HUT70176A - Amylin antagonists and agonists - Google Patents

Abstract

The invention features amylin analogs which behave as amylin antagonists and agonists. The invention also features the use of the amylin antagonist for the treatment of Type II diabetes mellitus, and the use of the amylin agonists for the treatment of both Type I diabetes mellitus and hypercalcemia. The invention also features the use of amylin antagonists and agonists for the control of food intake.

Description

The present invention relates to specific amylin analogues having amylin antagonist or agonist activity and their use in the treatment of diabetes mellitus and hypercalcaemia and in the regulation of food intake.

Amiline, which is a diabetes-related peptide [Cooper et al., Proc. Natl. Acad. Sci. USA 85: 7763-7766 (1988)] or the island / insulin amyloid polypeptide (Westermark et al., Proc. Natl. Acad. Sci. USA, 84, 3881-3885 (1987)], is a polypeptide amide of 37 amino acids which was originally isolated from amyloid-rich pancreas in patients with insulinoma and non-insulin dependent diabetes mellitus (NIDD). Subsequently, rats were also isolated from normal pancreas [Asai et al., Biochem. Biophys. Gap. Commun. 164: 400-405 (1989)]. cDNA cloning [Ferrier et al., J. Mol. Endocrinol. 3, R1-R4 (1989)] and immunocytochemical studies have shown that amylin is synthesized in island cells and stored in insular secretory granules together with insulin. It is excreted together with insulin [Kanatsuka et al., FEBS Lett. 259: 199-201 (1989). Small amounts of amylin have also been detected in the stomach, intestine, lungs and dorsal root ganglion [Asai et al., Biochem. Biophys. Gap. Commun. 169: 788-795 (1990); and Ferrier et al., supra].

Following the isolation of amylin, biological studies have shown that amylin inhibits basal and insulin-stimulated glucose uptake and glycogen in the maxillary muscle. (Leighton et al., Natur. 335, 632-635 (1988)). This peripheral insulin resistance by amylin has also been demonstrated in vivo by euglycemic glucose clamp assays in dogs (Sowa et al., Diabetologia 33: 118-120 (1990)) and in the rat (Molina et al., Diabetes 39: 260-265 (1990)). In addition, these studies in rats showed that amylin weakened the inhibition of hepatic glucose secretion by insulin (Molina et al., Supra). Based on the above observations and the discovery that amylin inhibits basal insulin secretion [Ohsawa et al., Biochem. Biophys. Gap. Commun. 160, 961-967 (1989)] suggested that amylin may play a role in glucose metastasis and in the pathophysiology of non-insulin-dependent diabetes mellitus (NIDDM), a type ΙΙ diabetes mellitus.

Diabetes mellitus is a metabolic disease characterized by chronic hyperglycaemia, i.e., elevated blood glucose levels. This disease affects a significant percentage of the population. There are two main types of diabetes mellitus, commonly referred to as type I and type ΙΙ diabetes. In patients with type 1 diabetes mellitus, the number of active S cells in the pancreas in Langerhans is reduced, resulting in low concentrations of both insulin and amylin (Cooper, Medical Hypothesis 26, 284-288 (1991)). Patients with type 1 diabetes mellitus who are treated with insulin often develop hypoglycaemia as an adverse reaction. In patients with type II diabetes mellitus amylin · · ·

- 4 concentrations higher. Patients with type 2 diabetes mellitus show variable resistance to the normal biological effects of insulin. Elevated levels of amylamine, termed hyperamilinemia, are involved in inducing insulin resistance in several model systems, such as the genetically obese LA / N-cp rat (Huang et al., Hypertension 19, i-101-1109 (1992)), genetically obese diabetic yellow mice (Gill et al., Life Sci 48: 703-7188 (1991)), dexamethasone-induced diabetic rats (Jamal et al., J. Endocrin. 126, 425-429 (1990)], streptozocin-induced diabetic rats (Inoue et al., Diabetes 41, 723-727 (1992)) and ventromedial hypothalamus in injured rats and Zucker rats (Tokuyama et al., Endocrinology 128, 2739-27 (1991)). )].

Other studies have shown that amylin, like calcitonin, has the ability to induce serum calcium lowering activity in rats in vivo and in cell culture systems [Datta et al., Biochem. Biophys. Gap. Commun. 162: 876-881 (1989)]. Amylin has also been shown to act as an anorectic agent (Balasubramaniam et al., Peptides 12, 919-924 (1991)).

The present invention relates to amylin analogs which act as amylin antagonists and agonists.

The present invention relates, on the one hand, to amylin analogs, which are linear analogs of biologically active amylin and have the following general formula:

Ri

R ₂ -X-a8-a9-A10-A11-A12_ _of 13_ _{in the} 14_ 15_ _to 16_ _to 17_ _to 18_ _of 19_ _{in the} 20_ 21_

-a ²² -a ²³ -yz where

X is a chain of 0 to 5 amino acids the N-terminus of which is attached to R ₁ and R ₂ ;

Y is a chain of 0 to 4 amino acids the C-terminus of which is attached to Z;

R 1 and R ₂ are each independently hydrogen, C 1 -C 12 alkyl (e.g. methyl), C 6 -C 18 aryl (e.g. phenyl, naphthaleneacetyl), C 1 -C 12 acyl (e.g. formyl, acetyl, and myristoyl), C 7-18 aralkyl (e.g. benzyl) or C 7-18 alkylaryl (e.g. p-methylphenyl);

The meaning of θθ

below,

At,

Thi,

Phe, Bth,

Pcp, N-Me-Thr, Aib or Anb;

Meaning of A9

Thr,

below,

ANB,

Aib, Ser,

N-Me-Ser or N-Me-Thr;

Save A10 j

Gin,

below,

Asn,

N-Me-Gln,

You are Gly, Nva, Aib

ANB;

The meaning of AH

Arg, homo-Arg, diethyl-homo-Arg, Lys-e-NH-R (wherein R is hydrogen, straight or branched C 1 -C 10 alkyl or aryl), Orn or Lys;

^I2 is Leu, Ile, Val, Aib, Anb or N-Me-Leu;

Al ³ is Ala, Nal, Thi, Phe, Bth, Pcp, N-Me-Thr, Aib or Anb;

¹⁴ is Asn, Ala, Gln, Gly, N-Me-Asn, Nva, Aib or Anb;

¹⁵ is Phe or an optionally substituted aromatic amino acid;

I ¹ is Leu, Ile, Val, Aib, Anb or N-Me-Leu;

A ¹⁷ is Val, Ile, Aib, Anb or N-Me-Val;

¹⁸ is His, Thr, 3-Me-His, 1-Me-His, β-pyrazolyl-alanine, N-Me-His, Arg, homo-Arg, diethyl-homo-Arg, Lys-e-NH-R (wherein R is hydrogen, straight or branched C 1 -C 10 alkyl, or

aryl), below, Aib, Anb or Orn; ³⁹ meaning Ser, Thr, N-Me-Ser, N-Me-Thr, Aib; ANB obsession below; _the 20th meaning Ser, Thr, N-Me-Ser, N-Me-Thr, Aib; ANB obsession below; _a 21st meaning Asn, below, Gin, Gly, N-Me-Asn, Aib; ANB obsession below; _a 22 meaning Asn, below, Gin, Gly, N-Me-Asn, Aib; ANB obsession

below;

²³ is Phe, an optionally substituted aromatic amino acid, Leu, Ile, Val, Aib, Anb, Ala or N-Me-Leu, and

Z is -NHR 3 or -OR 3 (wherein R 3 is hydrogen, C 1 -C 12 alkyl, C 7 -C 10 phenylalkyl, C 3 -C 20 alkenyl, C 3 -C 20 alkynyl, phenyl or naphthyl).

In preferred embodiments, the analogs are antagonists. In a very preferred embodiment, the amylin antagonist is human amylin 8-23. amino acids corresponding to the N-α-acetyl derivative of the amino acids containing a C-terminal amidated carboxyl group, hereinafter referred to as Ν-α-ac-human amyl (8-23) -NH 2, having the formula:

N-a-Ac-Ala-Thr-Gln-Arg-Leu-Ala-Asn-Phe-Leu-Val-His-Ser-Ser-Asn-Asn-Phe-NH 2 (SEQ ID NO: 1)

In another preferred embodiment, the amylin antagonist has the formula:

N-a-Ac-Ala-Thr-Gln-Arg-Leu-Ala-Asn-Phe-Leu-Val-Arg-Ser-Ser-Asn-Asn-Leu-NH2 (SEQ ID NO: 2)

On the other hand, the present invention relates to amylin analogs which are linear analogs of biologically active amylin and have the following general formula:

^R 1

R2-XA ¹ -A ² -A ³ -A ⁴ -A ⁵ -A ⁶ -A ⁷ -A ⁸ -A ⁹ -A ¹⁰ -A ¹¹ -A ¹² -A ¹³ -A ¹⁴ -A ¹⁵ -A ¹⁶ - ¹⁷ -A ¹⁸ -A ¹⁹ -A ²⁰ -A ²¹ -A ²² -A ²³ -YZ where

X is a chain of 0 to 5 amino acids the N-terminus of which is attached to R 1 and R 2;

Y is a chain of 0 to 4 amino acids the C-terminus of which is attached to Z;

R 1 and R 2 are each independently hydrogen, C 1 -C 12 alkyl, C 6 -C 18 aryl, C 1 -C 12 acyl, C 7 -C 18 aralkyl, or C 7 -C 18 alkylaryl;

A ¹ is Lys, Arg, homo-Arg, diethyl-homo-Arg, Lys-ε-ΝΉR (where R is hydrogen, straight or branched).

C 1-10 alkyl or aryl) or Orn;

In ^{Figure 2} meaning Cys or Anb; In ^{Figure 3} meaning Asn, Ala, Gin, Gly, N-Me-Asn, Aib, Anb or Nva; In ^{Figure 4} meaning Thr, Ser, N-Me-Ser, N-Me-Thr, Ala, Aib or ANB; DIG meaning Alá, Nal, Thi, Phe, Bth, Pcp, N-Me-Ala, Aib or Anb; ⁶ meaning Thr, Ser, N-Me-Ser, N-Me-Thr, Ala, Aib or

ANB;

A ⁷ is Cys or Anb;

^A8 is Ala, Nal, Thi, Phe, Bth, Pcp, N-Me-Ala, Aib, or Anb;

⁹ is Thr, Ser, N-Me-Ser, N-Me-Thr, Ala, Aib or Anb;

A ¹⁰ is Gln, Ala, Asn, N-Me-Gln, Gly, Nva, Aib or Anb;

A ¹¹ is Arg, homo-Arg, diethyl-homo-Arg, Lys-e-NH-R (where R is hydrogen, straight or branched C 1-10 alkyl or aryl group), or Orn;

¹² is Leu, Ile, Val, Aib, Anb or N-Me-Leu;

A ¹⁸ is Ala, Nal, Thi, Phe, Bth, Pcp, N-Me-Ala, Aib, or Anb;

¹⁴ is Asn, Ala, Gln, Gly, N-Me-Asn, Aib, Anb or

Nva ·

¹⁵ is Phe or optionally substituted aromatic a-

qualify acid;

¹⁶ is Leu, Ile, Val, Aib, Anb or N-Me-Leu;

A ¹⁷ is Val, Ile, Aib, Anb or N-Me-Val;

¹⁸ is His, Thr, 3-Me-His, 1-Me-His, S-pyrazolylalanine, N-Me-His, Arg, homo-Arg, diethyl-homo-Arg, Lys-e-NH-R (wherein R is hydrogen, straight or branched C 1 -C 10 alkyl or aryl);

group), Orn, below, You're Aib ANB; A19 meaning Ser, Thr, N-Me-Ser, N-Me-Thr, below, Aib obsession ANB; _the 20th meaning Ser, Thr, N-Me-Ser, N-Me-Thr, below, Aib obsession ANB; The ^21st meaning Asn, below, Gin, Gly, N-Me-Asn, Aib; ANB obsession Nva; The ²² meaning Asn, below, Gin, Gly, N-Me-Asn, Aib; ANB obsession

Nva;

²³ is Phe, an optionally substituted aromatic amino acid, Leu, Ile, Val, Aib, Anb, Ala or N-Me-Leu, and

Z is -NHR 3 or -OR 3 (wherein R 3 is hydrogen, C 1 -C 12 alkyl, C 7 -C 10 phenylalkyl, C 3 -C 20 alkenyl, C 3 -C 20 alkynyl, phenyl or naphthalene).

In a very preferred embodiment, the amylin analog is human amylin 1-23. which contains a C-terminally amidated carboxyl group, hereinafter referred to as human amyl (1-23) -NH2

Lys-Cys-Asn-Thr-Ala-Thr-Cys-Ala-Thr-Gln-Arg-Leu-Ala-Asn-Phe-Leu-Val-His-Ser-Ser-Asn-Asn-Phe-NH ₂ (3 SEQ ID NO:

In another very preferred embodiment, the amylin analog is rat amylin 1-23. amino acid containing a C-terminally amidated carboxyl group, hereinafter referred to as rat amylin (1-23) -NH ₂ , having the formula:

Lys-Cys-Asn-Thr-Ala-Thr-Cys-Ala-Thr-Gln-Arg-Leu-Ala-Asn-Phe-Leu-Val-His-Ser-Ser-Asn-Asn-Leu-NH 2 (4. SEQ ID NO:

In a more preferred embodiment, the amylin analog is rat amylin 1-23. corresponding to the α-amino-n-butyric acid substitution at the 2 and 7 position and the C-terminally amidated carboxylic group, hereinafter referred to as [Anb ² > ⁷ ] rat amylline (1-23) -NH ₂ is called, which has the formula:

Lys-Anb-Asn-Thr-Ala-Thr-Anb-Ala-Thr-Gln-Arg-Leu-Ala-Asn-Phe-Leu-Val-His-Ser-Ser-Asn-Asn-Leu-NH ₂ (5 SEQ ID NO:

The present invention further provides a method of treating type diabetes diabetes mellitus in man comprising administering to the subject an amylin antagonist of the invention in a therapeutically effective amount. In a very preferred embodiment of the method for treating type diabetes diabetes mellitus, Ν-α-ac-human amyl (8-23) -NH2 is added.

The present invention further relates to a method of treating Type I diabetes mellitus in man comprising administering to the patient a therapeutically effective amount of an amyl agonist of the invention. effective amount.

The present invention further relates to a method of treating hypercalcaemia comprising administering to a patient a therapeutically effective amount of an amylin agonist of the invention.

The compounds of the present invention have a very wide range of biological activities related to glucose metabolism, blood calcium levels and appetite. The amylin antagonists of the invention attenuate the inhibition of insulin-stimulated glucose uptake by amylin. As a result, the amylin antagonists of the present invention reduce hyperglycemia from increased amylin concentration associated with type II diabetes mellitus. The amylin agonists of the present invention inhibit insulin-stimulated glucose uptake, thereby increasing blood glucose levels. As a result, the amylin agonists of the invention are useful in reducing the hypoglycemia frequently associated with the treatment of type 1 diabetes mellitus with insulin. The amylin agonists of the present invention also lower serum calcium levels and are therefore useful in the treatment of hypercalcaemia. In addition, amylin agonists have an appetite suppressant effect, while amylin antagonists increase appetite. Amylin agonists and antagonists can therefore also be used to regulate food intake. For example, amylin agonists are useful in the treatment of obesity-related problems.

• ·" ··· "" ··"·"·· · • * * * · · " EE " · " *

Most of the compounds of the present invention are particularly preferred because they represent a truncated version of the natural amylin peptide. The shorter peptide sequence not only facilitates the synthesis and purification of the compounds, but also improves selectivity and reduces manufacturing operations and costs.

Other features and advantages of the invention will be apparent from the following description of preferred embodiments.

First, the figures are briefly described.

Figure 1 shows a comparison of the primary structure of human amylin (hAMILIN) and rat amylin (rAMILINN).

Figure 2 shows the effect of human amylin and Na-ac-human amylin (8-23) -NH ₂ , separately and in combination, on glucose uptake in C ₂ Cl ₂ muscle cells.

Figures 3a and 3b show saline, rat amylin, α-α-ac-human amylin (8-23) -NH ₂ , and rat Ν-α-ac-human amylin (8-23) -NH ₂ + rat effects of amylin on plasma glucose concentration and plasma insulin concentration in Spargue Dawley rats.

Figure 4 shows the in vitro effect of human amylin and human amylin (1-23) -NH ₂ , alone and in combination, on insulin-stimulated glucose uptake in C ₂ C ₃₂ muscle cells.

Figures 5a and 5b show the in vivo effect of saline, rat amylin, human amylin (1-23) -NH ₂ , and human amylin (1-23) -NH ₂ + rat plasma glucose and plasma insulin concentration in Spargue Dawley rats.

• · * · · · «« · * • «· ··· <* · • ·· ·» ·· · ««

- 13 Figure 6 shows the rat amylin (l-23) _-NH2 and rat amylin effects (1-23) _NH2 in vitro separately insulin stimulated glucose uptake [Anb ^{2 »7]} C ₂ Ci _{2 in} muscle cells.

Figure 7a and 7b show the saline, rat amylin, [Anb ^{2 »7]} rat amylin (1-23) -NH2 and [Anb ^{2» 7]} rat amylin (l-23) -NH2 + in rat amylin plasma glucose and plasma insulin concentrations in Spargue Dawley rats.

Structure

The sequence of naturally occurring human amylin (hAmilin) and rat amylin (rAmilin) is shown in Figure 1 (Balasubramaniam et al., Peptides 12, 919-924 (1991)). The sequence homology between the amylines from these two species is very high. It is hypothesized that the cysteine residues at positions 2 and 7 in the natural hAmilin and rAmilin, which are present in both species, form an internal disulfide bond, resulting in a ring structure.

The amylin analogs of the invention are hAmilin and rAmilin 8-23. biologically active fragments of amino acids thereof and derivatives thereof; and hAmilin and rAmilin 1-23. based on biologically active fragments of the amino acid residues thereof and derivatives thereof. In the formula of the amylin analogs, A ^x symbols and the like, and in the peptide sequence Ser, Leu and the like, denote amino acid residues in the peptide sequence. When the amino acid residue is optically active, the abbreviations refer to the L configuration unless explicitly referred to as the D isomer. All peptide sequences herein are conventionally represented, i.e., the N-terminal amino acid is on the left and the C-terminal amino acid is on the right. The short lines between two amino acid residues represent a peptide bond. At the carboxyl-terminus of the peptide, the -O R and -NHR substituents replace the -OH group in the carboxyl-terminal amino acid, whereby the C-terminal residues have the formula -HN-CH (R) -COOR and -HN-CH (R) - It will be CONHR. When the carboxyl-terminal substituent is -NH2, the peptide is in the carboxylic acid amide form.

For the sake of simplicity, both conventional and non-conventional abbreviations are used throughout the description of the invention for the various amino acids. These abbreviations, known to those skilled in the art, include:

Asp = D = aspartic acid

Bottom = A = Alanine

Arg = R = arginine

Asn = N = Asparagine

Cys = C = cysteine

Gly = G = glycine

Glu = E = glutamic acid

Gin = Q = Glutamine

His = H = Histidine

Ile = I = isoleucine

Leu = L = leucine

Lys = K = Lysine

Met = M = methionine

Phe = F = Phenylalanine * ···· · · * 4 «« · · · «·» · '·· ·· * · ·

- 15 Pro = Ρ = proline Ser = S = serine Thr = T = threonine Trp = W = tryptophan Tyr = Y = tyrosine Val = V = valine Orn = ornithine Nal = 2-naphthylalanine Nva = norvaline Thi = 2-thienyl -alanine Pcp = 4-chloro-phenylalanine Bth = 3-benzothienyl alanine Bip = 4,4 ¹ -biphenyl-alanine Tic = tetrahydroisoquinoline-3-carboxylic acid Aib = aminoisobutyric acid Anb = .alpha.-amino-n-butyric acid Dip = 2,2-diphenylalanine

The compounds of the invention may also be in the form of pharmaceutically acceptable salts. Preferred are pharmaceutically acceptable organic acids such as acetic acid, lactic acid, maleic acid, citric acid, malic acid, ascorbic acid, succinic acid, benzoic acid, salicylic acid, methanesulfonic acid, toluenesulfonic acid, trifluoroacetic acid or pamoic acid; and polymeric acids such as tannic acid or carboxymethylcellulose; and salts with inorganic acids such as hydrogen halides such as hydrochloric acid, sulfuric acid or phosphoric acid.

Analysis

The structure-activity relationship between amylin and amylin analogues was studied in both an in vitro model (using a mouse cell line of? 2? 12) and an in vivo model (using Spargue Dawley rats).

In in vitro assays, insulin stimulated glucose uptake of the C2C12 cell line in a dose-dependent manner and was attenuated by rat amylin (100 pmol / L). However, rat amylin had no effect on basal glucose uptake in the above cell line. Cholera toxin did not affect insulin-stimulated glucose uptake, but completely inhibited the effect of the rat amylin inhibitor.

Some partial sequences of human and rat amylines were synthesized and their effects were investigated in in vitro and in vivo models.

Peptide Synthesis

Human and rat amylins were prepared according to the procedure of Balasubramaniam et al., Peptides 12: 919-924 (1991). Synthetic peptides were characterized by their sequence and mass spectral analysis and were found to have a purity of greater than 97% by analytical reverse phase chromatography.

The peptides were synthesized in an Applied Biosystem Model 430A synthesizer. HPLC was performed using a Waters Model 600 solvent delivery system coupled to a U6K injector, Model 481 spectrophotometer and Baseline 810 Data collection software on an IBM-XT computer. Protected amino acid derivatives (Peninsula, CA) were used in the synthesis17 · ··· · · 9 · · · · · 9

999 99 99 9 Reagent (Applied Biosystems, CA) and solvents (Fischer Scientific, OH) were commercially available and used without further purification. 0.45 mmol of para-methylbenzhydroxylamine (MBHA) resin (NH2 group) was added to the synthesizer reaction vessel and the amino acid derivatives were automatically coupled using a standard program provided by the manufacturer, modified by the inclusion of a double coupling procedure. All amino acids were coupled using 2.2 equivalents of preformed symmetric anhydride. However, Arg, Asn and Gln were coupled using 1-hydroxybenzotriazole esters (4.4 equivalents) to avoid deamination or lactam formation. At the end of the synthesis, the Ν-α-Boc group was removed and the peptide resin (1.3 g) was treated with hydrogen fluoride (10 mL) for 1 hour at -2 to 4 ° C. , 88 ml), p-cresol (0.8 g) and p-thiocresol (~ 0.2 g). Hydrogen fluoride was removed in vacuo and the residue was applied to a sintered glass frit, washed several times with diethyl ether, extracted twice with 15 mL of acetic acid and lyophilized. The crude peptide thus obtained (100 mg) was dissolved in 6 ml of 6M guanidine hydrochloride, diluted with 500 ml of distilled water and adjusted to pH 8 with ammonia. Then 0.1% potassium ferricyanide solution was added gradually with stirring until the yellow color of the solution remained. The solution was stirred for an additional 30 minutes and then adjusted to pH 5 with acetic acid. The solution ··· 10 g wet weight of anion exchange resin "·» · · • • ···· * · · · · · * (AG-3, Cl ^- form) was stirred for 30 minutes, 0.45 μιη- filtered through a semi-preparative reverse phase column and purified according to the procedure of Balasubramaniam et al., Peptides 12, 919-924 (1991). The total human and rat amylline yields obtained were between 10 and 20%.

In vitro studies

C2C12 cells were cultured at 37 ° C in a humidified 5% CO 2 atmosphere with low glucose (1 g / L) DMEM medium containing 20% fetal calf serum and 0.5% chicken embryo extract (culture medium). Cells were seeded in 75 cm ² flasks cells / flask at a density of lxlO ^eighth When the culture became contiguous (3-4 days), the cells were trypsinized (0.25% trypsin) and washed with culture medium. The final cell pellet was suspended in culture medium and seeded on 24-well (16 mm diameter) plates at 2.5-10 ⁴ cells / well and cultured to 70% confluency (3 days). To induce fusion, mononuclear myoblasts were treated with medium containing 10% horse serum (fusion medium) instead of 20% FBS. The fusion medium was changed daily to avoid premature detachment of the cells, and the cells were fused to day 9 (6 days in fusion medium) almost completely into multi-core muscle tubes. The medium was changed 1 day before the start of the experiment.

The uptake of 2-deoxy glucose into C2C12 muscle tubes was determined according to the method of Klip et al., Biochem. J. 242: 131-136 (1987)]. Briefly, cells were washed with PBS (phosphate buffered saline) and serum-free, ι · «· · · · ·

- Incubated in 19 high glucose (25 mM DMEM) media for 5 hours. At the end of the incubation, cells were washed with PBS and various concentrations (10 pmol / l 10 gmol / l) of amylin or amylin analogue were added and then incubated with 1 mmol / l 2-deoxy- [ ³ H] -glucose for 10 min. Non-carrier-mediated uptake was determined by incubating the cells with 15 μιηοΐ / ΐ cytocalazine B. The uptake was stopped by rapid evaporation of the solution and the cells were washed with ice-cold PBS. Cell-associated radioactivity was determined by lysing the cells with 1 M NaOH, neutralizing the aliquots, and counting the counts using a scintillation counter. The protein content of the aliquots was determined by the Lowry method.

After inoculation, undifferentiated mononuclear myoblasts multiplied logarithmically and reached 70% by day 3. Fused cells were detected on day 5 and contained> 90% multinucleated muscle medium by day 9 (6 days in fusion medium). In 6-day cells, glucose uptake increased by 30% in response to insulin, compared to 68-115% in 9-day cells. These results are similar to previous observations [Klip et al., Supra]. The low insulin response of 6-day cells is probably due to the presence of non-differentiated myoblasts with low insulin receptor density as demonstrated in the Lg muscle cell line (Bequinot et al., Endocrinology 18: 446-455 (1986)). Because of the above results, and based on the observation that insulin stimulated glucose uptake in 9-day cells, 9-day C2C12 cells were used to examine the effect of amylin or amylin analogs on insulin-stimulated glucose uptake. The maximal insulin-stimulated response was observed at a concentration of 100 nM, and this remained on this plateau at further increasing doses. Insulin-stimulated glucose uptake into C2C12 muscle tubes is likely to occur mainly through facilitated diffusion, since cytocalazine B (15 μτηοΐ / ΐ) inhibited the insulin-stimulated glucose uptake by> 90%.

Carrier tests

Spargue Dawley rats (Zivic Miller, Zelienople, PA) used in the above study were housed individually in air-conditioned (22-24 ° C) compartments, and were given Purina rat food and water ad libitum for 12 hours.

The approx. 300 g Spargue Dawley rats were fasted overnight (18-22 hours). The rats were then anesthetized with 40 mg / kg sodium pentobarbital and catheters were implanted into the jugular vein. 0.1 ml of saline was injected through the jugular vein, 50 μg of rat amyline in 0.1 ml of saline, or 100 μg of peptide fragment / analog in 0.1 ml of saline, and another 0.1 ml of saline was added rapidly. To test for antagonistic effects, 50 μg of rat amylin was injected 2 minutes after injection of 100 peptide fragments / analog in 0.1 ml saline. Thirty minutes after peptide injection, 4-5 ml of blood was collected from the jugular vein and collected in heparinized tubes containing 0.1 μΐ aprotinin 21 ············ * · · · *. Plasma was obtained by centrifugation. Plasma glucose and insulin concentrations were determined by glucose oxidase (Model 27 glucose analyzer, Yellow Springs Instruments, Yellow Springs, OH) and radioimmunoassay kit (Peninsula Laboratories, Belmont, CA).

blood vessels

Figure 2 shows that one of the antagonists of the invention, α-α-ac-human amylin (8-23) -NH2, had no significant effect on insulin-stimulated glucose uptake when tested separately in vitro. Figure 2 also shows that the presence of 1 μτηοΐ / ΐ α-α-ac-human amylline (8-23) -NH2 significantly shifted the dose response curve of human amylin inhibitor to insulin-stimulated glucose uptake with human amylin. (i.e., higher concentrations of human amylin) thereby increasing the IC 50 value from 0.20 nmol / l to 350 nmol / l.

The in vivo effect of α-α-ac-human amylline (8-23) -NH2 is approx.

300 g of anesthetized (45 mg / kg) Spargue Dawley rats were examined after fasting for at least 20 hours. The following samples were injected into the cannulated jugular vein in each animal: (1) 100 μΐ saline (n = 5), (2) 50 μg rat amylin, (3) 100 μg Ν-α-ac-human amylin (8-23). -NH2, and after 2 minutes 50 μς rat amylin. Thirty minutes after injection, 4-5 ml of blood was collected from each animal in heparinized tubes and the plasma was separated by centrifugation. Plasma glucose and insulin concentrations were determined sequentially and the results are shown in Figures 3a and 3b, respectively.

• · · · · 9 · · · * • · · · · · · · · · · · · · · · · · ·

- 22 Figure 3a shows that rat amylin significantly increased plasma glucose concentration compared to saline control, whereas Ν-α-ac-human amylin (8-23) -NH2 significantly decreased plasma glucose concentration compared to saline control, probably by antagonizing the action of endogenous amylin. Figure 3a also shows that Na-ac-human amylin (8-23) -NH2 significantly attenuated the increase in plasma glucose levels in rat amylin (8- 23) -NH 2 was all given to rat amylin (i.e., plasma glucose levels dropped to approximately control levels). The p values refer to Figures 3a and 3b, and the rest of the values obtained with ANOVA for n = 5-8.

These results confirm that Na-ac-human amylin (8-23) -NH2 is ^a potent antagonist of human amylin in vitro and that rat amine is in vivo.

Figure 4 depicts insulin-stimulated glucose uptake in the in vitro assay in the same way as human amylin (1-23) -NH2. Figure 4 also shows that human amylin (1-23) -NH2 has a dose-response inhibitory effect on insulin-stimulated glucose uptake by C2C12 cells and is comparable to that of intact human amine.

Figure 5a shows that human amylin (1-23) -NH2 attenuated rat amylin-induced hyperglycemia.

As shown in Figure 6, [Anb ² < ⁷ ] rat amylin (l-23) -NH2 is qualitatively similar to rat amylin (l-23) -NH2. ···· ··· «« ·· · ·

- 23 inhibited insulin-stimulated glucose uptake in the in vitro study. Figure 6 and Figure 4 show that rat amylline (1-23) -NH ₂ exhibited a dose-response inhibitory effect comparable to that of intact human amylin on insulin-stimulated glucose uptake by C ₂ Cl ₂ cells. Figures 6 and 4 also show that the potency of the [Anb ² ' ⁷ ] rat amylin (1-23) -NH _{2 is} comparable to that of human amylin.

Figure 7 shows that [Anb ² ' ⁷ ] rat amylline (1-23) -NH _{2 has} no significant effect on amylline-induced hyperglycemia, but shows a tendency towards attenuation.

The results obtained are shown in Table 1 together with data from the literature.

• · · · ·

Table 1

peptides ^C 2 ^C 12 Effect on insulin-stimulated glucose uptake Anesthetized Rat Plasma Plasma Glucose ¹ ' ² Ca ²⁺ 1. Human amylin (IF) inhibits (Figure 2) increase ³ reduce ⁴ 2. rat amylin (R) inhibits ^ increase ³ reduce ⁴ 3. HA (1-23) -NH ₂ inhibit (for HA 1. similarly, Figure 4) 2. reduces n.v. setpoint (Figure 5a) attenuates amine-induced hyperglycemia 4. RA (1-23) -NH ₂ inhibit (similar to HA, Figure 6) N.V. N.V. 5. [Anb ² » ⁷ ] RA- (1-23) -NH ₂ inhibit (for HA 1. similarly, Figure 6) Second reduces n.v. setpoint (Figure 7a) no effect on amine-induced hyperglycemia 6. α-α-Ac-HA-1 (8-23) -NH ₂ 2 . no effect 1. . weakens it amylin (Figure 2) 2. reduces n.v. setpoint (Figure 3a) attenuates amyline-induced hyperglycemia

· ♦ ¹ Current investigations;

²¹⁰⁰ gg peptide analog effect on basal value or 50 ptg rat amylin induced hyperglycemia;

³ Molina et al., Diabetes 39: 260-265 (1990); and Young et al., Am. J. Physiology 259, E457-461 (1990);

⁴ Data et al., Biochem. Biophys. Gap. Commun. 162: 876-881 (1989);

⁵ Sheriff et al., Biochim. Biophys. Acta 1136, 219-

222 (1992)

n.v .: not tested

The agonist and antagonist activity of other amylin analogs of the invention may also be determined by the methods described above.

Application

Amylin inhibits insulin-stimulated glucose uptake and glycogen synthesis and enhances hepatic glucose secretion. Therefore, some insulin / amylin ratio appears to be required to maintain normal plasma glucose levels.

The amylin agonists and antagonists of the invention are potentially useful in the treatment of type I and type 2 diabetes mellitus. As type I diabetes mellitus increases with amylin concentration and blood glucose levels, administration of the amylin antagonists of the invention decreases to normal or clinically acceptable blood glucose levels26 ·

in sufficient quantities to produce therapeutic results. The

In people with type 1 diabetes, both insulin and amylin levels are lower and treatment with insulin may lead to the development of hypoglycaemia. Administration of the amylin agonists of the invention in an amount sufficient to raise blood glucose levels to a normal or clinically acceptable level in response to insulin-induced hypoglycaemia, together with a therapeutic amount of insulin, may result in a therapeutic result.

The amylin agnists of the present invention reduce serum calcium and are thus useful in the treatment of hypercalcaemia in human medicine. The amylin agonists of the invention also have an appetite suppressant activity, while the amylin antagonists increase appetite. The amylin agonists and antagonists of the invention may therefore also be used to regulate food intake. For example, the amylin agonists of the invention are useful in the treatment of obesity.

The peptides of the invention may be administered in the form of sustained release formulations in human medicine by one of the conventional routes of administration (e.g., orally, parenterally, transdermally or transmucosally) or by using biologically degradable biocompatible polymers.

Sequence match

- 27 SEQ ID NO: 1

Length of sequence: 16

Type of sequence: amino acid

Thread Type:

Topology: linear

Sequence description:

Ν-α-Ac-Ala Thr Gin Arg Leu Al Asn Phe Leu Val His Ser Ser

10

Asn Asn Phe-NH ₂

SEQ ID NO: 2

Length of sequence: 16

Type of sequence: amino acid

Thread Type:

Topology: linear

Sequence description:

Ν-α-Ac-Ala Thr Gin Arg Leu Al Asn Phe Leu Val Arg Ser Ser

10

Asn Asn Leu-NH ₂

SEQ ID NO: 3

- 28 Sequence Length: 23

Type of sequence: amino acid

Topology: linear

Sequence description:

Lys Cys Asn Thr Alá Thr Cys

Alá Thr Gin Arg Leu Alá Asn Phe

Leu Val His Ser Ser Asn Asn Phe-NH2

SEQ ID NO: 4

Length of sequence: 23

Type of sequence: amino acid

Thread Type:

Topology: linear

Sequence description:

Lys Cys Asn Thr Alá Thr Cys

Leu Val His Ser Ser Asn Asn

Alá Thr Gin Arg Leu Alá Asn Phe

15

Leu-NH2

SEQ ID NO: 5

Length of sequence: 23

Type of sequence: amino acid

Thread Type:

topology:

linear

- 29 Sequence description:

Lys Anb Asn Thr Alá Thr Anb Alá Thr Gin Arg Leu

Alá Asn

Phe

Leu Val His Ser Ser Asn Asn Leu-NH ^

SEQ ID NO: 6

Length of sequence: 37

Type of sequence: amino acid

Thread Type:

Topology: linear

Sequence description:

Lys Cys Asn Thr Alá Thr Cys Alá Thr Gin Arg Leu Alá Asn Phe 15 5 10 Leu With His Ser Ser Asn Asn Phe Gly below Ile Leu Ser Ser Thr 20 25 30 Asn With Gly Ser Asn Thr Tyr

SEQ ID NO: 7

Length of sequence: 37

Type of sequence: amino acid

Thread Type:

topology:

linear

- 30 Sequence description:

Lys Cys Asn Thr Alá Thr Cys Alá Thr Gin Arg Leu Alá Asn Phe 5 10 15 Leu With Arg Ser Ser Asn Asn Phe Gly Pro Val Leu Pro Pro Thr 20 25 30 Asn With Gly Ser Asn Thr Tyr

Claims (13)

Claims - 31 1. Amylin analogues having the amino acid sequence: where meaning A chain of 0-5 amino acids having the N-terminus It is linked to R 1 and R 2; meaning A chain of 0-4 amino acids the C-terminus of which is attached to Z; ^R 1 and R ₂ are each independently hydrogen, C 1 -C 12 alkyl (e.g., methyl), C 6 -C 18 aryl (e.g., phenyl, naphthaleneacetyl), C 1 -C 12 acyl (e.g. formyl, acetyl and myristoyl), C 7-18 aralkyl (e.g. benzyl), or C 7-18 alkylaryl (e.g. p-methylphenyl); The meaning of θθ below, At, Thi, Phe, Bth, Pcp, N-Me-Thr, Aib or Anb; Meaning of ⁹ Thr, below, ANB, Aib, Ser, N-Me-Ser or N-Me-Thr; ₁₀ j colorless Designation Gin, below, Asn, N-Me-Gln, You are Gly, Nva, Aib ANB; The meaning of AH Arg, homo-Arg, diethyl-homo-Arg, Lys-6-NH-R (wherein R is hydrogen, straight or branched C 1 -C 10 alkyl or aryl). · · · · · ····· · »· ·· · ♦ · - 32 ports), Orn or Lys; Meaning of ¹² Leu, Ile, With, Aib; Anb or N-Me-Leu; The meaning of A13 below, At, Thi, Phe, Bth, Pcp, N-Me-Thr, Aib A14 A15 or Anb; meaning ANB; meaning Asn, below, Gin, Gly, You're N-Me-Asn, Nva, Aib Phe or an optionally substituted aromatic amino acid; The ^16th meaning Leu, Ile, With, Aib; A * ⁷ meaning With, Ile, Aib; ANB _the 18th meaning His, Thr, 3-Me -His, Anb or N-Me-Leu; or N-Me-Val; 1-Me-His, S-pyrazolyl-alanine, N-Me-His, Arg, homo-Arg, diethyl-homo-Arg, Lys-e-NH-R (wherein R is hydrogen, straight or branched C 1 -C 10 alkyl or aryl), Ala, Aib, Anb or Orn; A19 meaning below; Ser, Thr, N-Me-Ser, _the 20th meaning Ser, Thr, N-Me Ser, below; _a 21st meaning Asn, below, Gin, Gly, below; _a 22 meaning Asn, below, Gin, Gly, below; _the 23rd meaning Phe, in that particular case sav, Leu, Ile, With, Aib; ANB, Z N-Me-Thr, Aib; ANB obsession N-Me-Thr, Aib; ANB obsession N-Me-Asn, Aib; ANB obsession N-Me-Asn, Aib; ANB obsession substituted aromatic aminoAla or N-Me-Leu, · and -NHR3 or -OR3 (wherein R3 is hydrogen, C1-C12 alkyl, C7-C10 phenylalkyl, C3-C20 alkenyl, 3-20 · · - (C 33 -alkynyl, phenyl or naphthalene), and pharmaceutically acceptable salts thereof. The amylin analogs of claim 1 which are antagonists. The amylin antagonist of claim 2, which is human amylin 8 to 23. amino acid sequence corresponding to the Ν-α-acetyl derivative of the amino acid sequence containing the C-terminally amidated carboxyl group (Ν-α-ac-human amine (8-23) -NH2)> ^{and its} amino acid sequence: N-a-Ac-Ala-Thr-Gln-Arg-Leu-Ala-Asn-Phe-Leu-Val-His-Ser-Ser-Asn-Asn-Phe-NH 2, and pharmaceutically acceptable salts thereof. The amylin analogue of claim 2, wherein the amino acid sequence is: And the pharmaceutically acceptable salts thereof. 5. Amylin analogs having the amino acid sequence: ^R i R ₂ -XA ¹ -A ² -A ³ -A ⁴ -A ⁵ -A ⁶ -A ⁷ -A ⁸ -A ⁹ -A ¹⁰ -A ¹¹ -A ¹² -A ¹³ -A ¹⁴ -A ¹⁵ -A ¹⁶ -A ¹⁷ -A ¹⁸ -A ¹⁹ -A ²⁰ -A ²¹ -A ²² -A ²³ -YZ where X is a chain of 0 to 5 amino acids the N-terminus of which is attached to R 1 and R 2; Y is a chain of 0 to 4 amino acids the C-terminus of which is attached to Z; R 1 and R 2 are each independently hydrogen, C 1 -C 12 alkyl, C 6 -C 18 aryl, C 1 -C 12 acyl, C 7 -C 18 aralkyl, or C 7 -C 18 alkylaryl; A ¹ is Lys, Arg, homo-Arg, diethyl homo-Arg, Lys-e-NHR (where R is hydrogen, straight or branched C 1 -C 10 alkyl or aryl) or Om; A ² is Cys or Anb; A ³ is Asn, Ala, Gln, Gly, N-Me-Asn, Aib, Anb or Nva; A ⁴ is Thr, Ser, N-Me-Ser, N-Me-Thr, Ala, Aib or Anb; ^A5 is Ala, Nal, Thi, Phe, Bth, Pcp, N-Me-Ala, Aib, or Anb; A ⁸ is Thr, Ser, N-Me-Ser, N-Me-Thr, Ala, Aib, or Anb; A ⁷ is Cys or Anb; ^A8 is Ala, Nal, Thi, Phe, Bth, Pcp, N-Me-Ala, Aib, or Anb; ⁹ is Thr, Ser, N-Me-Ser, N-Me-Thr, Ala, Aib or Anb; A ¹⁰ is Gln, Ala, Asn, N-Me-Gln, Gly, Nva, Aib or Anb; A ¹¹ is Arg, homo-Arg, diethyl homo-Arg, Lys-e-NH-R (wherein R is hydrogen, straight or branched C 1 -C 10 alkyl or aryl) or Orn; ¹² is Leu, Ile, Val, Aib, Anb or N-Me-Leu; - 35 ^ ¹ is Ala, Nal, Thi, Phe, Bth, Pcp, N-Me-Ala, Aib, or Anb; ¹⁴ is Asn, Ala, Gln, Gly, N-Me-Asn, Aib, Anb or Nva; The ¹ ^ is Phe or an optionally substituted aromatic amino acid; ¹⁶ is Leu, Ile, Val, Aib, Anb or N-Me-Leu; A ¹⁷ is Val, Ile, Aib, Anb or N-Me-Val; ¹⁸ is His, Thr, 3-Me-His, 1-Me-His, β-pyrazolyl-alanine, N-Me-His, Arg, homo-Arg, diethyl-homo-Arg, Lys-e-NH-R (wherein R is hydrogen, straight or branched C 1 -C 10 alkyl or aryl); group), Orn, Ala, Aib or Anb; A19 meaning ANB; Ser, Thr, N-Me-Ser, N-Me-Thr, below, Aib obsession _a 2 0 meaning ANB; Ser, Thr, N-Me-Ser, N-Me-Thr, below, Aib obsession _a 21st meaning Nva · Asn, below, Gin, Gly, N-Me-Asn, Aib; ANB obsession _a 22 meaning Nva · Asn, below, Gin, Gly, N-Me-Asn, Aib; ANB obsession _the 23rd meaning Phe, in that particular case substituted aromatic amino- sav, Leu, Ile, With, Aib, Anb, You're down N-Me Leu; and Z is -NHR 3 or -OR 3 (wherein R 3 is hydrogen, C 1 -C 12 alkyl, C 7 -C 10 phenylalkyl, C 3 -C 20 alkenyl, C 3 -C 20 alkynyl, phenyl or naphthyl), · ♦ · · And the pharmaceutically acceptable salts thereof. The amylin analog of claim 5 which is human amylin 1-23. amino acid and has a C-terminal amidated carboxyl group (human amylin (1-23) -NH ₂ ) and has the amino acid sequence: Lys-Cys-Asn-Thr-Ala-Thr-Cys-Ala-Thr-Gln-Arg-Leu-Ala-Asn-Phe-Leu-Val-His-Ser-Ser-Asn-Asn-Phe-NH ₂ , and and pharmaceutically acceptable salts thereof. The amylin analog of claim 5, which is rat amylin 1-23. corresponding to its amino acids and having a C-terminal amidated carboxyl group (rat amylin (1-23) -NH ₂ ) and having the amino acid sequence: Lys-Cys-Asn-Thr-Ala-Thr-Cys-Ala-Thr-Gln-Arg-Leu-Ala-Asn-Phe-Leu-Val-His-Ser-Ser-Asn-Asn-Leu-NH ₂ , and and pharmaceutically acceptable salts thereof. The amylin analog of claim 5, which is amyl 1-23 of rat amylin. corresponding to α-amino-n-butyric acid substitution at the 2- and 7-positions and a C-terminally amidated carboxyl group ([Anb ² ' ⁷ ] rat amyl (1-23) -NH ₂ ), and amino acid sequence: Lys-Anb-Asn-Thr-Ala-Thr-Anb-Ala-Thr-Gln-Arg-Leu-Ala-Asn-Phe-Leu-Val-His-Ser-Ser-Asn-Asn-Leu-NH ₂ , and and pharmaceutically acceptable salts thereof. 9. A method of treating type diabetes diabetes mellitus in man comprising administering a therapeutically effective amount of an amylin antagonist according to claim 2. • ·· « 4 • · «« * • t · • • 1 • * • 44 44 · 44 • 4 • 10. The method of claim 9, wherein the amylin antagonist is α-α-ac-human amylin (8-23) NH2. 11. A method of treating Type I diabetes mellitus in man comprising administering a therapeutically effective amount of an amylin agonist analog according to claim 5 in combination with a therapeutically effective amount of insulin. 12. A method of treating hypercalcemia in a human, comprising administering a therapeutically effective amount of an amylin agonist analogue according to claim 5 having an agonist activity. 13. A method of regulating food intake in a human comprising administering a therapeutically effective amount of an amylin analog according to claim 1 or 5.