CZ5795A3 - Amyline analogs and method of controlling reception of food by a human being - 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 amylin analogs and their use in the treatment of diabetes mellitus and hypercalcemia and in the regulation of food intake by administration of the analog. More specifically, the present invention relates to specific amylin anologues that act as amylin antagonists and agonists and their use in the treatment of diabetes mellitus and hypercalcemia and in the regulation of food intake.

BACKGROUND OF THE INVENTION

Amylin, also known as a polypeptide related to: diabetes (Cooper et al., Proc. Natl. Acad. Sci., USA, 85, 7763 (1988)) or as an amyloid islet / insulinoma polypeptide. (Westermark et al., Proc. Natl. Acad. Sci., USA 84, 3881 (1987).), Is a 37-residue polypeptide amide originally isolated from the amyloid-rich pancres of diabetic insulin patients and non-diabetic diabetic patients. insulin (NIDD). It was subsequently isolated from normal rat pancreas (Asai et al., Biochem. Biophys. Res. Commun. 164: 400 (1989)). CDNA cloning studies (Ferrier et al .: J. Mol. Endocrinol. 3, R1 (1989)) and immunocytochemical studies (Lukinius et al .: Diabetologia 32. 240 (1989)) have shown that amylin is synthesized in islet cells and that it is stored in islet secretory granules along with insulin. It is secreted with insulin (Kanatsuka et al., FEBS Lett. 259, 199 (1989)). Small amounts of amylin have also been delegated in the stomach, intestine, lungs and dorsal root ganglion (Asai et al., Biochem. Biophys. Res. Commun. 169. 788 (1990) and Ferrier et al., Supra).

Biological investigations following amylin isolation have shown that amylin inhibits basal and insulin-stimulated glucose uptake as well as glycogen synthesis by calf muscles (Leighton et al., Nature 335, 632 (1988)). This resistance to peripheral insulin by amylin has also been shown in vivo by euglycemic glucose studies in dogs (Sowa et al .: Diabetologia 33, 118 (1990)) and rats (Molina et al .: Diabetes 39. 260 (1990)). These studies in rats have further shown that amylin enhances the inhibition of hepatic glucose expenditure by insulin (Molina et al., Supra). Based on these observations and the finding that amylin inhibits basal insulin secretion (Ohsawa et al., Biochem. Biophys. Res. Commun. 160, 961 (1989)), it has been suggested that amylin may play a role in glucose metabolism and the pathology of non-insulin-dependent diabetes mellitus (NIDDM), commonly known as type II diabetes mellitus.

Diabetes mellitus is a metabolic disease characterized by chronic hyperglycaemia, ie elevated blood sugar levels. This disease affects a significant percentage of the population. There are two main categories of diabetes mellitus, commonly referred to as type I and type II. Patients with type I diabetes mellitus lose active β-cells in the islets of Langerhans in the pancreas, resulting in low levels of both insulin and amylin. Coper: Medical Hypothesis 26, 284 (1991). Patients with type I diabetes mellitus who are treated with insulin often tend to develop hypoglycaemia as a side effect. Amylin levels are elevated in patients with type II diabetes mellitus. Patients with type II diabetes mellitus show significant resistance to the normal biological effects of insulin. Elevated levels of amylin, known as hyperamyllinemia, lead to insulin resistance in many model systems, including genetically obese LA / N-cp rats (Huang et al., Hypertension 19, i-101 (1992).), Genetically obese diabetic Yellow mice (Gill et al .: Life Sci. 48, 703 (1991).), Dexamethasone-induced diabetic rats (Jamal et al., J. Endocrin. 126, 425 (1990)), streptozocin-induced diabetic rats (Inoue and et al., Diabetes 41, 723 (1992).) and ventromedial hypothalamic injured rats and Zucker rats (Tokuyama et al., Endocrinology 128, 2739 (1991)).

Further studies have shown that amylin, like calcitonin, may exhibit calcium-lowering effects in rat serum in vivo as well as in cell culture systems (Datta et al., Biochem. Biophys. Res. Commun. 162. 876 (1989)). . Amylin has also been shown to act as an anorectic agent. Balasubramaniam et al., Peptides 12, 919 (1991).

SUMMARY OF THE INVENTION

The present invention generally relates to amylin analogs that behave as amylin antagonists and agonists.

In one aspect, the invention relates to amylin analogs which are linear analogs of a biologically active amylin of the following general formula I

R 1 ^- (R 2) X ^- A ^and -A ⁹ -A ¹⁰ -A ¹¹ -A ¹² -A ¹³ -A ¹⁴ -A ¹⁵ -A ¹⁶ -A ¹⁷ -A ^is -A ¹⁹ -A ²⁰ -A ^il -A ²² -A ² -YZ.

»(I) r.

in which

X is a chain of up to 5 amino acids including the N-terminal amino acid that is linked to R _x and R ₂ ,

Y represents a chain of up to 4 amino acids including a C-terminal amino acid that is bound to Z, R _x and R ₂ independently represent hydrogen, C 1 -C 12 alkyl (e.g. methyl), C 6 -C 6 aryl 18 carbon atoms (e.g., phenyl, naphthaleneacetyl), acyl of 1 to 12 carbon atoms (e.g., formyl, acetyl and myristoyl), aralkyl of 7 to 18 carbon atoms (e.g., benzyl) or alkaryl of 7 to 18 atoms carbon (e.g., p-methylphenyl),

A ⁸ is Ala, Nal, Thi, Phe, Bth, Pcp, N-Ile-Thr, Aib, or Anb,

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

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

Af / i, v

Anb,

A ¹¹ is Arg, homo-Arg, diethyl-homo-Arg, Lys-e-NH-R (where R is hydrogen, alkyl having 1 to 10 carbon atoms, straight or branched chain or an aryl group)

Orn or Lys,

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

A ¹ * means Ala, Nal, Thi, Phe, Bth, Pcp, N-Me-Thr, Aib, or Anb,

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

Anb,

A ¹⁶ is Phe, or any aromatic amino acid with or without substituents substituents

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

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

A ¹⁸ is His, Thr, 3-Me-His, 1-Me-His, β-pyrosolyl-alanine, N-Me-Hiss (Arg) / bromo-A'r'g 'diethyl-homo-Arg,' Lys- e-NH-R- (wherein · - --- -R represents a hydrogen atom, a C 1 -C 10 alkyl group, a straight-chain or straight-chain alkyl group or an aryl group), Ala,

Aib, Anb or Orn, A ¹⁸ means Ser, Thr, N-Me-Ser, Nib-Thr, Aib, Anb or Ala, • A ²⁰ means Ser, Thr, N-Me-Ser, Nib-Thr, Aib, Anb or Ala, A ²¹ means Asn, Ala, Gin, Gly, N-Me-Asn Anb or Nva, A ²² means' Asn, Ala, Gin, Gly, N-Me-Asn, Aib, Anb or Nva, A ^2S means Phe, any aromatic amino acid with or without substituents , Leu, Ile, Val, Aib, .Anb, Ala or: N-Me- -Leu and

Z represents a group of formula NHR ₃ or _J 0R _(in which R represents a hydrogen ^- atom, an alkyl group having 1 to 12 carbon atoms ..., phenylalkyl of 7 to 10 carbon atoms, alkenyl having 3 to 20 carbon atoms, alkynyl of 3 to 20 carbon atoms, phenyl or naphthyl group ^1.

In preferred embodiments, these analogs are antagonists.

In a highly preferred embodiment, the amylin antagonist responds

Am-α-acetyl derivative of amino acids 8 to 23 of human amylin \ with an amidated carboxy group at the C-terminus, referred to herein as Ν-α-ac-human amylin (S-23) -NH _{2 of the} following formula III \

N-a-Ac-Al-Thr-Gln-Arg-Leu-Ala-Asn-Phe-Leu-Val-His-Ser-Ser-Asn-1

Asn-Phe-NH ₂ '(III). In another preferred embodiment, the amylin antagonist has the following formula (IV)

N-α-Ac-Ala-Thr-Gln-Arg-Leu-Ala-Asn-Phe-Leu-Val-Arg-Ser-Ser-1

-Asn-Asn-Leu-NHa (IV). 1

In another aspect, the invention relates to amylin analogs that are linear biologically active analogs. $ i

amylin of the following formula II

X - A ¹ Ά ~ A — A ⁴ A ^^ **

-A ¹⁸ -A ¹⁹ -A ^2θ -A ²¹ -A ²² -A ²³ -Υ-Ζ, (I) in which

X is a chain of up to 5 amino acids including the N-terminal amino acid that is linked to R _x and R ₂ ,

Y is a chain of up to 4 amino acids including a C-terminal amino acid that is attached to Z, R ₁ and R ₂ independently represent hydrogen, C 1 -C 12 alkyl, C 6 -C 18 aryl, allyl C 1 -C 12, C 7 -C 18 aralkyl or alkaryl.

a group having 7 to 18 carbon atoms,

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

pin) or Orn, A ² means Cys or Anb, A ³ means Asn, Ala, Gin, Gly, N-Me-Asn, Aib, Anb, or Nva, A ¹ means Thr, Ser, N-Me-Ser, N-Me-Thr, Ala Aib or

'7 * ·

Anb,

A ⁵ is Ala, Nal, Thi, Phe, Bth, Pcp, N-Me-Ala, Aib, or Anb,

A ⁶ is Thr, Ser, N-Me-Ser, N-Me-Thr, Ala, Aib or

Anb,

A ⁷ is Cys, or Anb,

A® means Ala, Nal, Thi, Phe, Bth, Pcp, N-Me-Ala, Aib, or Anb,

A * 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, alkyl having 1 to 10 carbon atoms, straight chain or branched arylovouskupinu) or Orn,

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

A ^1S means Ala, Nal, Thi, Phe, Bth, Pcp, N-Me-Ala, Ala, f *

Aib or Anb,

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

Nva,

A ^{1 S} is Phe or any aromatic amino acid with or without substituents,

^A18 is Leu, Ile, Val, Aib; Anb or N-Me-Leu,

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

A ¹⁸ is His, Thr, 3-Me-His, 1-Me-His, B-pyrozolylalanin, N-Me-His, Arg, homo-Arg, diethyl-homo-Arg, Lys-e-NH-R (where R2 is not hydrogen, straight chain or straight chain alkyl of 1 to 10 carbon atoms or aryl), Orn, Ala, Aib or Anb,

A ^1? means Ser, Thr, N-Me-Ser, N-Me-Thr, Ala, Aib, or Anb;

A ²⁰ is Ser, Thr, N-Me-Ser, N-Me-Thr, Ala, Aib or

Anb,

A ²¹ means Asn, Ala, Gln, Gly, N-Me-Asn, Aib, Anb or Nva.

A ²² is Asn, Ala, Gln, Gly, N-Me-Asn, Aib, Anb, or

Nva,

A ²³ is Phe, any aromatic amino acid with or without substituents, Leu, Ile, Val, Aib, Anb, Ala or N-Me-Leu

Z is NHR ₃ or OR ₃ in which R ₃ is hydrogen, C 1 -C 12 alkyl, C 7 -C 10 phenylalkyl, C 3 -C 20 alkenyl, C 3 alkynyl up to 20 carbon atoms, phenyl or naphthyl.

In one highly preferred embodiment, the amylin analog corresponds to amino acids 1 to 23 of a human amylin having an amidated carboxy group at the C-terminus, referred to herein as human amylin (1-23) -NH _{and the} following formula V

Lys-Cys-Asn-Thr-Ala-Thr-Cys-Ala-Thr-Gln-Arg-Leu-Ala-Asn-PheLeu-Val-His-Ser-Ser-Asn-Asn-Phe-NH ₂ (V).

In another highly preferred embodiment, the amylin analog corresponds to amino acids 1 to 23 of a rat amylin having an amidated C-terminus carboxy group, referred to herein as rat amylin (1-23) -NH _s , of the following formula VI

Lys-Cys-Asn-Thr-Ala-Thr-Cys-Ala-Thr-Gln-Arg-Leu-Ala-Asn-PheLeu-Val-Arg-Ser-Ser-Asn-Asn-Leu-NH ₂ (VI).

In yet another highly preferred embodiment, the amylin analog corresponds to an amino acid derivative of amino acids 1 to 23 of rat amylin provided that at positions 2 and 7 it is substituted with α-amino butyric acid (Anb) and has an amidated carboxy group at the C-terminus as [Anb ² ' ⁷ ] rat amylin (1-23) -NH _{2 of the} following formula VII

Lys-Anb-Asn-Th-Ala-Thr-Anb-Ala-Thr-Gln-Arg-Leu-Ala-Asn-PheLeu-Val-Arg-Ser-Ser-Asn-Asn-Leu-NH ₂ (VII.

In another aspect, the invention relates to a method of treating diabetic mellitus type II in humans by administering a therapeutic amount of an amylin antagonist of the invention. According to a highly preferred method of treating type II diabetes mellitus, Na-ac-human amylin (8-23) -NH _{2 is administered} .

In another aspect, the invention relates to a method of treating type I diabetes mellitus in humans by administering a therapeutic amount of an amylin agonist of the invention together with a therapeutic amount of insulin.

In yet another aspect, the invention relates to a method of treating hypercalcemia by administering a therapeutic amount of an amylin agonist of the invention.

The compounds of the invention exhibit a wide variety of biological activities, including those relating to glucose metabolism, blood calcium levels and taste. The amylin antagonists of the invention enhance the inhibition of insulin-stimulated glucose uptake. As a result, the amylin antagonists of the invention act. in reducing hyperglycemia resulting from elevated levels of amylin associated with type II diabetes mellitus. The amylin agonists of the invention inhibit insulin-stimulated glucose uptake, resulting in increased blood sugar levels. As a result, the amylin agonists of the invention are useful in reducing hypoglycaemia, which often accompanies insulin type I diabetes mellitus treatment. The amylin agonists of the invention inhibit insulin-stimulated glucose uptake, resulting in increased blood sugar levels. As a result, the amylin agonists of the invention are useful in reducing hypoglycemia, which often accompanies insulin-type diabetes mellitus treatment. The amylin agonists of the invention also reduce blood calcium levels and are thus useful in the treatment of hypercalcemia. In addition, amylin agonists exhibit appetite suppressing effects, while amylin antagonists enhance appetite. Thus, amylin agonists and antagonists are useful in regulating food intake. For example, amylin agonists are useful in treating overweight problems.

Many of the compounds of the invention are particularly preferred because they are truncated versions of the natural amylin peptide. A shorter peptide not only facilitates the synthesis and purification of the compounds, but also improves selectivity, reduces the number of manufacturing processes and reduces costs.

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

F

First, the figures will be briefly described.

Figure 1 compares the primary structures of human amylin (h-amylin) and rat amylin (r-amylin).

Figure 2 shows the effect of human amylin and N-α-ac-human amylin (8-23) NH ₂ , separately and together, on glucose uptake by either C ₂ C ₁₂ muscle cells. _b ,

Figures 3a and 3b show the in vivo effects of saline solution of rat amylin, β-α-ac-human amylin (8-23) -NH ₂ and Na-ac-human amylin (8-23) -NH ₂ plus rat amylin to plasma glucose and insulin levels in Sprague Dawley rats. F

Figure 4 shows the in vitro effect of human amylin and human? Amylin (1-23) -NH ₂ , separately, on insulin-stimulated glucose uptake in C ₂ C ₁₂ muscle cells.

Ř

Figures 5a-a 5b show the in vivo effects of saline, rat amylin, human amylin (1-23) -NH ₂ and human amylin (1-23) -NH ₂ plus rat amylin on plasma glucose and insulin levels in Sprague Dawley rats .

Figure 6 separately shows the in vitro effects of rat amylin (1-23) -NH ₂ and [Anb ^s ' ⁷ ] rat amylin (1-23) -NH ₂ on insulin-stimulated glucose uptake in C ₂ C ₁₂ muscle cells.

Figures 7a and 7b show the in vivo effects of saline, rat amylin, [Anb ² ' ⁷ ] rat amylin (T-23) -NH 2' and [Anb ^2,7 ] rat amylin (1-23) -NHa plus rat amylin on plasma glucose and insulin levels in Sprague Dawley rats.

The sequences of naturally occurring human amylin ( ⁿ -amylin * ¹ ) and rat amylin (r-amylin) are shown in Figure 1. Balasubramaniam et al., Peptides 12, 919 (1991). Among these amylins, there is a high degree of sequence horology. Naturally occurring h-amylin and r-amylin (their cysteine residues at positions 2 and 7 present in both forms) are believed to form an internal disulfide bond, resulting in a cyclic structure.

The amylin analogs of the invention are based on biologically active subfragments containing amino acids 8 to 23 h-amylin and r-amylin. and derivatives thereof, and on biologically active, subfragments containing amino acids 1 to 23 h-amylin and r-amylin and derivatives thereof. In the above amylin formulas, the symbols A 'and the like and Ser, Leu and the like, as used herein in the peptide sequences, are amino acid residues. If the amino acid is optically active, then it is the L-form. unless the D-form is explicitly mentioned. All peptide sequences listed herein are written according to the usual convention, wherein the amino acid at the N-terminus is on the left and the amino acid at the C-terminus is on the right. A short line between amino acid residues indicates a peptide bond. The substituent -OR or -NHR at the carboxy terminus of the peptide replaces -OH at the carboxyl terminus of the amino acid residue to give the -NH-CH (R) -COOR group, and -NH-CH (R) -CONHR at the C-terminus residues. If there is an -NH ₂ substituent on the terminal carboxyl, then the peptide is in the form of an amidated carboxy group.

As mentioned above and for the sake of convention in describing the present invention, conventional and unconventional abbreviations are used for various amino acids. These abbreviations are known to those skilled in the art, but for clarity, a list is given:

Asp = D = aspartic acid

Ala-A = alanine

Arg = R = arginine

Asn = N = asparagine

Cys = C = cysteine

Gly = G = glycine

Glu = E = glutamic acid

Gln = Q = glutamine

His = H = histidine

Ile = I = isoleucine

Leu = L = leucine

Lys = K = lysine

Het = M = methionine

Phe = F = phenylalanine

Pro = P = Proline

Ser = S = $ serine

Thr = T = threonine

Trp = w = tryptophan

Tyr = Y = tyrosine

Val = V = valine

Orn = ornithine

NaI = 2-naphthylalanine

Nva = norvaline

Thi = 2-thienylalanine

Pcp = 4-chlorophenylalanine

Bth = 3-benzothienylalanine

Bip = 4,4'-biphenylalanine

Tic = tetrahydroisoquinoline-3-carboxylic acid ’

Aib = aminoisobutyric acid

Anb = α-aminobutyric acid

Dip = 2,2 = Diphenylalanine The compounds of this invention can be obtained in the form of pharmaceutically acceptable salts. Examples of preferred salts are those with therapeutically acceptable organic acids such as acetic, lactic, maleic, citric, malic, ascorbic, succinic, benzoic, salicylic, methane-12-sulfonic, toluenesulfonic, trifluoroacetic or pamoic (1,1'-roethylene- bis [2-hydroxy-3-naphthoic acid]), salts with polymeric acids such as tannin or carboxymethylcellulose, and salts with inorganic acids such as hydrohalic acids such as hydrochloric, sulfuric, phosphoric and the like.

. I. ¹

The interrelationships between the structure and activity of amylin and amylin analogs were studied in both in vitro models (mouse muscle cell lines, C ₂ C ₁₂ ) and in vivo models (Sprague Dawley rats).

F

In in vitro studies, insulin stimulated glucose uptake by the C ₂ C ₁₂ cell line in a dose-dependent manner. This uptake was enhanced by rat amylin (100 µM) However, rat amylin showed no effect on basal. Glucose intake through this cell line ·: Cholera toxin has no effect on insulin-stimulated glucose uptake but blocks the inhibitory effect of rat amylin.

Some partial sequences of human and rat amylin and their analogs have been synthesized. Their effects have been investigated in in vitro and in vivo models.

Human and rat amylin were synthesized according to the procedure described by Balasubramaniam et al., Peptides 12, 919 (1991). Synthetic peptides were characterized by sequence and mass spectra analysis. Reversed phase analytical chromatography revealed their purity to be greater than 97% by weight. *

Peptide synthesis was performed on an Applied Biosystem Model 430A synthesizer. HPLC was performed on a Waters Model 600 system with a U6K injector, a Model 481 spectrophotometer, and Baseline 810 Data acquisition software on an IMB-XT computer. Protected amino acid derivatives (Peninsula, Ka.), Synthesis reagents (Applied Biosystms, Ka.) And solvents (Fischer Scientific, Oh.) Were obtained commercially and used without further purification. Place the methylbenzhydroxylamine (MBHA) resin (0/45 mmol, NH ₂ group) in the εε synthesizer reaction vessel. Amino acid derivatives are automatically condensed using a standard program provided by the manufacturer modified to include a double condensation process. All amino acids were condensed using 2.2 equivalents of preformed, symmetrical anhydrides. However, Arg, Asn and Gln were condensed in the form of preformed 1-hydroxybenzotriazole esters (4.4 equivalents) to avoid deamidation or lactam formation. At the end of the synthesis, the Ν-α-Boc group was removed and the peptide resin (1.3 g) was reacted with hydrogen fluoride (10 ml) containing dimethylsulfide (0.8 ml), p-cresol (0.8 g) and p- thiocresol (0.2 g) for one hour at -2 to 4 ° C. The HF was removed in vacuo and the residue was transferred to a fritted filter funnel, washed repeatedly with diethyl ether, extracted with acetic acid (2 x 15 mL) and lyophilized. The thus obtained crude peptide ¹ ··. Dissolve (100 mg) in 6M guanidine hydrochloride (6 ml), dilute with 500 ml distilled water and adjust the pH to 8 with ammonia. Then add a solution of 0.1% potassium hexacyanoferrate (w / w to v / v). parts) with stirring until the yellow color persists. After stirring for an additional 30 minutes, the pH of the solution was adjusted to 5 with acetic acid. The solution was then stirred with anion exchange resin (AG = Cl, 10 g dry weight) for 30 minutes, filtered. It is pumped onto a reverse phase semiprep column and purified as described by Balasubramaniam et al., Peptides 12, 919 (1991). The total yield of the thus obtained rat and human amylin varied between 10 and 20% by weight.

C ₂ C ₁₂ cells are cultured at 37 ° C in a humidified atmosphere with 5% CO ₂ in low glucose (1 g / L) DMEM medium containing 20% w / w. fetal bovine serum and 0.5 wt. chicken embryo extract (growth medium). Cells are seeded into 75 cm ² flasks at a density of i. ^{10 6} cells per flask. Once cells are confluent (3-4 days), they are treated with trypsin (0.25% (w / w) trypsin) and washed with growth medium. The resulting cell pellet was suspended in growth medium, seeded at a density of 2.5 to 10 ⁴ cells / well in 24 well plates σ (16 mm diameter) and allowed to grow ..se 70% confluency (fusion) (3 days). To induce attachment, the mononuclear myoblasts are exposed to a medium containing 10 wt. mare sera instead of 20 wt. FBS (connection medium). This medium is changed every day to avoid premature cell tearing. Cells are almost completely connected to multinucleated royotubes on day 9 (day 6 in this medium). Medium was changed the day before the experiment. * ^; fc.il. '

The uptake of 2-deoxyglucose in the Cs ₁₂ muscle tubes is determined as is. described by Klip et al., Biochem. J. 242.,

131 (1987). Briefly, cells are washed with PBS (phosphate buffered saline) and incubated for 5 hours in DMEM medium without high glucose (25mM) serum. At the end of incubation, cells are washed with PBs7. Adding different doses of amylin or amylin analogs , (10 µM to 10 µΜ) and incubate with 2-deoxy- [ ³ H] -glucose (1 mM) for 10 minutes. The non-carrier-mediated intake is determined by incubating the cells with cytochalasin B (15 µΜ). Intake was determined by rapid suction of the solution, the cells were washed with ice-cold PBS, and cell-related radioactivity was determined by lysing the cells in 1M NaOH, aliquots. neutralized and measured in a pulse counter. Protein content in each aliquot is determined by the Lowry method. ~. .- ** ·

I 'i

After seeding, undifferentiated mono nuclear myoblasts grew logarithmically and reached 70% confluency at 3 days. Fusion cells are detected on day 5, on day 9 they contain more than -90% more nuclear myotubes (6 days in fusion environment). 6 day old cells showed a 30% increase in glucose uptake in response to insulin compared to a 68-115% increase in 9 day old cells. These results are similar to previous observations (Klip et al., Supra). The low insulin response by 6 days old cells is probably due to the presence of undifferentiated myoblasts with low insulin receptor density as evident in muscle cell line _L6 (Beguinot et al .: Endocrinology 18, 446 (1986).). In view of these findings and observations,

That insulin stimulates glucose uptake in 9-day-old cells in a dose-dependent manner, we have used 9 day-old c ₂ C ₁₂ cells to test the effects of amylin or amylin analogues on insulin-stimulated glucose uptake. Maximum insulin stimulation responses were observed at 100 nM and maintained the same level with further dose escalation. Apparently, 2e insulin stimulated glucose uptake in myotubes C ₂ C _i2 occurs mainly facilitating diffusion because cytochalasin B (15 μΜ) inhibited more than 90% of insulin-stimulated 2-deoxyglucose receiving cells.

Sprague Dawley rats (Miller Resin, Zelienople, Pa.) Used in this invention are housed individually in air-conditioned rooms (22-24 ° C) with a 12 hour light / 12 hour dark cycle with access to Purina rat food and water ad libitum.

Sprague Dawley rats weighing 300 g are attracted overnight (18-22 hours). The rats are then anesthetized with pentobarbital (40 mg / kg). A catheter is implanted into the jugular vein. Saline (0.1 ml), rat amylin (50 µg) in saline (0.1 ml) or peptide fragments are injected via the jugular vein. analogues (100 µg) in saline (0.1 ml) and then rinsed with an additional 0.1 ml of saline. In the case of antagonistic effects, two minutes followed by injection of peptide fragments / analogs (100 µg) in saline (0, 1 ml) with rat amylin (50 μς) in saline (0.1 ml) 30 minutes after injection of the peptide, blood (4-5 ml) is drawn via the jugular vein and a db heparinized tube containing aprotinin (10 μΐ) is inserted. The plasma glucose and insulin levels are determined by a glucose oxidase method (Glucose Analyzer Model 27, Yellow Spring Instruments, Yellow Springs, OH) and a radioimmunoassay kit (Peninsula Laboratories, Belmont, Ka.).

As seen in Figure 2, one of the antagonists of the invention, Ν-α-ac-human amylin (8-23) -NH ₂ , shows no significant effect on insulin-stimulated glucose uptake in the in vitro assay when tested separately. Again, Figure 2 shows that the presence of Na-ac-human amylin (8-23) -NH ₂ (1 μΜ) with human 16 amylin significantly shifts the response curve at the human amylin inhibition dose to insulin-stimulated glucose uptake to the right (i.e., higher). concentration of human amylin), thereby increasing the IC ₅₀ value from 0.20 nM to 350 nM.

The in vivo effects of Ν-α-ac-human amylin (8-23) -NH ₂ were studied in anesthetized (45 mg / kg) Sprague Dawley rats (300 g) fasted overnight (20 hours or longer). By cannulated jugular vein, each rat was injected with the following samples: (1) 100 μΐ of the lower solution (n = 5), (2) rat amylin (50 Mg), (3) Ν-α-ac-human amylin (8-23) -NH ₂ (100 Mg) and (4) Ν-α-ac-human amylin (8-23) -NH ₂ (100 µν) followed by (after two minutes) rat amylin (50 M9) * After 30 minutes from 4 to 5 ml of heparinized 2-tube blood is taken from each rat. The plasma is separated by centrifugation. Plasma levels of glucose and insulin are then determined. The results are shown in Figures 3a and 3b.

Figure 3a shows that rat amylin significantly increases plasma glucose levels compared to saline control, while Ν-α-ac-human amylin (8-23) -NH ₂ significantly lowers glucose levels in plasma. plasma control, possibly by antagonizing the effects of endogenous amylin. Referring to Figure 3a, Ν-α-ac-human amylin (8-23) -NH ₂ significantly enhances plasma glucose levels by rat amylin in rats treated as Ν-α-ac-human amylin (8-23). Thus, -NH ₂ and rat amylin (i.e., plasma glucose levels fell to values close to control values). The values of p in Figs. 3a and 3b et seq. Relate to the values obtained using ANOVA sn 5 to 8.

These observations confirm that Ν-α-ac-human amylin (8-23) -NH _S is a potential antagonist of human amylin in vitro and rat amylin in vivo.

Referring to Figure 4, human amylin (1-23) -NH ₂ inhibits insulin-stimulated glucose uptake in an in vitro assay in a manner similar to human amylin. Referring still to Figure 4, human 17 amylin (1-23) -NH ₂ exhibits a dose-dependent inhibitory effect on insulin-stimulated glucose uptake by C ₂ C ₁₂ cells at an intensity comparable to intact human amylin.

Referring to Figure 5a, human amylin (1-23) -NH ₂ potentiates rat amylin-induced hyperglycemia.

Referring to Figure 6, [Anb ^1.7 ] rat amylin (1-23) -NHs inhibits insulin-stimulated glucose uptake in an in vitro assay in a manner qualitatively similar to that of rat amylin (1-23) -NH2. It can be seen from Figures 6 and 4 that rat amylin (1-23) -NH2 exhibits a dose-dependent inhibitory effect on insulin-stimulated glucose uptake in C2C12 cells with an intensity comparable to that of intact human amylin. From the last two figures 6 and 4, it can be seen that [Anb ² ' ⁷ ] rat amylin (1-23) -NH 2 also exhibits an intensity comparable to that of human amylin;

From Figure 7, it can be seen that [Anb ² ' ⁷ ] rat amylin (1-23) -NH _s has no significant effect on amylin-induced hyperglycemia, but a tendency to increase was observed.

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

'I t.

Table 1

peptides C ₂ C ₁₂ effect on insulin-stimulated glucose intake ¹ anesthetized krvsv glucose ^1-2 calcium in the plasma ²⁴ in the plasma 1. human amylin inhibits (fig.2) increases ³ decreases ⁴ (HA) 2. rat amylin (RA) inhibits ⁵ increases ₃ decreases ⁴ 3. HA (1-23) -NH ₂ inhibits (similarly 1. Reduces N.D. as human amylin) (Fig. 4) bázální (Fig. 5a) 2. Increases _____ amylin induced hyper- glycemia 4th RA (l-23) _-NH2 inhibits (similar to human amy- (Fig. 6) ' N.D, N.D. r * "* 5th ^{[ANB '7]} / RA (1- inhibits (similarly 1. Reduces N.D. basal -23) -NH _s as human amylin (Fig. 6) (Fig. 7a) 2. has no effect to amylin induced hyperglycemia 6. N-α-Ac-HA 1. No influence 1. Reduces N.D. (8-23) NH ₂ 2. Increases influence basal ⁴¹ ' amylin (fig. 2) (Fig. 3a) 2. 2vysuje amylin induced hyper- glycemia

¹ This study.

² Effect of 100 µg peptide analogs on hyperglycemia-induced basal or 50 µg rat amylin.

³ Molina et al., Diabetes 39, 260 (1990) and Young et al., Am.

J. Physiology 259: E457 (1990).

⁴ Data et al., Biochem. Biophys. Res. Commun. 162. 876 (1989).

⁵ Sheriff et al., Biochim. Biophys. Acta 1136,219 (1992).

The effect of an agonist or antagonist of other amylin analogs of the invention can be determined by the assays described above.

Thus, amylin inhibits insulin-stimulated glucose uptake and glycogen synthesis and increases hepatic glucose output. Thus, an appropriate ratio of insulin to amylin appears to be needed to maintain normal blood glucose levels.

The amylin agonists and antagonists of the invention have useful applications in the treatment of dibates mellitus types I and II. Because people with type II diabetes have elevated plasma levels of amylin and glucose, administration of the amylin antagonist of the invention in an amount sufficient to lower blood glucose levels to normal or clinically acceptable levels gives therapeutic results. People with type 1 diabetes mellitus have elevated levels of both insulin and amylin and tend to develop hypoglycaemia when treated with insulin. Administration of an amylin agonist of the invention in an amount sufficient to raise blood glucose levels to normal or clinically acceptable levels in response to insulin-induced hypoglycemia, together with a therapeutic amount of insulin, provides therapeutic results.

The amylin agonists of the invention reduce serum calcium levels and can be administered to humans for the treatment of hypercalcemia. The amylin agonists of the invention exhibit appetite suppressing effects, while amylin antagonists increase appetite. Thus, the amylin agonists and antagonists of the invention are useful in regulating food intake. For example, the amylin agonists of the invention may be administered in the treatment of obesity.

The peptides of the invention may be administered to a human by one of the traditional routes of administration (eg, orally, parenterally, transdermally, or transmucosally) or in the form of a sustained release formulation using a biodegradable biocompatible polymer.

Claims (7)

Amylin analogs of general formula I R _x - (R ₂ ) Χ-Α Α Α Α Α Α Α Α Α Α Α Α Α -A 'A ²³ -YZ (I), in which X is a chain of up to 5 amino acids including the N-terminal amino acid that is linked to R _x and R ₂ , Y is a chain of up to 4 amino acids including a C-terminal amino acid that is bound to Z, R ₁ and R ₂ independently represent hydrogen, C 1 -C 12 alkyl (eg methyl), C 6 -C 6 aryl 18 carbon atoms (e.g., phenyl, naphthalenacetyl), acyl of 1 to 12 carbon atoms (e.g., formyl, acetyl and myristoyl), aralkyl of 7 to 18 carbon atoms (e.g., benzyl), or alkaryl of 7 to 18 atoms carbon (e.g., p-methylphenyl), A ⁸ is Ala, Nal, Thi, Phe, Bth, Pcp, N-Me-Thr, Aib, or Anb, A ⁹ is Thr, Ala, Anb, Aib, Ser, N-Me-Ser, or N-Me-Thr, A ¹⁰ is Gln, Ala, Asn, N-Me-Gln, Gly, Nva, Aib, or Anb, A ¹¹ is Arg, homo-Arg, diethyl-homo-Arg, Lys *. f -e-NH-R (where R represents a hydrogen atom, a straight or branched chain alkyl group having 1 to 10 carbon atoms, or an aryl group), Orn or Lys, A ¹² is Leu, Ile, Val, Aib, Anb, or N-Me-Leu; A ¹³ is Ala, Nal, Thi, Phe, Bth, Pcp, N-Me-Thr, Aib, or Anb, A ¹⁴ is Asn, Ala, Gln, Gly, N-Me-Asn, Nva, Aib, or Anb, A ¹⁶ is Phe, or any aromatic amino acid with or without substituents substituents A ¹⁶ is Leu, Ile, Val, Aib, Anb, or N-Me-Leu; A ¹ 'means Val, Ile, Aib,' Anb or N-Me-Val, A ¹⁸ is His, Thr, 3-Me-His, 1-Me-His, S-pyrozolylalanin, N-Me-His, Arg, homo-Arg, diethy1-homo-Arg, Lys-e-NH-R (where R represents a hydrogen atom, a straight-chain or straight-chain alkyl group having 1 to 10 carbon atoms or an aryl group), Ala, Aib, Anb or Orn, A ¹ * means Ser, Thr, N-Me-Ser, N-Me-Thr, Aib, Anb or Ala, A ²⁰ is Ser, Thr, N-Me-Ser, N-Me-Thr, Aib, Anb, or Ala, And ^ai is Asn, Ala, Gln, Gly, N-Me-Asn, Aib, Anb, or Nva, ^Aaa represents Asn, Ala, Gln, Gly, N-Me-Asn, Aib, Anb or Nva '. -f- ~ i A 'means Phe, any aromatic amino acid with or without substituents, Leu, Ile, Val, Aib, Anb, Ala or N-Me-Leu and Z is NHR ₃ or OR ₃ in which R ₃ is hydrogen, C 1 -C 12 alkyl, C 7 -C 10 phenylalkyl, C 3 -C 20 alkenyl, Or a pharmaceutically acceptable salt thereof. The amylin analogs of claim 1 which are antagonists. The amylin analogue according to claim 2, which corresponds to the Na-acetyl derivative of amino acids 8 to 23 of human amylin with an amidated carboxy group at the c-terminus (Na-ac-human amylin (8-23) -NH ₂ ) of formula III N-α-Ac-Ala-Thr-Gln-Arg-Leu-Ala-Asn-Phe-Leu-Val-His-Ser-Ser-Asn-Asn-Phe-NH 3 or a pharmaceutically acceptable salt thereof. (III) The amyllium analog of claim 2 of formula IV Na-Ac-Ala-Thr-Gln-Arg-Leu-Ala-Asn-Phe-Leu-Val-Arg-Ser-Ser-Asn-Asn-Leu-NH ₂ '. (IV) or a pharmaceutically acceptable salt thereof. 5. Amylin analogs of formula II R ₁ - (R ₂₎ XA ¹ -A ² -A ⁴ -A ^s -A ⁵ -A ⁷ -A -A ^{e e e} -A -A ¹⁰ -A ¹² -A ¹² -A ¹³ -A ¹ * - A ^ls -A ¹ * -A ¹⁷ ' -Α ^1β -Α ^1ΐ -Α ^2Ο -Α ²¹ -Α ²² -Α ²³ -Υ-Ζ (I) in which X žnaínená chain of up to five amino acids including N-terminal amino acid which is bonded to R _3, and R _2, Y is a chain of up to four amino acids including a C-koncovéí ^'amino acid which is bound to Z, the R _x and R ₂ are independently hydrogen, alkyl having 1 to 12 carbon atoms, aryl of C 6 -C 18, C 1 -C 12 allyl, C 7 -C 18 aralkyl or C 7 -C 18 alkaryl, < A ¹ is Lys, Arg, homo-Arg, diethyl-homo-Arg, Lys-e-NH-R (where R is hydrogen, C 1 -C 10 alkyl, branched or straight chain, or aryl) or Orn , ' A ² is Cys or Anb, A ³ means · Asn, Ala, Gin, Gly, N-Me-Asn, Aib, Anb, or Nva, A 'means Thr, Ser, N-Me-Ser, N-Me-Thr, Ala, Aib, or Anb, * A ⁵ is Ala, Nal, Thi, Phe, Bth, Pcp, N-Me-Ala, Aib, or Anb, A ⁶ is Thr, Ser, N-Me-Ser, N-Me-Thr, Ala, Aib, or Anb, A ⁷ is Cys, or Anb, A® is Ala, Nal, Thi, Phe, Bth, Pcp, N-Me-Ala, Aib or Anb, A * 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, alkyl having 1 to 10 carbon atoms, straight or branched chain or an aryl group), or Orn, A ¹² is Leu, Ile, Val, Aib, Anb, or N-Me-Leu; A ¹³ is Ala, Nal, Thi, Phe, Bth, Pcp, N-Me-Ala, Aib, or Anb, A ¹⁴ is Asn, Ala, Gln, Gly, N-Me-Asn, Aib, Anb, or, Nva, ._________ _____ A ¹⁵ is Phe, or any aromatic amino acid with or without substituents substituents A ¹⁶ is Leu, Ile, Val, Aib, Anb, or N-Me-Leu; A ¹⁷ is Val, Ile, Aib, Anb, or N-Me-Val; A ^1e represents His, Thr, 3-Me-His, 1-Me-His, β-pyrosolylalanine, N-Me-His, Arg, homo-Arg, diethyl-homo-Arg, Lys-e-NH-R (wherein R represents a hydrogen atom, a straight-chain or straight-chain alkyl group having 1 to 10 carbon atoms or an aryl group), Orn, Ala, Aib or Anb, And ¹⁹ means Ser, Thr, N-Me-Ser N-Me-Thr Ala, Aib or Anb, A ²⁰ means Ser, Thr, N-Me-Ser N-Me-Thr Ala, Aib or Anb, A ²¹ means ( Asn, Ala, Gin, Gly, N-Me-Asn Aib, Anb or and Nva, and ²² means Asn, < Ala, Gin, Gly, N-Me-Asn Aib, Anb or Nva ,. ^{23 23 -} means Phe, any aromatic amino acid with or without substituents, Leu, Ile, Val, Aib, Anb, Ala or N-Me-Leu and, 3 represents a group of the general formula NHR ₃ or OR ₃ in which R ₃ represents a hydrogen atom, an alkyl group of from 1 to 12 carbon atoms, a phenylalkyl group of from 7 to 10 carbon atoms; . C 3 -C 20 pins, a tonyl group, a non-tertiaryl group or a pharmaceutically acceptable salt thereof. The amylin analogue according to claim 5, which corresponds to amino acids 1 to 23 of human amylin with an amidated carboxyl group at the C-terminus of human amylin (1-23) -Nt-is) of the formula v L ys-C ys-Asr-T hr-A1 and-Thr-Cys-Ala-Thr-0ln-Arg-Leu-Ala-AsnPhe-Leu-Vai-His-Ser-Ser-Asn-Asη-Phe-N rte Or a pharmaceutically acceptable hall thereof; ' ^k 7, the phthrayin esters of claim 3, correspond to amino acids 1 at 23 of rat and my 3 amidated carbo. amyim, - .. rt- ·: .... formulas Vl t, y s- C y = -Asn - Τ hr - A ia - T hr - C ye - A1 a - T hr - □ 1 n - A rg - oeu - A1 a - A snf 'he - eu - Va1 - rt rg - 3 er - 3 er - rt sn ~ rt sn - L eu - t It is a pharmaceutically acceptable salt thereof. An amine analogue according to claim S, which corresponds to an amino acid derivative of amylin and 23-amylin, with the proviso that the amylin derivative is an amine derivative. in position iaííimsvia οη3λ: -. ..Knaa 2 and 7 is substituted with n-amino-butyric acid and is ma; karhoxyskupinu amidated at the C-terminus íCŘnb ^{2 7} J rat amy11n '. 1-23) -Ni-u> in the lens S S 11 S (Ul I? 2 ye - rt η u-rt sn - T hr — rt1 a – 7 hr - An b- A1 a - T hr - G1 n - A g g - L eu --- is - i-en en rthe-Leu Va 1 -Ar--; 2: ..... ·: -.? .....- sn-ni-2 ··. / I £: neno; v; pharmaceutically acceptable. A composition for regulating food intake in a human body, comprising administering to said human a therapeutic amount of an amylin analogue according to claim 1 or 5. 1 Lys Leu Asn Cys Wall Wall Asn His Gly Thr Ser Ser 5 Ala 20 May Ser 35 Asn Thr Asn Thr Cys Asn Tyr Ala Phe Thr Gly 10 Gin 25 Ala Arg Ile Leu Leu Ala Ser Asn Ser 15 Dec Phe 30 Thr h-amylin (VIII) 1 5 10 15 Dec Lys Cys Asn Thr Ala Thr Gin Arg Asn Phe 20 May 25 30 Leu Val 'Arg' Ser ^- Ser ^- Asn Asn - Leu Gly-Pro-Val-Leu Pro For Thr Asn Val Gly Ser Asn Thr Tyr r-amylin (IX) Giant. 1 sj I J Giant. 2 inhibition of insulin (100 nM) stimulated glucose uptake (% maximum) 110 inhibition of insulin (100 nM) stimulated glucose uptake (% of maximum) Giant. 4 peptides, log [M] 0 ^ 5 200 plasma glucose (mg / dl) 100 ALIGN! II Giant. 3A Giant. 3B a = p <0.05 versus control b = not significant versus control c = p <0.05 versus rat amylin I = saline solution II = rat amylin (50 μφ) III = N-α-ac-human amylin (8-23) -nh2 (100 μς) IV = N-α-ac-human amylin (8023) -NH = (100 Mg) plus rat amylin (50 Mg) a = p <0.05 versus control b = p <0.05 versus rat amylin; a = p <0.05 versus control b = not relevant to control c = p <0.05 versus rat amylin a = p <0.05 versus control b = not significant to control Giant. 5B c = p <0.05 against rat amylin I = saline solution II = rat amylin (50 yg) III = human amylin (1-23) -NH ² (50 gg) IV = human amylin (1 '23) -NH ² (100 μς) v = human amylin (1-23) -NH ² (100 Mg) plus blood amylin PM J Giant. 6 Δ Τ 'Ν Fig. Insulin (gjedn./mlJ Giant. 7B I = saline solution II = rat amylin (50 pg) III = [Amb ² ' ⁷ ] rat amylin (1-23) -ΝΗ ² (100 gg) XV = [Amb ^s ' ⁷ ] rat amylin (1-23) -NH ² (100 gg) plus rat amylin (50 gg) a = p <0.05 vs control b = not significant vs control c = p <0, 05 against rat amylin