United States Patent (19)
`Gaeta et al.
`
`54 AMYLIN AGONIST PEPTDES AND USES
`THEREFOR
`
`75) Inventors: Laura S. L. Gaeta, Foster City;
`Howard Jones, Poway; Elisabeth
`Albrecht, San Diego, all of Calif.
`73) Assignee: Amylin Pharmaceuticals, Inc., San
`Diego, Calif.
`
`(21) Appl. No.: 447,849
`22 Filed:
`May 23, 1995
`Related U.S. Application Data
`63 Continuation of Ser. No. 794.266, Nov. 19, 1991, aban
`doned, which is a continuation-in-part of Ser. No. 667,040,
`Mar. 8, 1991, abandoned.
`(51) Int. Cl. ............. A61K 38/16; A6K 38/28:
`CO7K 14/00
`52 U.S. Cl. ................................. 514/12: 514/2; 514/4;
`514/866; 530/324
`58 Field of Search ................................. 530/324; 514/2,
`514/4, 806, 12
`
`56
`
`
`
`References Cited
`U.S. PATENT DOCUMENTS
`5,124,314 6/1992 Cooper ..................................... 514/4
`5,175,145 12/1992 Cooper ........................................ 51474
`5,367,052 11/1994 Cooper et al. .......................... 530/307
`FOREIGN PATENT DOCUMENTS
`0309100 8/1988 European Pat. Off..
`OTHER PUBLICATIONS
`Clark, A., et al., Lancet i: 231-234 (1987).
`Cooper, G.J.S., et al. Proc. Natl. Acad. Sci. (USA)
`84:8628-8632 (1987).
`Cooper G.J.S., et al., in Diabetes 1988, ed. Larkins, R.,
`Zimmet, P. & Chisholm, D. (Elsevier, Amsterdam), pp.
`493-496 (1989).
`Glenner et al., Biochem, Biophys. Res
`155:608-614 (1988).
`Westermark et al., Proc. Natl. Acad. Sci. (USA) 87:
`5036-5040 (1990).
`Dayhoff et al., "Atlas of Protein Sequence and Structure",
`vol. 5 pp. 89-99, 1972.
`
`Commun,
`
`US005686411A
`Patent Number:
`11
`45 Date of Patent:
`
`5,686,411
`Nov. 11, 1997
`
`Goodman & Gilman, "The Pharmacological Basis of Thera
`peutics" 6th Ed. pp. 1514-1519, 1980.
`O'Brien, et al., Islet Amyloid Polypeptide and Insulin Secre
`tion From Isolated Perfused Pancreas of Fed, Fasted, Glu
`cose-Treated, and Dexamethasone-Treated Rats, Diabetes
`40: 1701-1706 (1991).
`Doherty, Endogenous Vasoactive Peptides, Annual Reports
`In Medicinal Chemistry 26: 83-92 (1991).
`Ohagi, et al., Sequences of Islet Amyloid Polypeptide Pre
`cursors of An Old-World Monkey, The Pig-Tailed Macaque
`(Macaca-nemestrina), and The Dog (Canis familiaris), Dia
`betologia 34: 555-558 (1991).
`Gustavsson, et al., Normal Transthyretin And Synthetic
`Transthyretin Fragments Form Amyloid-Like Fibrils In
`Vitro, Biochem, Biophys. Res. Commun. 175: 1159–1164
`(1991).
`Bell, Molecular Defects. In Diabetes-Mellitus, Diabetes 40:
`413-422 (1991).
`Johnson, et al., Newly Identified Pancreatic Protein Islet
`Amyloid Polypeptide-What Is Its Relationship To Diabe
`tes?, Diabetes 40: 310-314 (1991).
`Steiner, et al., Is Islet Amyloid Polypeptide A Significant
`Factor In Pathogenesis Or Pathophysiology of Diabetes?,
`Diabetes 40: 305-309 (1991).
`Johnson, et al., Amyloid In The Pancreatic-Islets of The
`Cougar (Felis-concolor) Is Derived From Islet Amyloid
`Polypeptide (IAPP), Comp. Biochem. Physiol. 98: 115-119
`(1991).
`Porte, Beta Cells InType II Diabetes Mellitus, Diabetes 40:
`166-180 (1991).
`Stridsberg and Wilander, Islet Amyloid Polypeptide
`(IAPP)-A Short Review, Acta Oncologica 30: 451–456
`(1991).
`Hilbich, et al., Aggregation and Secondary Structure of
`Synthetic Amyloid Beta-A4 Peptides of Alzheimer's Dis
`ease, J. Mol. Bio. 218: 149-163 (1991).
`Primary Examiner-Cecilia J.Tsang
`Assistant Examiner-Bennett Celsa
`Attorney, Agent, or Firm-Lyon & Lyon, L.L.P.
`57
`ABSTRACT
`Agonist analogues of amylin and related pharmaceutical
`compositions, and methods of treatment of diabetes and
`other insulin-requiring states, as well as methods of treat
`ment of hypoglycemia, are provided.
`
`45 Claims, 3 Drawing Sheets
`
`MYLAN INST. EXHIBIT 1082 PAGE 1
`
`MYLAN INST. EXHIBIT 1082 PAGE 1
`
`

`

`U.S. Patent
`
`Nov. 11, 1997
`
`Sheet 1 of 3
`
`5,686,411
`
`FIGURE
`
`- 'Llys-Cys-Asn-Thr-Ala-Thr-eys-Ala-Thr-Gln-Arg-Leu-Ala
`Asn-Phe-Leu-Val-His-Ser-ser-Asn-Asn-Phe-Gly-Ala
`Ille-Leu-S er-Ser-Thr-Asn-val-Gly-Ser-Asn-Thr-Tyr-NH,
`
`MYLAN INST. EXHIBIT 1082 PAGE 2
`
`MYLAN INST. EXHIBIT 1082 PAGE 2
`
`

`

`U.S. Patent
`
`Nov. 11, 1997
`
`Sheet 2 of 3
`
`5,686,411
`
`FIGURE 2
`
`Amylin
`
`human
`cat
`dog
`
`rat
`
`KCNTATCATORLANFLVHSSNNFGAILSSTNVGSNTY-NH
`---------------- IR-------ee- P--------
`-----------------RT--------P--------
`
`-----------------R----L-Pv-PP ww
`
`a bar
`
`OSe
`hamster
`guinea pig
`
`man armrms or --- mass---R----L-PW-PP- - ------
`----as-r------------N--L-PV--P--------
`------------T----R-H-L-A-LP-D------
`
`MYLAN INST. EXHIBIT 1082 PAGE 3
`
`MYLAN INST. EXHIBIT 1082 PAGE 3
`
`

`

`U.S. Patent
`
`Nov. 11, 1997
`
`Sheet 3 of 3
`
`5,686,411
`
`FIGURE 3
`
`A-X-Asn-Thr-Ala-Thr-Y-Ala-Thr-Gln-Arg-Leu
`B-Asn-Phe-Leu-C-D-E-F-G-Asn-H-Gly-I-J-
`Leu-K-L-Thr-M-val-Gly-Ser-Asn-Thr-Tyr-Z
`
`15
`
`20
`
`25
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`MYLAN INST. EXHIBIT 1082 PAGE 4
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`

`

`5,686.411
`
`1.
`AMYLN AGONST PEPTDES AND USES
`THEREFOR
`
`This is a continuation of application Ser. No. 07/794.266
`filed on Nov. 19, 1991, now abandoned, which is a
`continuation-in-part of U.S. application Ser. No. 07/667,040
`filed Mar. 8, 1991 (abandoned), which is hereby incorpo
`rated by reference.
`
`10
`
`15
`
`Introduction to the
`
`BACKGROUND
`1. Field of the Invention
`The field of the invention is medicine, particularly the
`treatment and prevention of hypoglycemic conditions and
`other conditions in which enhanced amylin action is of
`benefit, including insulin-requiring states such as diabetes
`mellitus. More specifically, the invention relates to the
`preparation and use of agonist analogues of the peptide
`hormone amylin.
`2. Description of Related Art and
`Invention
`Diabetes mellitus is a serious metabolic disease that is
`defined by the presence of chronically elevated levels of
`blood glucose (hyperglycemia). This state of hyperglycemia
`is the result of a relative or absolute lack of activity of the
`peptide hormone, insulin. Insulin is produced and secreted
`by the B cells of the pancreas. Insulin is reported to promote
`glucose utilization, protein synthesis, and the formation and
`storage of neutral lipids. Glucose, the principal source of
`carbohydrate energy, is stored in the body as glycogen, a
`form of polymerized glucose, which may be converted back
`into glucose to meet metabolism requirements. Under nor
`mal conditions, insulin is secreted at both a basal rate and at
`enhanced rates following glucose stimulation, all to main
`tainmetabolic homeostasis by the conversion of glucose into
`glycogen.
`The term diabetes mellitus encompasses several different
`hyperglycemic states. These states include Type 1 (insulin
`dependent diabetes mellitus or IDDM) and Type 2 (non
`insulin-dependent diabetes mellitus or NIDDM) diabetes.
`The hyperglycemia present in individuals with Type I dia
`betes is associated with deficient, reduced, or nonexistent
`levels of insulin which are insufficient to maintain blood
`glucose levels within the physiological range. Treatment of
`Type 1 diabetes involves administration of replacement
`45
`doses of insulin, generally by the parenteral route. The
`hyperglycemia present in individuals with Type II diabetes
`is initially associated with normal or elevated levels of
`insulin; however, these individuals are unable to maintain
`metabolic homeostasis due to a state of insulin resistance in
`peripheral tissues and liver and, as the disease advances, due
`to a progressive deterioration of the pancreatic B cells which
`are responsible for the secretion of insulin. Thus, initial
`therapy of Type 2 diabetes may be based on diet and lifestyle
`changes augmented by therapy with oral hypoglycemic
`agents such as sulfonylureas. Insulin therapy is often
`required, however, especially in the latter stages of the
`disease, in attempting to produce some control of hyperg
`lycemia and minimize complications of the disease. Thus,
`many Type 2 diabetics ultimately require insulin in order to
`survive.
`Amyloid is the name given to extracellular deposits off
`sheet protein filaments. Deposits of amyloid material have
`been reported to be found in pancreas of patients with Type
`2 diabetes mellitus. Other studies have indicated that the
`degree of amyloid depositions increases with the degree of
`hyperglycemia in humans and the severity of Type 2 diabe
`
`2
`tes. Chemical analysis of pancreatic amyloid led to the
`surprising and unexpected discovery of the peptide
`hormone, amylin. Clark, A., et al., Lancet ii. 231-234
`(1987). This peptide was discovered to be comprised of 37
`amino acids, none of which are acidic residues, to have a
`disulfide linkage between the cysteine residues at positions
`2 and 7, and to be C-terminally amidated. Amylin is the
`major protein constituent of the amyloid which is reported to
`be found in the pancreatic Islets of Langerhans in patients
`with type 2 diabetes mellitus.
`It has been reported that the presence of both the intramo
`lecular cystine bridge and the carboxy terminal amide group
`in the peptide structure of the synthetic molecule yield the
`greatest biological activity to inhibit glycogen synthesis in
`skeletal muscle. E.g., Cooper, G. J. S., et al., Proc. Natl.
`Acad. Sci. (U.S.A.) 84:8628-8632 (1987); Cooper G. J. S.,
`et al., in Diabetes 1988, ed. Larkins, R., Zimmet, P. &
`Chisholm, D. (Elsevier, Amsterdam), pp. 493-496 (1989).
`The amino acid sequence of amylin (see FIG. 1) has 46%
`homology with human calcitonin gene related peptide 2
`(CGRP-2).
`One report states that a limited segment of the amylin
`molecule, residues 20-29, is a potential contributor toward
`amyloid fibril formation in the islets of Langerhans in Type
`2 diabetes mellitus. Glenner et al., Biochem. Biophys. Res
`Commun. 155:608-614 (1988). It has also been reported that
`amino acid sequence differences between amylins from
`certain mammalian species occur in this region, and further
`investigation has focused on identifying residues linked to
`amyloid formation. Westermarket al., Proc. Natl. Acad. Sci.
`(USA) 87: 5036-5040 (1990). The study of Westermarket
`al. reported attempts to synthesize various 20-29 amino acid
`segments of amylin sequences from different species foll
`lowed by a comparison of their ability to form amyloid
`fibrils. It was proposed that the residues 25-29 of human
`amylin were the most strongly amyloidogenic and that the
`proline-for-serine substitution in position 28, as in several
`rodent species, significantly inhibited fibril formation in the
`studied decapeptides.
`Amylin is a complex peptide, and the synthesis of bio
`active preparations of amylin is laborious. Amylin has also
`been found to have limited solubility and limited stability in
`solution. We have found that rat amylin has a higher
`solubility and stability in solution than human amylin. This
`may be due in some measure, although this is not known, to
`the different aggregation properties of the amylins from
`different species. Only the human, non-human primate, and
`cat species of amylin have been reported to aggregate to
`form islet amyloid in vivo. The sequences of amylin now
`reported to have been isolated from a number of species are
`set forth in FIG. 2.
`In Type I diabetes, amylin levels are severely reduced or
`are nonexistent when compared to normal controls. In the
`disease state of Type I diabetes mellitus, the B-cells, which
`are the producers of insulin and amylin, have been destroyed
`by an autoimmune process. Amylin has been proposed to be
`useful in the treatment of diabetes mellitus and
`hypoglycemia, including insulin-induced hypoglycemia. It
`has also been proposed that the co-administration of insulin
`with amylin is a superior therapy to the existing adminis
`tration of insulin alone, and that coadministration of amylin
`with glucagon for the treatment of hypoglycemia is a
`superior therapy to the existing administration of glucagon
`alone. It would be useful to provide, for such purposes and
`others, less complicated compounds that have the activities
`of native human amylin, as well as compounds which may
`show enhanced solubility and/or stability overnative human
`amylin. Such compounds are described and claimed herein.
`
`25
`
`30
`
`35
`
`50
`
`55
`
`65
`
`MYLAN INST. EXHIBIT 1082 PAGE 5
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`MYLAN INST. EXHIBIT 1082 PAGE 5
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`

`

`5.686.411
`
`3
`SUMMARY OF THE INVENTION
`The present invention is directed to novel analogues of the
`peptide hormone amylin. These compounds mimic the
`effects of amylin, and are referred to as amylin agonists or
`as agonist analogues of amylin.
`The invention is also directed to pharmaceutical compo
`sitions comprising the agonist analogues of the present
`invention, and to methods of treatment and prevention of
`hypoglycemic conditions and other conditions in which
`enhanced amylin action is of benefit, including insulin
`requiring states such as diabetes mellitus, comprising
`administering an agonist analogue of amylinto an animal
`(alone or in conjunction with an insulin or a glucagon).
`Definitions
`As used herein, the following terms have the following
`meanings unless expressly stated to the contrary:
`The term "alkyl" refers to both straight- and branched
`chain alkyl groups. The term "lower alkyl" refers to both
`straight- and branched-chain alkyl groups having a total of
`from 1 to 6 carbon atoms and includes primary, secondary
`and tertiary alkyl groups. Typical lower alkyls include, for
`example, methyl, ethyl, n-propyl, isopropyl, n-butyl,
`isobutyl, t-butyl, n-penty, n-hexyl, and the like.
`The term "aryl" refers to carbocyclic aromatic groups of
`6 to 14 cartbon atoms such as phenyl and naphthyl, as well
`as heterocyclic aromatic groups containing 1 to 3 heteroa
`toms (nitrogen, oxygen, sulfur, etc.) such as pyridyl,
`triazolopyrazine, pyrimidine and the like.
`The term "aralkyl" refers to an “aryl" group of 6 to 10
`carbon atoms directly attached to an "alkyl" group of 1 to 4
`carbon atoms and includes for example benzyl,
`p-chlorobenzyl, p-methylbenzyl, and 2-phenylethyl.
`The term "cycloalkyl" refers to cyclic alkyl groups of 5 to
`8 carbon atoms.
`
`BRIEF DESCRIPTION OF THE DRAWINGS
`FIG. 1 depicts the amino acid sequence of human amylin.
`FIG. 2 depicts a comparison of amino acid sequences of
`amylins isolated from several mammals.
`FIG. 3 depicts the amino acid sequence of novel amylin
`agonist peptides.
`DETALED DESCRIPTTION OF THE
`NVENTON
`According to the present invention, novel agonist ana
`logues of amylin are provided. These analogues are useful as
`agonists of amylin, including as hyperglycemics, and may
`be represented by FIG. 3.
`In one aspect, the present invention is directed to agonist
`analogues of FIG. 3, wherein A is hydrogen Lys, Ser, Ala,
`des-o-amino Lys, or acetylated Lys; B is Ala, Seror Thr; C
`is Val, Leu or Ile; D, is His or Arg; E, is Seror Thr; F, is
`Ser. Thr, Gln or Asn; G is Asn., Gln or His; H is Phe, Leu
`or Tyr; I is Ala or Pro; J is Ile, Val, Ala or Leu; K is Ser,
`Pro, Leu, Ile or Thr; L is Ser, Pro or Thr; M is Asn. Asp
`or Gin; X and Y are independently selected residues having
`side chains which are chemically bonded to each other to
`form an intramolecular linkage; and Z is hydroxy, amino,
`alkylamino, dialkylamino, cycloalkylamino, arylamino,
`aralkylamino, alkyloxy, aryloxy or aralkyloxy; provided that
`(a) when A is Lys, B is Ala, C is Val, D is His, E is Ser,
`F is Ser, G is Asn., H is Phe, I, is Ala, J is Ile, K is Ser,
`L is Ser, and M is Asn; (b) when A is Lys, B is Ala, C,
`is Ile, D is Arg, E is Ser, F is Ser, G is Asn. His Leu,
`I is Ala, J is Ile, K is Ser. L is Pro, and M is Asn; (c)
`
`55
`
`65
`
`10
`
`15
`
`25
`
`35
`
`45
`
`4
`when A is Lys, B is Ala, C is Val, D is Arg, E is Thr,
`F is Ser, G is Asn., H is Leu, I is Ala, J is Ile, K is Ser,
`L is Pro, and M is Asn; (d) when A is Lys, B is Ala, C
`is Val, D is Arg, E, is Ser, F, is Ser, G is Asn., H is Leu,
`It is Pro, J is Val, K is Pro, L is Pro, and M is Asn; (e)
`when A is Lys, B is Ala, C is Val, D is His, E is Ser, F,
`is ASn, G is Asn., H is Leu, I is Pro, J is Val, K is Ser,
`L is Pro and M is Asn; or (f) when A is Lys, B is Thr,
`C is Val, D, is Arg, E is Ser, F, is Ser, G is His, H is Leu,
`It is Ala, J is Ala, K is Leu, L is Pro and M is Asp; then
`one or more of any of A to M is not an L-amino acid and
`Z is not amino,
`Suitable side chains for X and Y include groups derived
`from alkyl sulfhydryls which may form disulfide bonds;
`alkyl acids and alkylamines which may form cyclic lactams;
`alkyl aldehydes or alkylhalides and alkylamines which may
`condense and be reduced to form an alkyl amine bridge; or
`side chains which may be connected to form an alkyl,
`alkenyl, alkynyl, ether or thioether bond. Preferred alkyl
`chains include lower alkyl groups having from about 1 to
`about 6 carbon atoms.
`An additional aspect of the presentinvention is directed to
`agonist analogues of FIG. 3 which are not bridged, and
`wherein X and Y are independently selected from Ala, Ser,
`Cys, Val, Leu and Ile or alkyl, aryl, or aralkyl esters and
`ethers of Ser or Cys.
`Biologically active derivatives of the above FIG. 3 ago
`nist analogues are also included within the scope of this
`invention in which the stereochemistry of individual amino
`acids may be inverted from (L)/S to (D)/R at one or more
`specific sites.
`Also included within the scope of this invention are the
`agonist analogues modified by glycosylation of Asn., Ser
`and/or Thr residues.
`Biologically active agonist analogues of amylin are
`included within the scope of this invention which contain
`less peptide character. Such peptide mimetics may include,
`for example, one or more of the following substitutions for
`-CO-NH- amide bonds: depsipeptides (-CO-O-),
`iminomethylenes (-CH-NH-), trans-alkenes
`(-CH=CH-), B-enaminonitriles (-CO=CH-CN)-
`NH-), thioamides (-CS-NH-), thiomethylenes (-S-
`CH- or -CH2-S-), methylenes (-CH-C-) and
`retro-amides (-NH-CO-).
`Compounds of this invention form salts with various
`inorganic and organic acids and bases. Such salts include
`salts prepared with organic and inorganic acids, for example,
`HCl, HBr, HSO, HPO, trifluoroacetic acid, acetic acid,
`formic acid, methanesulfonic acid, toluenesulfonic acid,
`maleic acid, fumaric acid and camphorsulfonic acid. Salts
`prepared with bases include, for example, ammonium salts,
`alkali metal salts (such as sodium and potassium salts) and
`alkali earth salts (such as calcium and magnesium salts).
`Acetate, hydrochloride, and trifluoroacetate salts are pre
`ferred.
`The salts may be formed by conventional means, as by
`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.
`The compounds of the invention include various stereoi
`somers. In the preferred compounds of this invention, the
`chiral centers on the peptide backbone are all S.
`Compounds of the present invention may be prepared by
`using certain conventional coupling reactions known in the
`
`MYLAN INST. EXHIBIT 1082 PAGE 6
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`MYLAN INST. EXHIBIT 1082 PAGE 6
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`10
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`
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`
`5,686.411
`6
`5
`The preparation of certain agonist analogues of amylin
`peptide art. The analogues of this invention are prepared by
`successively adding the desired amino acid to a growing
`within the invention is described in Examples 1 to 17 herein.
`peptide chain. Typically, ano-N-carbamoyl protected amino
`In addition, other agonist analogues which may be prepared
`acid and an amino acid attached to the growing peptide chain
`according to the above procedures are set forth in Table II
`on a resin Support are reacted at room temperature in an inert
`herein. The compounds of the invention may also be pre
`solvent such as N-methylpyrrolidone, dimethylformamide
`pared using recombinant DNA techniques, using methods
`or methylene chloride in the presence of coupling agents
`now known in the art. See, e.g., Sambrook et al., Molecular
`such as dicyclohexylcarbodiimide 1-hydroxybenzotriazole
`Cloning: A Laboratory Manual, 2d Ed., Cold Spring Harbor
`in the presence of a base such as diisopropylethylamine. The
`(1989).
`ot-N-carbamoyl protecting group is removed from the result
`The nomenclature of the compounds of the present inven
`ant peptide with a reagent such as trifluoroacetic acid or
`tion can be used to indicate both the peptide that the
`piperidine, and the coupling reaction repeated with the next
`sequence is based on and the modifications made to any
`desired N-protected amino acid. Suitable N-protecting
`basic peptide amylin sequence, such as human amylin. An
`groups are known in the art, with t-butyloxycarbonyl herein
`amino acid preceded by a superscript number indicates that
`preferred.
`the named amino acid replaces the amino acid normally
`Certain preferred methods for synthesis are described in
`present at the amino acid position of the superscript in the
`the commonly-assigned copending and commonly assigned
`basic amino acid sequence. For example, "Arg'Pro-h-
`patent application Ser. No. 667,040 ("Synthetic Preparation
`amylin" refers to a peptide based on the sequence of
`of Amylin and Amylin Analogs", filed Mar. 8, 1991). These
`"h-amylin" or "human-amylin" having the following sub
`methods provide for solid phase synthesis of a peptide which
`stitutions: Arg replacing His at residue 18, Pro replacing Ala
`comprises amylin or an amylin analog which has enhanced
`at residue 25 and Pro replacing Ser at residue 28. The term
`biological activity and is substantially free of deletion and
`"des-Lys-h-amylin" refers to a peptide based on the
`other contaminating peptides wherein said peptide is syn
`sequence of human amylin, with the first, or N-terminal,
`thesized using successive synthesis cycles, whereby in each
`amino acid deleted.
`such synthesis cycle, a designated amino acid is added to a
`The agonist analogues of amylin of this invention are
`growing peptide chain attached to an insoluble resin support
`useful in view of their pharmacological properties. In
`by formation of a peptide linkage between an O-amino group
`particular, compounds of this invention possess activity as
`of the growing peptide chain and on O-carboxyl of the
`amylin agonist agents, as will be evidenced by activity in the
`designated amino acid; and wherein each synthesis cycle
`receptor binding assay and the soleus muscle assay
`comprises: (a) treating the growing peptide chain under
`described in Examples 18 and 19, respectively. Amylin
`O-amino deprotecting conditions to remove an O-amino
`agonist activity of compounds may also be assessed by the
`group; (b) activating the ot-carboxyl group of the ot-amino
`ability to induce hyperlactemia and/or hyperglycemia in
`protected designated amino acid; (c) contacting the growing
`mammals. In addition to the description of compounds
`peptide chain and the designated amino acid under coupling
`pursuant to FIG. 3, certain preferred compounds are setforth
`conditions to form a peptide linkage between the free
`in Table I. The preferred compounds des-Lys-h-amylin,
`O-amino for the peptide chain and the activated o-carboxyl
`Pro-h-amylin,
`Pro-h-amylin, Arg-Pro-h-
`of the designated amino acid; and (d) repeating steps (b) and
`amylin, and des-Lys'Arg''Pro-h-amylin, all show amy
`(c) if the coupling efficiency of step (c) is less than about
`lin activity in vivo in treated test animals, provoking marked
`97%. It is preferred to repeat steps (b) and (c)if the coupling
`hyperlactemia followed by hyperglycemia. In addition to
`efficiency is less than about 99%. In another preferred
`having activities characteristic of amylin, certain of the
`aspect, steps (b) and (c) are repeated in each synthesis cycle.
`preferred compounds of the invention have also been found
`Optionally, the coupling efficiency is measured after each
`to possess more desireable solubility and stability charac
`coupling step.
`teristics when compared to human amylin. These preferred
`Suitable coupling conditions include use of a solvent
`compounds include ProVal-Pro-h-amylin, -,
`system which maximizes swelling of the solid support,
`29Pro-h-amylin, and 'Arg'Pro-h-amylin.
`minimizes secondary structure elements of the peptide chain
`Compounds described herein which are especially pre
`during synthesis cycles, and minimizes intrapeptide and
`ferred include 'Arg'Pro-h-amylin, des-Lys-Arg
`interpeptide hydrogen bonding. Preferably the synthesis
`2sPro-h-amylin, Arg'''Pro-h-amylin, des
`cycle includes a capping step after the coupling step(s)
`Lys Arg-Pro-h-amylin, -Pro-h-amylin, des
`wherein unreacted O-amino groups of the peptide chain are
`Lys'Pro-h-amylin, and Proval-Pro-h-amylin.
`rendered unreactive. The synthesis cycle is successively
`Still further amylin agonist peptide compounds are listed in
`repeated using appropriate protected O-amino acids to give
`Table II. They include:
`amylin or an amylin analog of specified sequence. After
`LeuProval-Pro-h-amylin;
`completions of the successive synthesis cycles, said amylin
`LeuProval Pro-h-amylin;
`or amylin analog is cleaved from the solid support. It is
`preferred that the cysteine residues of the peptide chain are
`des-Lys-Leu-Proval Pro-h-amylin;
`selectively deprotected and an intramolecular disulfide bond
`Arg'Leu'Proval Pro-h-amylin;
`is formed before cleaving the peptide bond from the solid
`"Arg'Leu-Pro-h-amylin;
`Support.
`Arg-Leu-Pro-h-amylin;
`Suitable O-amino protective groups include
`7Ile-Leu-??Pro-h-amylin;
`t-butoxycarbonyl and 9-fluorenylmethoxycarbonyl. In one
`preferred aspect, when t-butoxycarbonyl is used as the
`7Ile::Pro-h-amylin;
`O-amino protecting group, the O-carboxyl groups are acti
`des-Lys"Ile:Leu'Pro-h-amylin;
`wated using dicyclohexylcarbodiimide and
`'Ile'Arg'Leu-h-amylin;
`1-hydroxybenzotriazole to form 1-hydroxybenzotriazole
`'Ile'Arg'LeuvalPro-h-amylin;
`esters. A particularly preferred solvent system comprise
`'Ile'Arg'LeuProval-Pro-h-amylin;
`N-methylpyrrollidone.
`
`45
`
`50
`
`55
`
`65
`
`MYLAN INST. EXHIBIT 1082 PAGE 7
`
`MYLAN INST. EXHIBIT 1082 PAGE 7
`
`

`

`5,686,411
`
`10
`
`7
`Thrhis'Leu'Ala'LeuPro-Asp-h-amylin;
`Thr'His'Leu'Ala'Pro'Asp-h-amylin;
`des-Lys"Thr'His'Leu'Ala'Pro'Asp-h-amylin;
`ThrArgHis'Leu’Alaipro'Asp-h-amylin;
`Thr'ArgHis'LeuProAsp-h-amylin; and,
`1Thr1'Arg? His? Leu?'Pro2°Ala8.2°Pro-Asp-h-
`anyin.
`The compounds of this invention can be combined with
`pharmaceutical carriers to prepare pharmaceutical forms
`suitable for parenteral administration. Experimental
`responses of the compounds support the clinical application
`of such pharmaceutical compositions in the treatment of
`diabetes mellitus and other insulin-requiring states, as well
`as in the prevention and treatment of episodes of hypogly
`15
`cemia. The compounds of this invention can also be com
`bined with insulin for the treatment of diabetes mellitus and
`other insulin-requiring states. By "insulin" is meant a
`polypeptide or its equivalent useful in regulation of blood
`glucose levels. A general description of such insulins is
`provided in Goodman and Gilman's The Pharmacological
`Basis of Therapeutics, 8th Ed., Pergamon Press (1990). Such
`insulins can be fast acting, intermediate acting, or long
`acting. Various derivatives of insulin exist and are useful in
`this invention. See, e.g. U.S. Pat. Nos. 5,049,547, 5,028,
`587, and 5,016,643. Insulin peptides are also useful (see,
`e.g., U.S. Pat. No. 5,008,241), as are analogues (see, e.g.,
`U.S. Pat. Nos. 4,992,417 and 4.992,418). Such compositions
`can be administered by any standard route, including nasal
`administration (see, e.g., U.S. Pat. Nos. 4.988.512 and
`4,985.242, and 2 BioWorld Today, No. 125 (1991)). The
`compounds of this invention are also useful in combination
`with a glucagon for the prevention and treatment of
`hypoglycemia. See Young et al., U.S. application Ser. No.
`07/640,478, filed Jan. 10, 1991, entitled "Hyperglycemic
`Compositions,” which is incorporated herein by reference.
`Compositions or products of the invention may conve
`niently be provided in the form of solutions suitable for
`parenteral (including intravenous, intramuscular and
`subcutaneous) or nasal or oral administration. In many
`cases, it will be convenient to provide an agonist analogue
`of amylin and an insulin or glucagon in a single composition
`or solution for administration together. In other cases, it may
`be more advantageous to administer an insulin or a glucagon
`separately from said agonist analogue. A suitable adminis
`tration format may best be determined by a medical prac
`tioner for each patient individually. Suitable pharmaceuti
`cally acceptable carriers and their formulation are described
`in standard formulation treatises, e.g., Remington's Phar
`maceutical 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). Suitable formulations including insulin or
`glucagon are known in the art.
`The agonist preparations of the invention may be stabi
`lized at neutral pH. Since the products of the invention are
`amphoteric they may be utilized as free bases, as acid
`addition salts or as metal salts. The salts must, of course, be
`pharmaceutically acceptable, and these will include metal
`salts, particularly alkali and alkaline earth metal salts, e.g.,
`potassium or sodium salts. A wide variety of pharmaceuti
`cally acceptable acid addition salts are available, as
`described above. These include those prepared from both
`organic and inorganic acids, preferably mineral acids. Typi
`cal acids which may be mentioned by way of example
`include citric, succinic, lactic, hydrochloric and hydrobro
`
`45
`
`8
`mic acids. Such products are readily prepared by procedures
`well known to those skilled in the art.
`The products of the invention will normally be provided
`as parenteral compositions for injection or infusion. They
`can, for example, be suspended in an inert oil, suitably a
`vegetable oil such as sesame, peanut, or olive oil.
`Alternatively, they can be suspended in an aqueous isotonic
`buffer solution at a pH of about 5.6 to 7.4. Useful buffers
`include sodium citrate-citric acid and sodium phosphate
`phosphoric acid. 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.
`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.
`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).
`The therapeutically useful compositions of 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 pro
`vided in dosage unit form containing an amount of an
`agonist compound with or without insulin or glucagon
`which will be effective in one or multiple doses to control or
`reestablish blood sugar at the selected level. Therapeutically
`effective amounts of an agonist analogue of amylin as
`described hereinfor the treatment of hypoglycemia are those
`that increase blood sugar levels, preferably to above 80
`mg/dl. Therapeutically effective amounts of such agonist
`analogues for the treatment of diabetes mellitus and other
`insulin-requiring states are those sufficient to provide for
`reduced incidence of insulin overdose or undesired hypogly
`cemia. As will be recognized by those in the field, an
`effective amount of therapeutic