is assembled on 4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Example 32. Used in analysis are Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide is then carried out to determine the retention time of the product peptide. Electrospray Mass Spectrometry (M): calculated 3253.5.
EXAMPLE 87
Preparation of Peptide Having SEQ ID NO. 94









His Gly Glu Gly Thr Phe Thr Ser
[SEQ. ID. NO. 94]



Asp Leu Ser Lys Gln Leu Glu Glu


Glu Ala Val Arg Leu Naphthylala


Ile Glu Phe Leu Lys Asn-NH2






The above-identified amidated peptide is assembled on 4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Example 32. Used in analysis are Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide is then carried out to determine the retention time of the product peptide. Electrospray Mass Spectrometry (M): calculated 3289.5.
EXAMPLE 88
Preparation of Peptide Having SEQ ID NO. 95









His Gly Glu Gly Thr Phe Thr Ser
[SEQ. ID. NO. 95]



Asp Leu Ser Lys Gln Met Glu Glu


Glu Ala Val Arg Leu Phe


tButylgly Glu Trp Leu Lys


Asn-NH2






The above-identified amidated peptide is assembled on 4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Example 32. Used in analysis are Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide is then carried out to determine the retention time of the product peptide. Electrospray Mass Spectrometry (M): calculated 3183.4.
EXAMPLE 89
Preparation of Peptide Having SEQ ID NO. 96









His Gly Glu Gly Thr Phe Thr Ser
[SEQ. ID. NO. 96]



Asp Leu Ser Lys Gln Leu Glu Glu


Glu Ala Val Arg Leu Phe Ile Asp


Phe Leu Lys Asn-NH2






The above-identified amidated peptide is assembled on 4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Example 32. Used in analysis are Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide is then carried out to determine the retention time of the product peptide. Electrospray Mass Spectrometry (M): calculated 3237.6.
EXAMPLE 90
Preparation of Peptide Having SEQ ID NO. 97









His Gly Glu Gly Thr Phe Thr Ser
[SEQ. ID. NO. 97]



Asp Ala Ser Lys Gln Leu Glu Glu


Glu Ala Val Arg Leu Phe Ile Glu


Phe Leu Lys Asn Gly Gly Pro Ser


Ser-NH2






The above-identified amidated peptide is assembled on 4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Example 32. Used in analysis are Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide is then carried out to determine the retention time of the product peptide. Electrospray Mass Spectrometry (M): calculated 3637.9.
EXAMPLE 91
Preparation of Peptide Having SEQ ID NO. 98









His Gly Glu Gly Thr Phe Thr Ser
[SEQ. ID. NO. 98]



Asp Ala Ser Lys Gln Met Glu Glu


Glu Ala Val Arg Leu Phe Ile Glu


Trp Leu Lys Asn Gly-NH2






The above-identified amidated peptide is assembled on 4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Example 32. Used in analysis are Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide is then carried out to determine the retention time of the product peptide. Electrospray Mass Spectrometry (M): calculated 3309.7.
EXAMPLE 92
Preparation of Peptide Having SEQ ID NO. 99









His Gly Glu Gly Thr Phe Thr Ser
[SEQ. ID. NO. 99]



Asp Ala Ser Lys Gln Met Glu Glu


Glu Ala Val Arg Leu Phe Ile Glu


Trp Leu Lys Asn Gly Gly hPro Ser


Ser Gly Ala hPro hPro-NH2






The above-identified amidated peptide is assembled on 4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Example 37. Double couplings are required at residues 36 and 31. Used in analysis are Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide is then carried out to determine the retention time of the product peptide. Electrospray Mass Spectrometry (M): calculated 3711.1.
EXAMPLE 93
Preparation of C-Terminal Carboxylic Acid Peptides Corresponding to the Above C-Terminal Amide Sequences for SEQ ID NOS. 7, 40-61, 68-75, 78-80 and 87-96
Peptides having the sequences of SEQ ID NOS. 7, 40-61, 68-75, 78-80 and 87-96 are assembled on the so called Wang resin (p-alkoxybenzylalacohol resin (Bachem, 0.54 mmole/g)) using Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Example 32. Used in analysis are Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide is then carried out to determine the retention time of the product peptide. Electrospray Mass Spectrometry provides an experimentally determined (M).
EXAMPLE 94
Preparation of C-Terminal Carboxylic Acid Peptides Corresponding to the Above C-Terminal Amide Sequences for SEQ ID NOS. 62-67, 76, 77 and 81-86
Peptides having the sequences of SEQ ID NOS. 62-67, 76, 77 and 81-86 are assembled on the 2-chlorotritylchloride resin (200-400 mesh), 2% DVB (Novabiochem, 0.4-1.0 mmole/g)) using Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Example 32. Used in analysis are Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent A in Solvent A over 30 minutes) of the lyophilized peptide is then carried out to determine the retention time of the product peptide. Electrospray Mass Spectrometry provides an experimentally determined (M).
EXAMPLE 95
Preparation of Peptide Having SEQ ID NO. 100









Ala Gly Glu Gly Thr Phe Thr Ser
[SEQ. ID. NO. 100]



Asp Leu Ser Lys Gln Leu Glu Glu


Glu Ala Val Arg Leu Phe Ile Glu


Phe Leu Lys Asn-NH2






The above amidated peptide was assembled on 4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.). In general, single-coupling cycles were used throughout the synthesis and Fast Moc (HBTU activation) chemistry was employed. Deprotection (Fmoc group removal) of the growing peptide chain was achieved using piperidine. Final deprotection of the completed peptide resin was achieved using a mixture of triethylsilane (0.2 mL), ethanedithiol (0.2 mL), anisole (0.2 mL), water (0.2 mL) and trifluoroacetic acid (15 mL) according to standard methods (Introduction to Cleavage Techniques, Applied Biosystems, Inc.) The peptide was precipitated in ether/water (50 mL) and centrifuged. The precipitate was reconstituted in glacial acetic acid and lyophilized. The lyophilized peptide was dissolved in water). Crude purity was about 75%.
Used in purification steps and analysis were Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA in ACN).
The solution containing peptide was applied to a preparative C-18 column and purified (10% to 40% Solvent B in Solvent A over 40 minutes). Purity of fractions was determined isocratically using a C-18 analytical column. Pure fractions were pooled furnishing the above-identified peptide. Analytical RP-HPLC (gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide gave product peptide having an observed retention time of 19.2 minutes. Electrospray Mass Spectrometry (M): calculated 3171.6; found 3172.
EXAMPLE 96
Preparation of Peptide Having SEQ ID NO. 101









His Gly Ala Gly Thr Phe Thr Ser
[SEQ. ID. NO. 101]



Asp Leu Ser Lys Gln Leu Glu Glu


Glu Ala Val Arg Leu Phe Ile Glu


Phe Leu Lys Asn-NH2






The above amidated peptide was assembled on 4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Example 95. Used in analysis were Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC (gradient 36% to 46% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide gave product peptide having an observed retention time of 14.9 minutes. Electrospray Mass Spectrometry (M): calculated 3179.6; found 3180.
EXAMPLE 97
Preparation of Peptide Having SEQ ID NO. 102









His Gly Glu Ala Thr Phe Thr Ser
[SEQ. ID. NO. 102]



Asp Leu Ser Lys Gln Leu Glu Glu


Glu Ala Val Arg Leu Phe Ile Glu


Phe Leu Lys Asn-NH2






The above amidated peptide was assembled on 4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Example 95. Used in analysis were Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC (gradient 37% to 47% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide gave product peptide having an observed retention time of 12.2 minutes. Electrospray Mass Spectrometry (M): calculated 3251.6; found 3253.3.
EXAMPLE 98
Preparation of Peptide Having SEQ ID NO. 103









His Gly Glu Gly Thr Phe Thr Ser
[SEQ. ID. NO. 103]



Ala Leu Ser Lys Gln Leu Glu Glu


Glu Ala Val Arg Leu Phe Ile Glu


Phe Leu Lys Asn-NH2






The above amidated peptide was assembled on 4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Example 95. Used in analysis were Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC (gradient 35% to 45% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide gave product peptide having an observed retention time of 16.3 minutes. Electrospray Mass Spectrometry (M): calculated 3193.6; found 3197.
EXAMPLE 99
Preparation of Peptide Having SEQ ID NO. 104









Ala Gly Glu Gly Thr Phe Thr Ser
[SEQ. ID. NO. 104]



Asp Leu Ser Lys Gln Met Glu Glu


Glu Ala Val Arg Leu Phe Ile Glu


Trp Leu Lys Asn-NH2






The above-identified amidated peptide is assembled on 4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Example 95. Used in analysis are Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide is then carried out to determine the retention time of the product peptide. Electrospray mass Spectrometry (M): calculated 3228.6.
EXAMPLE 100
Preparation of Peptide Having SEQ ID NO. 105









His Gly Ala Gly Thr Phe Thr Ser
[SEQ. ID. NO. 105]



Asp Leu Ser Lys Gln Met Glu Glu


Glu Ala Val Arg Leu Phe Ile Glu


Trp Leu Lys Asn-NH2






The above-identified amidated peptide is assembled on 4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Example 95. Used in analysis are Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide is then carried out to determine the retention time of the product peptide. Electrospray Mass Spectrometry (M): calculated 3234.7.
EXAMPLE 101
Preparation of Peptide Having SEQ ID NO. 106









His Gly Glu Ala Thr Phe Thr Ser
[SEQ. ID. NO. 106]



Asp Leu Ser Lys Gln Met Glu Glu


Glu Ala Val Arg Leu Phe Ile Glu


Trp Leu Lys Asn-NH2






The above-identified amidated peptide is assembled on 4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Example 95. Used in analysis are Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide is then carried out to determine the retention time of the product peptide. Electrospray Mass Spectrometry (M): calculated 3308.7.
EXAMPLE 102
Preparation of Peptide Having SEQ ID NO. 107









His Gly Glu Gly Thr Phe Thr Ser
[SEQ. ID. NO. 107]



Ala Leu Ser Lys Gln Met Glu Glu


Glu Ala Val Arg Leu Phe Ile Glu


Trp Leu Lys Asn-NH2






The above-identified amidated peptide is assembled on 4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Example 95. Used in analysis are Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide is then carried out to determine the retention time of the product peptide. Electrospray Mass Spectrometry (M): calculated 3250.7
EXAMPLE 103
Preparation of Peptide Having SEQ ID NO. 108









His Gly Glu Gly Thr Phe Thr Ser
[SEQ. ID. NO. 108]



Asp Ala Ser Lys Gln Met Glu Glu


Glu Ala Val Arg Leu Phe Ile Glu


Trp Leu Lys Asn-NH2






The above-identified amidated peptide is assembled on 4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Example 95. Used in analysis are Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide is then carried out to determine the retention time of the product peptide. Electrospray Mass Spectrometry (M): calculated 3252.6.
EXAMPLE 104
Preparation of Peptide Having SEQ ID NO. 109









Ala Ala Glu Gly Thr Phe Thr Ser
[SEQ. ID. NO. 109]



Asp Leu Ser Lys Gln Met Glu Glu


Glu Ala Val Arg Leu Phe Ile Glu


Trp Leu Lys Asn-NH2






The above-identified amidated peptide is assembled on 4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Example 95. Used in analysis are Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide is then carried out to determine the retention time of the product peptide. Electrospray Mass Spectrometry (M): calculated 3200.6.
EXAMPLE 105
Preparation of Peptide Having SEQ ID NO. 110









Ala Ala Glu Gly Thr Phe Thr Ser
[SEQ. ID. NO. 110]



Asp Leu Ser Lys Gln Leu Glu Glu


Glu Ala Val Arg Leu Phe Ile Glu


Phe Leu Lys Asn-NH2






The above-identified amidated peptide is assembled on 4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Example 95. Used in analysis are Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide is then carried out to determine the retention time of the product peptide. Electrospray Mass Spectrometry (M): calculated 3143.5.
EXAMPLE 106
Preparation of Peptide Having SEQ ID No. 111









Ala Gly Asp Gly Thr Phe Thr Ser
[SEQ. ID. NO. 111]



Asp Leu Ser Lys Gln Met Glu Glu


Glu Ala Val Arg Leu Phe Ile Glu


Trp Leu Lys Asn-NH2






The above-identified amidated peptide is assembled on 4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Example 95. Used in analysis are Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide is then carried out to determine the retention time of the product peptide. Electrospray Mass Spectrometry (M): calculated 3214.6.
EXAMPLE 107
Preparation of Peptide Having SEQ ID NO. 112









Ala Gly Asp Gly Thr Phe Thr Ser
[SEQ. ID. NO. 112]



Asp Leu Ser Lys Gln Leu Glu Glu


Glu Ala Val Arg Leu Phe Ile Glu


Phe Leu Lys Asn-NH2






The above-identified amidated peptide is assembled on 4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Example 95. Used in analysis are Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide is then carried out to determine the retention time of the product peptide. Electrospray Mass Spectrometry (M): calculated 3157.5.
EXAMPLE 108
Preparation of Peptide Having SEQ ID NO. 113









Ala Gly Asp Gly Ala Phe Thr Ser
[SEQ. ID. NO. 113]



Asp Leu Ser Lys Gln Met Glu Glu


Glu Ala Val Arg Leu Phe Ile Glu


Trp Leu Lys Asn-NH2






The above-identified amidated peptide is assembled on 4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Example 95. Used in analysis are Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide is then carried out to determine the retention time of the product peptide. Electrospray Mass Spectrometry (M): calculated 3184.6.
EXAMPLE 109
Preparation of Peptide Having SEQ ID NO. 114









Ala Gly Asp Gly Ala Phe Thr Ser
[SEQ. ID. NO. 114]



Asp Leu Ser Lys Gln Leu Glu Glu


Glu Ala Val Arg Leu Phe Ile Glu


Phe Leu Lys Asn-NH2






The above-identified amidated peptide is assembled on 4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Example 95. Used in analysis are Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide is then carried out to determine the retention time of the product peptide. Electrospray Mass Spectrometry (M): calculated 3127.5.
EXAMPLE 110
Preparation of Peptide Having SEQ ID NO. 115









Ala Gly Asp Gly Thr NaphthylAla
[SEQ. ID. NO. 115]



Thr Ser Asp Leu Ser Lys Gln Met


Glu Glu Glu Ala Val Arg Leu Phe


Ile Glu Trp Leu Lys Asn-NH2






The above-identified amidated peptide is assembled on 4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Example 95. Used in analysis are Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide is then carried out to determine the retention time of the product peptide. Electrospray Mass Spectrometry (M): calculated 3266.4.
EXAMPLE 111
Preparation of Peptide Having SEQ ID NO. 116









Ala Gly Asp Gly Thr Naphthylala
[SEQ. ID. NO. 116]



Thr Ser Asp Leu Ser Lys Gln Leu


Glu Glu Glu Ala Val Arg Leu Phe


Ile Glu Phe Leu Lys Asn-NH2






The above-identified amidated peptide is assembled on 4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Example 95. Used in analysis are Solvent A (0.1% TFA in water) and Solvent B (0.1% TEA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide is then carried out to determine the retention time of the product peptide. Electrospray Mass Spectrometry (M): calculated 3209.4.
EXAMPLE 112
Preparation of Peptide Having SEQ ID NO. 117









Ala Gly Asp Gly Thr Phe Ser Ser
[SEQ. ID. NO. 117]



Asp Leu Ser Lys Gln Met Glu Glu


Glu Ala Val Arg Leu Phe Ile Glu


Trp Leu Lys Asn-NH2






The above-identified amidated peptide is assembled on 4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Example 95. Used in analysis are Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide is then carried out to determine the retention time of the product peptide. Electrospray Mass Spectrometry (M): calculated 3200.6.
EXAMPLE 113
Preparation of Peptide Having SEQ ID NO. 118









Ala Gly Asp Gly Thr Phe Ser Ser
[SEQ. ID. NO. 118]



Asp Leu Ser Lys Gln Leu Glu Glu


Glu Ala Val Arg Leu Phe Ile Glu


Phe Leu Lys Asn-NH2






The above-identified amidated peptide is assembled on 4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Example 95. Used in analysis are Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide is then carried out to determine the retention time of the product peptide. Electrospray Mass Spectrometry (M): calculated 3143.5.
EXAMPLE 114
Preparation of Peptide Having SEQ ID NO. 119









Ala Gly Asp Gly Thr Phe Thr Ala
[SEQ. ID. NO. 119]



Asp Leu Ser Lys Gln Met Glu Glu


Glu Ala Val Arg Leu Phe Ile Glu


Trp Leu Lys Asn-NH2






The above-identified amidated peptide is assembled on 4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Example 95. Used in analysis are Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide is then carried out to determine the retention time of the product peptide. Electrospray Mass Spectrometry (M): calculated 3198.6.
EXAMPLE 115
Preparation of Peptide Having SEQ ID NO. 120









Ala Gly Asp Gly Thr Phe Thr Ala
[SEQ. ID. NO. 120]



Asp Leu Ser Lys Gln Leu Glu Glu


Glu Ala Val Arg Leu Phe Ile Glu


Phe Leu Lys Asn-NH2






The above-identified amidated peptide is assembled on 4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Example 95. Used in analysis are Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide is then carried out to determine the retention time of the product peptide. Electrospray Mass Spectrometry (M): calculated 3141.5.
EXAMPLE 116
Preparation of Peptide Having SEQ ID NO. 121









Ala Gly Asp Gly Thr Phe Thr Ser
[SEQ. ID. NO. 121]



Ala Leu Ser Lys Gln Met Glu Glu


Glu Ala Val Arg Leu Phe Ile Glu


Trp Leu Lys Asn-NH2






The above-identified peptide is assembled on 4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Example 95. Used in analysis are Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide is then carried out to determine the retention time of the product peptide. Electrospray Mass Spectrometry (M); calculated 3170.6.
EXAMPLE 117
Preparation of Peptide Having SEQ ID NO. 122









Ala Gly Asp Gly Thr Phe Thr Ser
[SEQ. ID. NO. 122]



Ala Leu Ser Lys Gln Leu Glu Glu


Glu Ala Val Arg Leu Phe Ile Glu


Phe Leu Lys Asn-NH2






The above-identified amidated peptide is assembled on 4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Example 95. Used in analysis are Solvent A (0.1% TFA in water) and Solvent 3 (0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide is then carried out to determine the retention time of the product peptide. Electrospray Mass Spectrometry (M): calculated 3113.5.
EXAMPLE 118
Preparation of Peptide Having SEQ ID NO. 123









Ala Gly Asp Gly Thr Phe Thr Ser
[SEQ. ID. NO. 123]



Glu Leu Ser Lys Gln Met Glu Glu


Glu Ala Val Arg Leu Phe Ile Glu


Trp Leu Lys Asn-NH2






The above-identified amidated peptide is assembled on 4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Example 95. Used in analysis are Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA in ACN) Analytical RP-HPLC (gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide is then carried out to determine the retention time of the product peptide. Electrospray Mass Spectrometry (M): calculated 3228.6.
EXAMPLE 119
Preparation of Peptide Having SEQ ID NO. 124









Ala Gly Asp Gly Thr Phe Thr Ser
[SEQ. ID. NO. 124]



Glu Leu Ser Lys Gln Leu Glu Glu


Glu Ala Val Arg Leu Phe Ile Glu


Phe Leu Lys Asn-NH2






The above-identified amidated peptide is assembled on 4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Example 95. Used in analysis are Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide is then carried out to determine the retention time of the product peptide. Electrospray Mass Spectrometry (M): calculated 3171.6.
EXAMPLE 120
Preparation of Peptide Having SEQ ID NO. 125









Ala Gly Asp Gly Thr Phe Thr Ser
[SEQ. ID. NO. 125]



Asp Ala Ser Lys Gln Met Glu Glu


Glu Ala Val Arg Leu Phe Ile Glu


Trp Leu Lys Asn-NH2






The above-identified amidated peptide is assembled on 4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Example 95. Used in analysis are Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide is then carried out to determine the retention time of the product peptide. Electrospray Mass Spectrometry (M): calculated 3172.5.
EXAMPLE 121
Preparation of Peptide Having SEQ ID NO. 126









Ala Gly Asp Gly Thr Phe Thr Ser
[SEQ. ID. NO. 126]



Asp Ala Ser Lys Gln Leu Glu Glu


Glu Ala Val Arg Leu Phe Ile Glu


Phe Leu Lys Asn-NH2






The above-identified amidated peptide is assembled on 4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Example 95. Used in analysis are Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide is then carried out to determine the retention time of the product peptide. Electrospray Mass Spectrometry (M): calculated 3115.4.
EXAMPLE 122
Preparation of Peptide Having SEQ ID NO. 127









Ala Gly Asp Gly Thr Phe Thr Ser
[SEQ. ID. NO. 127]



Asp Pentylgly Ser Lys Gln Met


Glu Glu Glu Ala Val Arg Leu Phe


Ile Glu Trp Leu Lys Asn-NH2






The above-identified amidated peptide is assembled on 4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Example 95. Used in analysis are Solvent A (0.1% TFA in water) and Solvent: B (0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide is then carried out to determine the retention time of the product peptide. Electrospray Mass Spectrometry (M): calculated 3230.4.
EXAMPLE 123
Preparation of Peptide Having SEQ ID NO. 128









Ala Gly Asp Gly Thr Phe Thr Ser
[SEQ. ID. NO. 128]



Asp Pentylgly Ser Lys Gln Leu


Glu Glu Glu Ala Val Arg Leu Phe


Ile Glu Phe Leu Lys Asn-NH2






The above-identified amidated peptide is assembled on 4-(2+-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Example 95. Used in analysis are Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide is then carried out to determine the retention time of the product peptide. Electrospray Mass Spectrometry (M): calculated 3198.6.
EXAMPLE 124
Preparation of Peptide Having SEQ ID NO. 129









Ala Gly Asp Gly Thr Phe Thr Ser
[SEQ. ID. NO. 129]



Asp Leu Ala Lys Gln Met Glu Glu


Glu Ala Val Arg Leu Phe Ile Glu


Trp Leu Lys Asn-NH2






The above-identified amidated peptide is assembled on 4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Example 95. Used in analysis are Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide is then carried out to determine the retention time of the product peptide. Electrospray Mass Spectrometry (M): calculated 3141.5.
EXAMPLE 125
Preparation of Peptide Having SEQ ID NO. 130









Ala Gly Asp Gly Thr Phe Thr Ser
[SEQ. ID. NO. 130]



Asp Leu Ala Lys Gln Leu Glu Glu


Glu Ala Val Arg Leu Phe Ile Glu


Phe Leu Lys Asn-NH2






The above-identified amidated peptide is assembled on 4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Example 95. Used in analysis are Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide is then carried out to determine the retention time of the product peptide. Electrospray Mass Spectrometry (M): calculated 3157.5.
EXAMPLE 126
Preparation of Peptide Having SEQ ID NO. 131









Ala Gly Asp Gly Thr Phe Thr Ser
[SEQ. ID. NO. 131]



Asp Leu Ser Ala Gln Met Glu Glu


Glu Ala Val Arg Leu Phe Ile Glu


Trp Leu Lys Asn-NH2






The above-identified amidated peptide is assembled on 4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Example 95. Used in analysis are Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA in ACN) Analytical RP-HPLC (gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide is then carried out to determine the retention time of the product peptide. Electrospray Mass Spectrometry (M): calculated 3100.4.
EXAMPLE 127
Preparation of Peptide Having SEQ ID NO. 132









Ala Gly Asp Gly Thr Phe Thr Ser
[SEQ. ID. NO. 132]



Asp Leu Ser Ala Gln Leu Glu Glu


Glu Ala Val Arg Leu Phe Ile Glu


Phe Leu Lys Asn-NH2






The above-identified amidated peptide is assembled on 4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Example 95. Used in analysis are Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide is then carried out to determine the retention time of the product peptide. Electrospray Mass Spectrometry (M): calculated 3157.6.
EXAMPLE 128
Preparation of Peptide Having SEQ ID NO. 133









Ala Gly Asp Gly Thr Phe Thr Ser
[SEQ. ID. NO. 133]



Asp Leu Ser Lys Ala Met Glu Glu


Glu Ala Val Arg Leu Phe Ile Glu


Trp Leu Lys Asn-NH2






The above-identified amidated peptide is assembled on 4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Example 95. Used in analysis are Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide is then carried out to determine the retention time of the product peptide. Electrospray Mass Spectrometry (M): calculated 3100.5.
EXAMPLE 129
Preparation of Peptide Having SEQ ID NO. 134









Ala Gly Asp Gly Thr Phe Thr Ser
[SEQ. ID. NO. 134]



Asp Leu Ser Lys Ala Leu Glu Glu


Glu Ala Val Arg Leu Phe Ile Glu


Phe Leu Lys Asn-NH2






The above-identified amidated peptide is assembled on 4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Example 95. Used in analysis are Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide is then carried out to determine the retention time of the product peptide. Electrospray Mass Spectrometry (M): calculated 3100.5.
EXAMPLE 130
Preparation of Peptide Having SEQ ID NO. 135









Ala Gly Asp Gly Thr Phe Thr Ser
[SEQ. ID. NO. 135]



Asp Leu Ser Lys Gln Ala Glu Glu


Glu Ala Val Arg Leu Phe Ile Glu


Trp Leu Lys Asn-NH2






The above-identified amidated peptide is assembled on 4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Example 95. Used in analysis are Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide is then carried out to determine the retention time of the product peptide. Electrospray Mass Spectrometry (M): calculated 3154.5.
EXAMPLE 131
Preparation of Peptide Having SEQ ID NO. 136









Ala Gly Asp Gly Thr Phe Thr Ser
[SEQ. ID. NO. 136]



Asp Leu Ser Lys Gln Ala Glu Glu


Glu Ala Val Arg Leu Phe Ile Glu


Phe Leu Lys Asn-NH2






The above-identified amidated peptide is assembled on 4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Example 95. Used in analysis are Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent E in Solvent A over 30 minutes) of the lyophilized peptide is then carried out to determine the retention time of the product peptide. Electrospray Mass Spectrometry (M): calculated 3115.5.
EXAMPLE 132
Preparation of Peptide Having SEQ ID NO. 137









Ala Gly Asp Gly Thr Phe Thr Ser
[SEQ. ID. NO. 137]



Asp Leu Ser Lys Gln Pentylgly


Glu Glu Glu Ala Val Arg Leu Phe


Ile Glu Trp Leu Lys Asn-NH2






The above-identified amidated peptide is assembled on 4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Example 95. Used in analysis are Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide is then carried out to determine the retention time of the product peptide. Electrospray Mass Spectrometry (M): calculated 3212.4.
EXAMPLE 133
Preparation of Peptide Having SEQ ID NO. 138









Ala Gly Asp Gly Thr Phe Thr Ser
[SEQ. ID. NO. 138]



Asp Leu Ser Lys Gln Pentylgly


Glu Glu Glu Ala Val Arg Leu Phe


Ile Glu Phe Leu Lys Asn-NH2






The above-identified amidated peptide is assembled on 4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Example 95. Used in analysis are Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide is then carried out to determine the retention time of the product peptide. Electrospray Mass Spectrometry (M): calculated 3173.4.
EXAMPLE 134
Preparation of Peptide Having SEQ ID NO. 139









Ala Gly Asp Gly Thr Phe Thr Ser
[SEQ. ID. NO. 139]



Asp Leu Ser Lys Gln Met Ala Glu


Glu Ala Val Arg Leu Phe Ile Glu


Trp Leu Lys Asn-NH2






The above-identified amidated peptide is assembled on 4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Example 95. Used in analysis are Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide is then carried out to determine the retention time of the product peptide. Electrospray Mass Spectrometry (M): calculated 3156.6.
EXAMPLE 135
Preparation of Peptide Having SEQ ID NO. 140









Ala Gly Asp Gly Thr Phe Thr Ser
[SEQ. ID. NO. 140]



Asp Leu Ser Lys Gln Leu Ala Glu


Glu Ala Val Arg Leu Phe Ile Glu


Phe Leu Lys Asn-NH2






The above-identified amidated peptide is assembled on 4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Example 95. Used in analysis are Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide is then carried out to determine the retention time of the product peptide. Electrospray Mass Spectrometry (M): calculated 3099.5.
EXAMPLE 136
Preparation of Peptide Having SEQ ID NO. 141









Ala Gly Asp Gly Thr Phe Thr Ser
[SEQ. ID. NO. 141]



Asp Leu Ser Lys Gln Met Glu Ala


Glu Ala Val Arg Leu Phe Ile Glu


Trp Leu Lys Asn-NH2






The above-identified amidated peptide is assembled on 4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Example 95. Used in analysis are Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide is then carried out to determine the retention time of the product peptide. Electrospray Mass Spectrometry (M): calculated 3156.6.
EXAMPLE 137
Preparation of Peptide Having SEQ ID NO. 142









Ala Gly Asp Gly Thr Phe Thr Ser
[SEQ. ID. NO. 142]



Asp Leu Ser Lys Gln Leu Glu Ala


Glu Ala Val Arg Leu Phe Ile Glu


Phe Leu Lys Asn-NH2






The above-identified amidated peptide is assembled on 4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Example 95. Used in analysis are Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA in ACN) Analytical RP-HPLC (gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide is then carried out to determine the retention time of the product peptide. Electrospray Mass Spectrometry (M): calculated 3099.5.
EXAMPLE 138
Preparation of Peptide Having SEQ ID NO. 143









Ala Gly Asp Gly Thr Phe Thr Ser
[SEQ. ID. NO. 143]



Asp Leu Ser Lys Gln Met Glu Glu


Ala Ala Val Arg Leu Phe Ile Glu


Trp Leu Lys Asn-NH2






The above-identified amidated peptide is assembled on 4-(2′-4′-dimethoxyphenyl).-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Example 95. Used in analysis are Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-IIPLC (gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide is then carried out to determine the retention time of the product peptide. Electrospray Mass Spectrometry (M): calculated 3156.6.
EXAMPLE 139
Preparation of Peptide Having SEQ ID NO. 144









Ala Gly Asp Gly Thr Phe Thr Ser
[SEQ. ID. NO. 144]



Asp Leu Ser Lys Gln Leu Glu Glu


Ala Ala Val Arg Leu Phe Ile Glu


Phe Leu Lys Asn-NH2






The above-identified amidated peptide is assembled on 4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Example 95. Used in analysis are Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide is then carried out to determine the retention time of the product peptide. Electrospray Mass Spectrometry (M): calculated 3099.5.
EXAMPLE 140
Preparation of Peptide Having SEQ ID NO. 145









Ala Gly Asp Gly Thr Phe Thr Ser
[SEQ. ID. NO. 145]



Asp Leu Ser Lys Gln Met Glu Glu


Glu Ala Ala Arg Leu Phe Ile Glu


Trp Leu Lys Asn-NH2






The above-identified amidated peptide is assembled on 4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Example 95. Used in analysis are Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide is then carried out to determine the retention time of the product peptide. Electrospray Mass Spectrometry (M): calculated 3186.6.
EXAMPLE 141
Preparation of Peptide Having SEQ ID NO. 146









Ala Gly Asp Gly Thr Phe Thr Ser
[SEQ. ID. NO. 146]



Asp Leu Ser Lys Gln Leu Glu Glu


Glu Ala Ala Arg Leu Phe Ile Glu


Phe Leu Lys Asn-NH2






The above-identified amidated peptide is assembled on 4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Example 95. Used in analysis are Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide is then carried out to determine the retention time of the product peptide. Electrospray Mass Spectrometry (M): calculated 3129.5.
EXAMPLE 142
Preparation of Peptide Having SEQ ID NO. 147









Ala Gly Asp Gly Thr Phe Thr Ser
[SEQ. ID. NO. 147]



Asp Leu Ser Lys Gln Met Glu Glu


Glu Ala Val Ala Leu Phe Ile Glu


Trp Leu Lys Asn-NH2






The above-identified amidated peptide is assembled on 4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Example 95. Used in analysis are Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide is then carried out to determine the retention time of the product peptide. Electrospray Mass Spectrometry (M): calculated 3129.5.
EXAMPLE 143
Preparation of Peptide Having SEQ ID NO. 148









Ala Gly Asp Gly Thr Phe Thr Ser
[SEQ. ID. NO. 148]



Asp Leu Ser Lys Gln Leu Glu Glu


Glu Ala Val Ala Leu Phe Ile Glu


Phe Leu Lys Asn-NH2






The above-identified amidated peptide is assembled on 4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Example 95. Used in analysis are Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide is then carried out to determine the retention time of the product peptide. Electrospray Mass Spectrometry (M): calculated 3072.4.
EXAMPLE 144
Preparation of Peptide Having SEQ ID NO. 149









Ala Gly Asp Gly Thr Phe Thr Ser
[SEQ. ID. NO. 149]



Asp Leu Ser Lys Gln Met Glu Glu


Glu Ala Val Arg Ala Phe Ile Glu


Trp Leu Lys Asn-NH2






The above-identified amidated peptide is assembled on 4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Example 95. Used in analysis are Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide is then carried out to determine the retention time of the product peptide. Electrospray Mass Spectrometry (M): calculated 3172.5.
EXAMPLE 145
Preparation of Peptide Having SEQ ID NO. 150









Ala Gly Asp Gly Thr Phe Thr Ser
[SEQ. ID. NO. 150]



Asp Leu Ser Lys Gln Leu Glu Glu


Glu Ala Val Arg Ala Phe Ile Glu


Phe Leu Lys Asn-NH2






The above-identified amidated peptide is assembled on 4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Example 95. Used in analysis are Solvent R (0.1% TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide is then carried out to determine the retention time of the product peptide. Electrospray Mass Spectrometry (M): calculated 3115.5.
EXAMPLE 146
Preparation of Peptide Having SEQ ID NO. 151









Ala Gly Asp Gly Thr Phe Thr Ser
[SEQ. ID. NO. 151]



Asp Leu Ser Lys Gln Met Glu Glu


Glu Ala Val Arg Leu Naphthylala


Ile Glu Trp Leu Lys Asn-NH2






The above-identified amidated peptide is assembled on 4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Example 95. Used in analysis are Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide is then carried out to determine the retention time of the product peptide. Electrospray Mass Spectrometry (M): calculated 3266.4.
EXAMPLE 147
Preparation of Peptide Having SEQ ID NO. 152









Ala Gly Asp Gly Thr Phe Thr Ser
[SEQ. ID. NO. 152]



Asp Leu Ser Lys Gln Leu Glu Glu


Glu Ala Val Arg Leu Naphthylala


Ile Glu Phe Leu Lys Asn-NH2






The above-identified amidated peptide is assembled on 4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Example 95. Used in analysis are Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide is then carried out to determine the retention time of the product peptide. Electrospray Mass Spectrometry (M): calculated 3209.4.
EXAMPLE 148
Preparation of Peptide Having SEQ ID NO. 153









Ala Gly Asp Gly Thr Phe Thr Ser
[SEQ. ID. NO. 153]



Asp Leu Ser Lys Gln Met Glu Glu


Glu Ala Val Arg Leu Phe Val Glu


Trp Leu Lys Asn-NH2






The above-identified amidated peptide is assembled on 4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Example 95. Used in analysis are Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide is then carried out to determine the retention time of the product peptide. Electrospray Mass Spectrometry (M): calculated 3200. 6.
EXAMPLE 149
Preparation of Peptide Having SEQ ID NO. 154









Ala Gly Asp Gly Thr Phe Thr Ser
[SEQ. ID. NO. 154]



Asp Leu Ser Lys Gln Leu Glu Glu


Glu Ala Val Arg Leu Phe Val Glu


Phe Leu Lys Asn-NH2






The above-identified amidated peptide is assembled on 4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Example 95. Used in analysis are Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide is then carried out to determine the retention time of the product peptide. Electrospray Mass Spectrometry (M): calculated 3143.5.
EXAMPLE 150
Preparation of Peptide Having SEQ ID NO. 155









Ala Gly Asp Gly Thr Phe Thr Ser
[SEQ. ID. NO. 155]



Asp Leu Ser Lys Gln Met Glu Glu


Glu Ala Val Arg Leu Phe


tButylgly Glu Trp Leu Lys


Asn-NH2






The above-identified amidated peptide is assembled on 4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Example 95. Used in analysis are Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide is then carried out to determine the retention time of the product peptide. Electrospray Mass Spectrometry (M): calculated 3216.5.
EXAMPLE 151
Preparation of Peptide Having SEQ ID NO. 156









Ala Gly Asp Gly Thr Phe Thr Ser
[SEQ. ID. NO. 156]



Asp Leu Ser Lys Gln Leu Glu Glu


Glu Ala Val Arg Leu Phe


tButylgly Glu Phe Leu Lys


Asn-NH2






The above-identified amidated peptide is assembled on 4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Example 95. Used in analysis are Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide is then carried out to determine the retention time of the product peptide. Electrospray Mass Spectrometry (M): calculated 3159.4.
EXAMPLE 152
Preparation of Peptide Having SEQ ID NO. 157









Ala Gly Asp Gly Thr Phe Thr Ser
[SEQ. ID. NO. 157]



Asp Leu Ser Lys Gln Met Glu Glu


Glu Ala Val Arg Leu Phe Ile Asp


Trp Leu Lys Asn-NH2






The above-identified amidated peptide is assembled on 4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Example 95. Used in analysis are Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide is then carried out to determine the retention time of the product peptide. Electrospray Mass Spectrometry (M): calculated 3200.6.
EXAMPLE 153
Preparation of Peptide Having SEQ ID NO. 158









Ala Gly Asp Gly Thr Phe Thr Ser
[SEQ. ID. NO. 158]



Asp Leu Ser Lys Gln Leu Glu Glu


Glu Ala Val Arg Leu Phe Ile Asp


Phe Leu Lys Asn-NH2






The above-identified amidated peptide is assembled on 4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Example 95. Used in analysis are Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide is then carried out to determine the retention time of the product peptide. Electrospray Mass Spectrometry (M): calculated 3143.5.
EXAMPLE 154
Preparation of Peptide Having SEQ ID NO. 159









Ala Gly Asp Gly Thr Phe Thr Ser
[SEQ. ID. NO. 159]



Asp Leu Ser Lys Gln Met Glu Glu


Glu Ala Val Arg Leu Phe Ile Glu


Ala Leu Lys Asn-NH2






The above-identified amidated peptide is assembled on 4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Example 95. Used in analysis are Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide is then carried out to determine the retention time of the product peptide. Electrospray Mass Spectrometry (M): calculated 3099.5.
EXAMPLE 155
Preparation of Peptide Having SEQ ID NO. 160









Ala Gly Asp Gly Thr Phe Thr Ser
[SEQ. ID. NO. 160]



Asp Leu Ser Lys Gln Leu Glu Glu


Glu Ala Val Arg Leu Phe Ile Glu


Ala Leu Lys Asn-NH2






The above-identified amidated peptide is assembled on 4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Example 95. Used in analysis are Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide is then carried out to determine the retention time of the product peptide. Electrospray Mass Spectrometry (M): calculated 3081.4.
EXAMPLE 156
Preparation of Peptide Having SEQ ID NO. 161









Ala Gly Asp Gly Thr Phe Thr Ser
[SEQ. ID. NO. 161]



Asp Leu Ser Lys Gln Met Glu Glu


Glu Ala Val Arg Leu Phe Ile Glu


Trp Ala Lys Asn-NH2






The above-identified amidated peptide is assembled on 4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Example 95. Used in analysis are Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide is then carried out to determine the retention time of the product peptide. Electrospray Mass Spectrometry (M): calculated 3172.5.
EXAMPLE 157
Preparation of Peptide Having SEQ ID NO. 162









Ala Gly Asp Gly Thr Phe Thr Ser
[SEQ. ID. NO. 162]



Asp Leu Ser Lys Glu Leu Glu Glu


Glu Ala Val Arg Leu Phe Ile Glu


Phe Ala Lys Asn-NH2






The above-identified amidated peptide is assembled on 4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Example 95. Used in analysis are Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide is then carried out to determine the retention time of the product peptide. Electrospray Mass Spectrometry (M): calculated 3115.5.
EXAMPLE 158
Preparation of Peptide Having SEQ ID NO. 163









Ala Gly Asp Gly Thr Phe Thr Ser
[SEQ. ID. NO. 163]



Asp Leu Ser Lys Gln Met Glu Glu


Glu Ala Val Arg Leu Phe Ile Glu


Trp Leu Ala Asn-NH2






The above-identified amidated peptide is assembled on 4-(2′-4′-dimethoxyphenyl) -Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Example 95. Used in analysis are Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide is then carried out to determine the retention time of the product peptide. Electrospray Mass Spectrometry (M): calculated 3157.5.
EXAMPLE 159
Preparation of Peptide Having SEQ ID NO. 164









Ala Gly Asp Gly Thr Phe Thr Ser
[SEQ. ID. NO. 164]



Asp Leu Ser Lys Gln Leu Glu Glu


Glu Ala Val Arg Leu Phe Ile Glu


Phe Leu Ala Asn-NH2






The above-identified amidated peptide is assembled on 4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Example 95. Used in analysis are Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide is then carried out to determine the retention time of the product peptide. Electrospray Mass Spectrometry (M): calculated 3100.4.
EXAMPLE 160
Preparation of Peptide Having SEQ ID NO. 165









Ala Gly Asp Gly Thr Phe Thr Ser
[SEQ. ID. NO. 165]



Asp Leu Ser Lys Gln Met Glu Glu


Glu Ala Val Arg Leu Phe Ile Glu


Trp Leu Lys Ala-NH2






The above-identified amidated peptide is assembled on 4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Example 95. Used in analysis are Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide is then carried out to determine the retention time of the product peptide. Electrospray Mass Spectrometry (M): calculated 3171.6.
EXAMPLE 161
Preparation of Peptide Having SEQ ID NO. 166









Ala Gly Asp Gly Thr Phe Thr Ser
[SEQ. ID. NO. 166]



Asp Leu Ser Lys Gln Leu Glu Glu


Glu Ala Val Arg Leu Phe Ile Glu


Phe Leu Lys Ala-NH2






The above-identified amidated peptide is assembled on 4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Example 95. Used in analysis are Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide is then carried out to determine the retention time of the produce peptide. Electrospray Mass Spectrometry (M): calculated 3114.5.
EXAMPLE 162
Preparation of Peptide Having SEQ ID NO. 167









Ala Gly Glu Gly Thr Phe Thr Ser
[SEQ. ID. NO. 167]



Asp Leu Ser Lys Gln Met Glu Glu


Glu Ala Val Arg Leu Phe Ile Glu


Trp Leu Lys Asn Gly Gly Pro Ser


Ser Gly Ala Pro Pro Pro-NH2






The above-identified amidated peptide is assembled on 4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Example 95. Used in analysis are Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA in ACN) Analytical RP-HPLC (gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide is then carried out to determine the retention time of the product peptide. Electrospray Mass Spectrometry (M): calculated 4033.5.
EXAMPLE 163
Preparation of Peptide Having SEQ ID NO. 168









His Gly Ala Gly Thr Phe Thr Ser
[SEQ. ID. NO. 168]



Asp Leu Ser Lys Gln Leu Glu Glu


Glu Ala Val Arg Leu Phe Ile Glu


Phe Leu Lys Asn Gly Gly Pro Ser


Ser Gly Ala Pro Pro Pro-NH2






The above-identified amidated peptide is assembled on 4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Example 95. Used in analysis are Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide is then carried out to determine the retention time of the product peptide. Electrospray Mass Spectrometry (M): calculated 3984.4.
EXAMPLE 164
Preparation of Peptide Having SEQ ID NO. 169









His Gly Glu Ala Thr Phe Thr Ser
[SEQ. ID. NO. 169]



Asp Leu Ser Lys Gln Met Glu Glu


Glu Ala Val Arg Leu Phe Ile Glu


Trp Leu Lys Asn Gly Gly Pro Ser


Ser Gly Ala Pro Pro-NH2






The above-identified amidated peptide is assembled on 4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Example 95. Used in analysis are Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide is then carried out to determine the retention time of the product peptide. Electrospray Mass Spectrometry (M): calculated 4016.5.
EXAMPLE 165
Preparation of Peptide Having SEQ ID NO. 170









His Gly Glu Gly Thr Phe Thr Ser
[SEQ. ID. NO. 170]



Ala Leu Ser Lys Gln Met Glu Glu


Glu Ala Val Arg Leu Phe Ile Glu


Trp Leu Lys Asn Gly Gly Pro Ser


Ser Gly Ala Pro-NH2






The above-identified amidated peptide is assembled on 4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Example 95. Used in analysis are Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide is then carried out to determine the retention time of the product peptide. Electrospray Mass Spectrometry (M): calculated 3861.3.
EXAMPLE 166
Preparation of Peptide Having SEQ ID NO. 171









Ala Gly Glu Gly Thr Phe Thr Ser
[SEQ. ID. NO. 171]



Asp Ala Ser Lys Gln Leu Glu Glu


Glu Ala Val Arg Leu Phe Ile Glu


Phe Leu Lys Asn Gly Gly Pro Ser


Ser Gly Ala Pro-NH2






The above-identified amidated peptide is assembled on 4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Example 95. Used in analysis are Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide is then carried out to determine the retention time of the product peptide. Electrospray Mass Spectrometry (M): calculated 3746.1.
EXAMPLE 167
Preparation of Peptide Having SEQ ID NO. 172









Ala Gly Glu Gly Thr Phe Thr Ser
[SEQ. ID. NO. 172]



Asp Leu Ser Lys Gln Met Glu Glu


Glu Ala Val Arg Leu Phe Ile Glu


Trp Leu Lys Asn Gly Gly Pro Ser


Ser Gly Ala-NH2






The above-identified amidated peptide is assembled on 4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Example 95. Used in analysis are Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide is then carried out to determine the retention time of the product peptide. Electrospray Mass Spectrometry (M): calculated 3142.1.
EXAMPLE 168
Preparation of Peptide Having SEQ ID NO. 173









His Gly Ala Gly Thr Phe Thr Ser
[SEQ. ID. NO. 173]



Asp Leu Ser Lys Gln Leu Glu Glu


Glu Ala Val Arg Leu Phe Ile Glu


Phe Leu Lys Asn Gly Gly Pro Ser


Ser Gly Ala-NH2






The above-identified amidated peptide is assembled on 4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Example 95. Used in analysis are Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide is then carried out to determine the retention time of the product peptide. Electrospray Mass Spectrometry (M): calculated 3693.1.
EXAMPLE 169
Preparation of Peptide Having SEQ ID NO. 174









His Gly Glu Ala Thr Phe Thr Ser
[SEQ. ID. NO. 174]



Asp Leu Ser Lys Gln Met Glu Glu


Glu Ala Val Arg Leu Phe Ile Glu


Trp Leu Lys Asn Gly Gly Pro Ser


Ser Gly-NH2






The above-identified amidated peptide is assembled on 4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Example 95. Used in analysis are Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide is then carried out to determine the retention time of the product peptide. Electrospray Mass Spectrometry (M): calculated 3751.2.
EXAMPLE 170
Preparation of Peptide Having SEQ ID NO. 175









His Gly Glu Gly Thr Phe Thr Ser
[SEQ. ID. NO. 175]



Ala Leu Ser Lys Gln Met Glu Glu


Glu Ala Val Arg Leu Phe Ile Glu


Trp Leu Lys Asn Gly Gly Pro Ser


Ser-NH2






The above-identified amidated peptide is assembled on 4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Example 95. Used in analysis are Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide is then carried out to determine the retention time of the product peptide. Electrospray Mass Spectrometry (M): calculated 3634.1.
EXAMPLE 171
Preparation of Peptide Having SEQ ID NO. 176









Ala Gly Glu Gly Thr Phe Thr Ser
[SEQ. ID. NO. 176]



Asp Leu Ser Lys Gln Met Glu Glu


Glu Ala Val Arg Leu Phe Ile Glu


Trp Leu Lys Asn Gly Gly Pro


Ser-NH2






The above-identified amidated peptide is assembled on 4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Example 95. Used in analysis are Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide is then carried out to determine the retention time of the product peptide. Electrospray Mass Spectrometry (M): calculated 3526.9.
EXAMPLE 172
Preparation of Peptide Having SEQ ID NO. 177









His Gly Ala Gly Thr Phe Thr Ser
[SEQ. ID. NO. 177]



Asp Leu Ser Lys Gln Leu Glu Glu


Glu Ala Val Arg Leu Phe Ile Glu


Phe Leu Lys Asn Gly Gly Pro


Ser-NH2






The above-identified amidated peptide is assembled on 4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Example 95. Used in analysis are Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide is then carried out to determine the retention time of the product peptide. Electrospray Mass Spectrometry (M): calculated 3477.9.
EXAMPLE 173
Preparation of Peptide Having SEQ ID NO. 178









His Gly Glu Ala Thr Phe Thr Ser
[SEQ. ID. NO. 178]



Asp Leu Ser Lys Gln Met Glu Glu


Glu Ala Val Arg Leu Phe Ile Glu


Trp Leu Lys Asn Gly Gly Pro-NH2






The above-identified amidated peptide is assembled on 4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Example 95. Used in analysis are Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide is then carried out to determine the retention time of the product peptide. Electrospray Mass Spectrometry (M): calculated 3519.9.
EXAMPLE 174
Preparation of Peptide Having SEQ ID NO. 179









His Gly Glu Gly Thr Phe Thr Ser
[SEQ. ID. NO. 179]



Ala Leu Ser Lys Gln Leu Glu Glu


Glu Ala Val Arg Leu Phe Ile Glu


Phe Leu Lys Asn Gly Gly-NH2






The above-identified amidated peptide is assembled on 4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Example 95. Used in analysis are Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide is then carried out to determine the retention time of the product peptide. Electrospray Mass Spectrometry (M): calculated 3307.7.
EXAMPLE 175
Preparation of Peptide Having SEQ ID NO. 180









Ala Gly Glu Gly Thr Phe Thr Ser
[SEQ. ID. NO. 180]



Asp Leu Ser Lys Gln Leu Glu Glu


Glu Ala Val Arg Leu Phe Ile Glu


Phe Leu Lys Asn Gly-NH2






The above-identified amidated peptide is assembled on 4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Example 95. Used in analysis are Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide is then carried out to determine the retention time of the product peptide. Electrospray Mass Spectrometry (M): calculated 3186.5.
EXAMPLE 176
Preparation of Peptide Having SEQ ID NO. 181









His Gly Ala Gly Thr Phe Thr Ser
[SEQ. ID. NO. 181]



Asp Leu Ser Lys Gln Met Glu Glu


Glu Ala Val Arg Leu Phe Ile Glu


Trp Leu Lys Asn Gly Gly tPro Ser


Ser Gly Ala tPro tPro tPro-NH2






The above-identified amidated peptide is assembled on 4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Example 95. Double couplings are required at residues 37,36 and 31. Used in analysis are Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide is then carried out to determine the retention time of the product peptide. Electrospray Mass Spectrometry (M): calculated 4121.1.
EXAMPLE 177
Preparation of Peptide Having SEQ ID NO. 182









His Gly Glu Ala Thr Phe Thr Ser
[SEQ. ID. NO. 182]



Asp Leu Ser Lys Gln Met Glu Glu


Glu Ala Val Arg Leu Phe Ile Glu


Trp Leu Lys Asn Gly Gly Pro Ser


Ser Gly Ala tPro tPro tPro-NH2.






The above-identified amidated peptide is assembled on 4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Example 95. Double couplings are required at residues 37, 36 and 31. Used in analysis are Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide is then carried out to determine the retention time of the product peptide. Electrospray Mass Spectrometry (M): calculated 4173.2.
EXAMPLE 178
Preparation of Peptide Having SEQ ID NO. 183









His Gly Glu Gly Thr Phe Thr Ser
[SEQ. ID. NO. 183]



Ala Leu Ser Lys Gln Met Glu Glu


Glu Ala Val Arg Leu Phe Ile Glu


Trp Leu Lys Asn Gly Gly NMeala


Ser Ser Gly Ala NMeala


NMeala-NH2






The above-identified amidated peptide is assembled on 4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Compound 1. Double couplings are required at residues 36 and 31. Used in analysis are Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide is then carried out to determine the retention time of the product peptide. Electrospray Mass Spectrometry (M): calculated 3796.1.
EXAMPLE 179
Preparation of Peptide Having SEQ ID NO. 184









Ala Gly Glu Gly Thr Phe Thr Ser
[SEQ. ID. NO. 184]



Asp Leu Ser Lys Gln Met Glu Glu


Glu Ala Val Arg Leu Phe Ile Glu


Trp Leu Lys Asn Gly Gly hPro Ser


Ser Gly Ala hPro-NH2






The above-identified amidated peptide is assembled on 4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin. (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Example 95. A double coupling is required at residue 31. Used in analysis are Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide is then carried out to determine the retention time of the product peptide. Electrospray Mass Spectrometry (M): calculated 3871.1.
EXAMPLE 180
Preparation of Peptide Having SEQ ID NO. 185









His Gly Ala Gly Thr Phe Thr Ser
[SEQ. ID. NO. 185]



Asp Leu Ser Lys Gln Met Glu Glu


Glu Ala Val Arg Leu Phe Ile Glu


Trp Leu Lys Asn Gly Gly Pro Ser


Ser Gly Ala-NH2






The above-identified amidated peptide is assembled on 4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Example 95. Used in analysis are Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide is then carried out to determine the retention time of the product peptide. Electrospray Mass Spectrometry (M): calculated 3750.2.
EXAMPLE 181
Preparation of Peptide Having SEQ ID NO. 186









His Gly Asp Ala Thr Phe Thr Ser
[SEQ. ID. NO. 186]



Asp Leu Ser Lys Gln Met Glu Glu


Glu Ala Val Arg Leu Phe Ile Glu


Trp Leu Lys Asn Gly Gly-NH2






The above-identified amidated peptide is assembled on 4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Example 95. Used in analysis are Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide is then carried out to determine the retention time of the product peptide. Electrospray Mass Spectrometry (M): calculated 3408.8.
EXAMPLE 182
Preparation of Peptide Having SEQ ID NO. 187









Ala Gly Glu Gly Thr Phe Thr Ser
[SEQ. ID. NO. 187]



Asp Leu Ser Lys Gln Met Glu Glu


Glu Ala Val Arg Leu Phe Ile Glu


Trp Leu Lys Asn Gly Gly Pro Ser


Ser Gly Ala Pro Pro Pro Ser-NH2






The above-identified amidated peptide is assembled on 4-(2′-4′-dimethoxyphenyl) -Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Example 95. Used in analysis are Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA in ACN) Analytical RP-HPLC (gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide is then carried out to determine the retention time of the product peptide. Electrospray Mass Spectrometry (M): calculated 4120.6.
EXAMPLE 183
Preparation of Peptide Having SEQ ID NO. 188









Ala Gly Ala Gly Thr Phe Thr Ser
[SEQ. ID. NO. 188]



Asp Leu Ser Lys Gln Leu Glu Glu


Glu Ala Val Arg Leu Phe Ile Glu


Phe Leu Lys Asn Gly Gly Pro Ser


Ser Gly Ala Pro Pro Pro Ser-NH2






The above-identified amidated peptide is assembled on 4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Example 95. Used in analysis are Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide is then carried out to determine the retention time of the product peptide. Electrospray Mass Spectrometry (M): calculated 4005.5.
EXAMPLE 184
Preparation of C-Terminal Carboxylic Acid Peptides Corresponding to the above C-Terminal Amide Sequences for Peptides having SEQ ID NOS. 100-166, 172-177, 179-180 and 185-188.
C-terminal carboxylic acid peptides corresponding to amidated having SEQ ID NOS. 100-166, 172-177, 179-180 and 185-188 are assembled on the so called Wang resin (p-alkoxybenzylalacohol resin (Bachem, 0.54 mmole/g)) using Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to that described in Example 95. Used in analysis are Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide is then carried out to determine the retention time of the product peptide. Electrospray Mass Spectrometry provides an experimentally determined (M).
EXAMPLE 185
Preparation of C-Terminal Carboxylic Acid Peptides Corresponding to the above C-Terminal Amide Sequences for Peptides having SEQ ID NOS. 167-171, 178 and 181-184.
C-terminal carboxylic acid peptides corresponding to amidated SEQ ID NOS. 167-171, 178 and 181-184 are assembled on the 2-chlorotritylchloride resin (200-400 mesh), 2% DVB (Novabiochem, 0.4-1.0 mmole/g)) using Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to that described in Example 95. Used in analysis are Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide is then carried out to determine the retention time of the product peptide. Electrospray Mass Spectrometry provides an experimentally determined (M).
EXAMPLE 186
Evaluation of Ability to Cross Placenta
I. Introduction
The purpose of this experiment was to determine whether this exendin-4, when delivered to the maternal circulation, is transported across the placenta and is detectable in amniotic fluid or fetal blood.
II. Materials and Methods
Animals:
Female Harlan Sprague Dawley rats (age 12 weeks, 17-21 days pregnant, approximately 300 grams) were housed at 22.8+/−0.8° C. in a 12:12 hour light: dark cycle. All experiments were performed during the light cycle. Animals were given free access to food and water until the start of the experiment.
Sample Collection:
Rats were anesthetized with 5% halothane and then maintained with 2% halothane during the surgical procedures. Body temperature was measured and controlled using a thermistor probe/controller (Model 73A, YSI, Yellow Springs, OH) and a heated operating table. Blood was collected from the tail vein immediately prior to a subcutaneous injection of exendin-4 (AC2993 Amylin Pharmaceuticals, Inc.) or vehicle (100 μl 0.15M NaCl) at t=0. At t=30 minutes, when plasma concentrations following a subcutaneous injection have been found to be maximal, another blood sample was taken. Immediately thereafter, a midline laparotomy was made to expose the uterine horns. Fluid was collected from the individual amniotic sacs by aspiration through a 16 g needle into a syringe. The amniotic fluids from individual fetuses were pooled from a given rat, but fluids from each rat were kept separate. Fetal blood was collected by heart puncture with a 28 g microfine needle and aspirated into a syringe. Amniotic fluid and fetal blood samples were collected within 10 minutes of when the laparotomy was made (t=30-40 min.). All blood and fluid samples were centrifuged. The plasma or supernatant was stored at −70° C. until assayed.
Treatment Groups:
There were 2 treatment groups:
Group A: Rats receiving exendin-4 dissolved at 21 μg/100 μl in 0.15M NaCl n=4.
Group B: Rats receiving exendin-4 dissolved at 210 μg/100 μl in 0.15 M NaCl n=5.
III. Results
Exendin-4 was not detected in any of the baseline samples, taken at t=0, when measured by a specific IRMA (immuno-radio-metric-assay) which has a LLQ (low limit of quantitation) of 15 pM. At t=30 plasma levels of exendin-4 in the mother rats that received 21 μg exendin-4 were 16.47 nM±2.45. Values obtained from amniotic fluid (6.1±5.3 pM) and fetal blood (12.7±6.5 pM) were 2700-fold and 1300-fold less than those in plasma and were generally below the lower limit of quantitation of the assay (FIG. 2). Similar results were obtained with the rats receiving 210 μg exendin-4 where plasma levels in the mother rats at t=30 were 232.16 nM±63.45 (FIG. 3). Values obtained from amniotic fluid (18.3±9.3 pM) and fetal blood (16.9±13.8 pM) were 12,680-fold and 13,750-fold less than those in plasma and were undetectable in over half of the samples.
IV. Discussion
The placenta is the organ responsible for nutrient and waste exchange between the fetus and the mother. Maternal and fetal circulations are separated by an epithelial layer that allows or denies diffusion or carrier mediated transport of substances across the interface. The risk of adverse effects on the fetus can be related to the extent to which the drug enters the fetal circulation. The data obtained here indicate that, even with high injected doses, which may exceed the per-kilogram doses administered to humans by up to 3000-fold, little or no exendin-4 appeared in the fetal circulation or amniotic fluid. Six out of 15 measurements were above the lower limit of quantitation, and in 9 of 15, exendin-4 was undetectable. In those samples in which exendin-4 was measurable, its presence may have been due to contamination from maternal blood (which need be present only at 1:1,000-1:10,000 to be measurable). Such contamination is possible following laparotomy of the dam and puncture of the fetus.
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 fall within the scope of the following claims.

## Claims

1. A method for treating gestational diabetes mellitus in a human in need thereof comprising subcutaneously injecting the human with about 1 microgram to about 50 micrograms per day of a peptide having the amino acid sequence of SEQ ID NO. 3, SEQ ID NO. 4, or SEQ ID NO. 5 to treat the gestational diabetes mellitus in the human in need thereof; wherein the peptide does not have the amino acid sequence of SEQ ID NO. 2; and
wherein SEQ ID NO. 3 is:
Xaa1 Xaa2 Xaa3 Gly Thr Xaa4 Xaa5 Xaa6 Xaa7 Xaa8 Ser Lys Gln Xaa9 Glu Glu Glu Ala Val Arg Leu Xaa10 Xaa11 Xaa12 Xaa13 Leu Lys Asn Gly Gly Xaa14 Ser Ser Gly Ala Xaa15 Xaa16 Xaa17 Xaa18-Z;

wherein:
Xaa1 is His, Arg, or Tyr;
Xaa2 is Ser, Gly, Ala, or Thr;
Xaa3 is Asp or Glu;
Xaa4 is Phe, Tyr, or naphthylalanine;
Xaa5 is Thr or Ser;
Xaa6 is Ser or Thr;
Xaa7 is Asp or Glu;
Xaa8 is Leu, Ile, Val, pentylglycine, or Met;
Xaa9 is Leu, Ile, pentylglycine, Val, or Met;
Xaa10 is Phe, Tyr, or naphthylalanine;
Xaa11 is Ile, Val, Leu, pentylglycine, tert-butylglycine, or Met;
Xaa12 is Glu or Asp;
Xaa13 is Trp, Phe, Tyr, or naphthylalanine;
Xaa14, Xaa15, Xaa16 and Xaa17 are independently Pro, homoproline, 3Hyp, 4Hyp, thioproline, N-alkylglycine, N-alkylpentylglycine, or N-alkylalanine;
Xaa18 is Ser, Thr, or Try; and
Z is —OH or —NH2;

wherein SEQ ID NO. 4 is:
Xaa1 Xaa2 Xaa3 Gly Xaa5 Xaa6 Xaa7 Xaa8 Xaa9 Xaa10 Xaa11 Xaa12 Xaa13 Xaa14 Xaa15 Xaa16 Xaa17 Ala Xaa19 Xaa20 Xaa21 Xaa22 Xaa23 Xaa24 Xaa25 Xaa26 Xaa27 Xaa28-Z1;

wherein:
Xaa1 is His, Arg, or Tyr;
Xaa2 is Ser, Gly, Ala, or Thr;
Xaa3 is Asp or Glu;
Xaa5 is Ala or Thr;
Xaa6 is Ala, Phe, Tyr, or naphthylalanine;
Xaa7 is Thr or Ser;
Xaa8 is Ala, Ser, or Thr;
Xaa9 is Asp or Glu;
Xaa10 is Ala, Leu, Ile, Val, pentylglycine, or Met;
Xaa11 is Ala or Ser;
Xaa12 is Ala or Lys;
Xaa13 is Ala or Gln;
Xaa14 is Ala. Leu, Ile, pentylglycine, Val, or Met;
Xaa15 is Ala or Glu;
Xaa16 is Ala or Glu;
Xaa17 is Ala or Glu;
Xaa19 is Ala or Val;
Xaa20 is Ala or Arg;
Xaa21 is Ala or Leu;
Xaa22 is Ala, Phe, Tyr, or naphthylalanine;
Xaa23 is Ile, Val, Leu, pentylglycine, tert-butylglycine, or Met;
Xaa24 is Ala, Glu, or Asp;
Xaa25 is Ala, Trp, Phe, Tyr, or naphthylalanine;
Xaa26 is Ala or Leu;
Xaa27 is Ala or Lys;
Xaa28 is Ala or Asn;
Z1 is —OH,
 —NH2,
 Gly-Z2,
 Gly Gly-Z2,
 Gly Gly Xaa31 -Z2,
 Gly Gly Xaa31 Ser-Z2,
 Gly Gly Xaa31 Ser Ser-Z2,
 Gly Gly Xaa31 Ser Ser Gly-Z2,
 Gly Gly Xaa31 Ser Ser Gly Ala-Z2,
 Gly Gly Xaa31 Ser Ser Gly Ala Xaa36-Z2,
 Gly Gly Xaa31 Ser Ser Gly Ala Xaa36 Xaa37-Z2, or
 Gly Gly Xaa31 Ser Ser Gly Ala Xaa36 Xaa37Xaa38-Z2;
Xaa31, Xaa36, Xaa37 and Xaa38 are independently Pro. homoproline, 3Hyp, 4Hyp. thioproline, N-alkylglycine, N-alkylpentylglycine, or N-alkylalanine; and
Z2 is —OH or —NH2;



provided that no more than three of Xaa3, Xaa5, Xaa6, Xaa8, Xaa10, Xaa11, Xaa12, Xaa13, Xaa14, Xaa15, Xaa16, Xaa17, Xaa19, Xaa20, Xaa21, Xaa24, Xaa25, Xaa26, Xaa27 and Xaa28 are Ala; and wherein SEQ ID NO. 5 is:

Xaa1 Xaa2 Xaa3 Xaa4 Xaa5 Xaa6 Xaa7 Xaa8 Xaa9 Xaa10 Xaa11 Xaa12 Xaa13 Xaa14 Xaa15 Xaa16 Xaa17 Ala Xaa19 Xaa20 Xaa21 Xaa22 Xaa23 Xaa24 Xaa25 Xaa26, Xaa27Xaa28-Z1;

wherein:
Xaa1 is His. Arg, Tyr, Ala, Norval, Val, or Norleu;
Xaa2 is Ser, Gly, Ala, or Thr;
Xaa3 is Ala, Asp, or Glu;
Xaa4 is Ala, Norval, Val, Norleu, or Gly;
Xaa5 is Ala or Thr;
Xaa6 is Phe, Tyr, or naphthylalanine;
Xaa7 is Thr or Ser;
Xaa8 is Ala, Ser or Thr;
Xaa9 is Ala, Norval, Val. Norleu, Asp, or Glu;
Xaa10 is Ala, Leu, Ile, Val, pentylglycine, or Met;
Xaa11 is Ala or Ser;
Xaa12 is Ala or Lys;
Xaa13 is Ala or Gln;
Xaa14 is Ala, Leu. Ile, pentylglycine, Val, or Met;
Xaa15 is Ala or Glu;
Xaa16 is Ala or Glu;
Xaa17 is Ala or Glu;
Xaa19 is Ala or Val;
Xaa20 is Ala or Arg;
Xaa21 is Ala or Leu;
Xaa22 is Phe, Tyr, or naphthylalanine;
Xaa23 is Ile, Val, Leu, pentylglycine, tert-butylglycine, or Met;
Xaa24 is Ala, Glu, or Asp;
Xaa25 is Ala, Trp, Phe, Tyr, or naphthylalanine;
Xaa26 is Ala or Leu;
Xaa27 is Ala or Lys;
Xaa28 is Ala or Asn;
Z1 is —OH,
 —NH2 ,
 Gly-Z2,
 Gly Gly-Z2,
 Gly Gly Xaa31 -Z2,
 Gly Gly Xaa31 Ser-Z2,
 Gly Gly Xaa31 Ser Ser-Z2,
 Gly Gly Xaa31 Ser Ser Gly-Z2,
 Gly Gly Xaa31 Ser Ser Gly Ala-Z2,
 Gly Gly Xaa31 Ser Ser Gly Ala Xaa36-Z2,
 Gly Gly Xaa31 Ser Ser Gly Ala Xaa36 Xaa37-Z2,
 Gly Gly Xaa31 Ser Ser Gly Ala Xaa36 Xaa37 Xaa38-Z2, or
 Gly Gly Xaa31 Ser Ser Gly Ala Xaa36 Xaa37 Xaa38 Xaa39-Z2;
wherein Xaa31, Xaa36, Xaa37 and Xaa38 are independently Pro, homoproline, 3Hyp, 4Hyp, thioproline, N-alkylglycine, N-alkylpentylglycine, or N-alkylalanine; and
Z2 is —OH or —NH2;



provided that no more than three of Xaa3, Xaa4, Xaa5, Xaa6, Xaa8, Xaa9, Xaa10, Xaa11, Xaa12, Xaa13, Xaa14, Xaa15, Xaa16, Xaa17, Xaa19, Xaa20, Xaa21, Xaa24, Xaa25, Xaa26, Xaa27 and Xaa28 are Ala; and provided also that, if Xaa1 is His, Arg, or Tyr, then at least one of Xaa3, Xaa4 and Xaa9 is Ala.

2. The method of claim 1, wherein the peptide has the amino acid sequence of SEQ ID NO. 3.

3. The method of claim 1, further comprising administering (i) an amylin; (ii) an amylin agonist peptide; or (iii) an amylin agonist peptide.

4. The method of claim 2, wherein the peptide having the amino acid sequence of SEQ ID NO. 3 is a peptide having the amino acid sequence of SEQ ID NO. 26.

5. A method for treating gestational diabetes mellitus in a subject in need thereof comprising peripherally administering to the subject a therapeutically effective amount of a peptide having the amino acid sequence of SEQ ID NO. 3, SEQ ID NO. 4, or SEQ ID NO. 5 to treat the gestational diabetes mellitus in the subject in need thereof;
wherein the peptide does not have the amino acid sequence of SEQ ID NO. 2; and
wherein SEQ ID NO. 3 is:
Xaa1 Xaa2 Xaa3 Gly Thr Xaa4 Xaa5 Xaa6 Xaa7 Xaa8 Ser Lys Gln Xaa9 Glu Glu Glu Ala Val Arg Leu Xaa10 Xaa11 Xaa12 Xaa13 Leu Lys Asn Gly Gly Xaa4Ser Ser Gly Ala Xaa15 Xaa16 Xaa17 Xaa18-Z;

wherein:
Xaa1 is His, Arg, or Tyr;
Xaa2 is Ser, Gly, Ala, or Thr;
Xaa3 is Asp or Glu;
Xaa4 is Phe, Tyr, or naphthylalanine;
Xaa5 is Thr or Ser;
Xaa6 is Ser or Thr;
Xaa7 is Asp or Glu;
Xaa8 is Leu, Ile, Val, pentylglycine, or Met;
Xaa9 is Leu, Ile, pentylglycine, Val, or Met;
Xaa10 is Phe, Tyr, or naphthylalanine;
Xaa11 is Ile, Val, Leu, pentylglycine, tert-butylglycine, or Met;
Xaa12 is Glu or Asp;
Xaa13 is Trp, Phe, Tyr, or naphthylalanine;
Xaa14, Xaa15, Xaa16 and Xaa17 are independently Pro, homoproline, 3Hyp, 4Hyp, thioproline, N-alkylglycine, N-alkylpentylglycine, or N-alkylalanine;
Xaa18 is Ser, Thr, or Try; and
Z is —OH or —NH2;

wherein SEQ ID NO. 4 is:
Xaa1 Xaa2 Xaa3 Gly Xaa5 Xaa6 Xaa7 Xaa8 Xaa9 Xaa10 Xaa11 Xaa12 Xaa13 Xaa14 Xaa15 Xaa16 Xaa17 Ala Xaa19 Xaa20 Xaa21 Xaa22 Xaa23 Xaa24 Xaa25 Xaa26 Xaa27 Xaa28-Z1;

wherein:
Xaa1 is His, Arg, or Tyr;
Xaa2 is Ser, Gly, Ala, or Thr;
Xaa3 is Asp or Glu;
Xaa5 is Ala or Thr;
Xaa6 is Ala, Phe, Tyr, or naphthylalanine;
Xaa7 is Thr or Ser;
Xaa8 is Ala, Ser, or Thr;
Xaa9 is Asp or Glu;
Xaa10 is Ala, Leu, Ile, Val, pentylglycine, or Met;
Xaa11 is Ala or Ser;
Xaa12 is Ala or Lys;
Xaa13 is Ala or Gln;
Xaa14 is Ala, Leu, Ile, pentylglycine, Val, or Met;
Xaa15 is Ala or Glu;
Xaa16 is Ala or Glu;
Xaa17 is Ala or Glu;
Xaa19 is Ala or Val;
Xaa20 is Ala or Arg;
Xaa21 is Ala or Leu;
Xaa22 is Ala, Phe, Tyr, or naphthylalanine;
Xaa23 is Ile, Val, Leu, pentylglycine, tert-butylglycine, or Met;
Xaa24 is Ala, Glu, or Asp;
Xaa25 is Ala, Trp, Phe, Tyr, or naphthylalanine;
Xaa26 is Ala or Leu;
Xaa27 is Ala or Lys;
Xaa28 is Ala or Asn;
Z1 is —OH,
 —NH2,
 Gly-Z2,
 Gly Gly-Z2,
 Gly Gly Xaa31-Z2,
 Gly Gly Xaa31 Ser-Z2,
 Gly Gly Xaa31 Ser Ser-Z2,
 Gly Gly Xaa31 Ser Ser Gly-Z2,
 Gly Gly Xaa31 Ser Ser Gly Ala-Z2,
 Gly Gly Xaa31 Ser Ser Gly Ala Xaa36-Z2,
 Gly Gly Xaa31 Ser Ser Gly Ala Xaa36 Xaa37-Z2, or
 Gly Gly Xaa31 Ser Ser Gly Ala Xaa36 Xaa37Xaa38-Z2;
Xaa31, Xaa36, Xaa37 and Xaa38 are independently Pro, homoproline, 3Hyp, 4Hyp, thioproline, N-alkylglycine, N-alkylpentylglycine, or N-alkylalanine; and
Z2 is —OH or —NH2;



provided that no more than three of Xaa3, Xaa5, Xaa6, Xaa8, Xaa10, Xaa11, Xaa12, Xaa13, Xaa14, Xaa15, Xaa16, Xaa17, Xaa19, Xaa20, Xaa21, Xaa24, Xaa25, Xaa26, Xaa27 and Xaa28 are Ala; and wherein SEQ ID NO. 5 is:
Xaa1 Xaa2 Xaa3 Xaa4 Xaa5 Xaa6 Xaa7 Xaa8 Xaa9 Xaa10 Xaa11 Xaa12 Xaa13 Xaa14 Xaa15 Xaa16 Xaa17 Ala Xaa19 Xaa20 Xaa21 Xaa22 Xaa23 Xaa24 Xaa25 Xaa26, Xaa27 Xaa28-Z1;
wherein:
Xaa1 is His, Arg, Tyr, Ala, Norval, Val, or Norleu;
Xaa2 is Ser, Gly, Ala, or Thr;
Xaa3 is Ala, Asp, or Glu;
Xaa4 is Ala, Norval, Val, Norleu, or Gly;
Xaa5 is Ala or Thr;
Xaa6 is Phe, Tyr, or naphthylalanine;
Xaa7 is Thr or Ser;
Xaa8 is Ala, Ser or Thr;
Xaa9 is Ala, Norval, Val. Norleu, Asp, or Glu;
Xaa10 is Ala, Leu, Ile, Val, pentylglycine, or Met;
Xaa11 is Ala or Ser;
Xaa12 is Ala or Lys;
Xaa13 is Ala or Gln;
Xaa14 is Ala, Leu, Ile, pentylglycine, Val, or Met;
Xaa15 is Ala or Glu;
Xaa16 is Ala or Glu;
Xaa17 is Ala or Glu;
Xaa19 is Ala or Val;
Xaa20 is Ala or Arg;
Xaa21 is Ala or Leu;
Xaa22 is Phe, Tyr, or naphthylalanine;
Xaa23 is Ile, Val, Leu, pentylglycine, tert-butylglycine, or Met;
Xaa24 is Ala, Glu, or Asp;
Xaa25 is Ala, Trp, Phe, Tyr, or naphthylalanine;
Xaa26 is Ala or Leu;
Xaa27 is Ala or Lys;
Xaa28 is Ala or Asn;
Z1 is —OH,
 —NH2,
 Gly-Z2,
 Gly Gly-Z2,
 Gly Gly Xaa31-Z2,
 Gly Gly Xaa31 Ser-Z2,
 Gly Gly Xaa31 Ser Ser-Z2,
 Gly Gly Xaa31 Ser Ser Gly-Z2,
 Gly Gly Xaa31 Ser Ser Gly Ala-Z2,
 Gly Gly Xaa31 Ser Ser Gly Ala Xaa36-Z2,
 Gly Gly Xaa31 Ser Ser Gly Ala Xaa36 Xaa37-Z2,
 Gly Gly Xaa31 Ser Ser Gly Ala Xaa36 Xaa37 Xaa38-Z2, or
 Gly Gly Xaa31 Ser Ser Gly Ala Xaa36 Xaa37 Xaa38 Xaa39-Z2;
wherein Xaa31, Xaa36, Xaa37 and Xaa38 are independently Pro, homoproline, 3Hyp, 4Hyp, thioproline, N-alkylglycine, N-alkylpentylglycine, or N-alkvlalanine; and
Z2 is —OH or —NH2;



provided that no more than three of Xaa3, Xaa4, Xaa5, Xaa6, Xaa8, Xaa9, Xaa10, Xaa11, Xaa12, Xaa13, Xaa14, Xaa15, Xaa16, Xaa17, Xaa19, Xaa20, Xaa21, Xaa24, Xaa25, Xaa26, Xaa27 and Xaa28 are Ala; and provided also that, if Xaa1 is His, Arg, or Tyr, then at least one of Xaa3, Xaa4 and Xaa9 is Ala.

6. The method of claim 5, wherein the peptide has the amino acid sequence of SEQ ID NO. 3.

7. The method of claim 5, wherein the peptide is continuously administered.

8. The method of claim 5, wherein the administration is by injection.

9. The method of claim 8, wherein the injection is a subcutaneous injection.

10. The method of claim 5, wherein the therapeutically effective amount is 1 microgram to 1 milligram per day.

11. The method of claim 5, wherein the therapeutically effective amount is 1 microgram to 100 micrograms per day.

12. The method of claim 5, wherein the therapeutically effective amount is 1 microgram to 50 micrograms per day.

13. The method of claim 5, further comprising administering (i) an amylin; (ii) an amylin agonist peptide; or (iii) an amylin and an amylin agonist peptide.

14. The method of claim 5, wherein the subject is a human.

15. The method of claim 6, wherein the peptide having the amino acid sequence of SEQ ID NO. 3 is a peptide having the amino acid sequence of SEQ ID NO. 26.

16. The method of claim 1, wherein the peptide has the amino acid sequence of SEQ ID NO. 4.

17. The method of claim 1, wherein the peptide has the amino acid sequence of SEQ ID NO. 5.

18. The method of claim 5, wherein the peptide has the amino acid sequence of SEQ ID NO. 4.

19. The method of claim 5, wherein the peptide has the amino acid sequence of SEQ ID NO. 5.