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Title:  Exendins, exendin agonists, and methods for their use
United States Patent:  6,989,366
Issued:  January 24, 2006
Inventors:  Beeley; Nigel Robert Arnold (Solana Beach, CA); Prickett; Kathryn S. (San Diego, CA); Bhavsar; Sunil (San Diego, CA)
Assignee:  Amylin PHarmaceuticals, Inc. (San Diego, CA)
Appl. No.:  187051
Filed:  June 28, 2002


 

George Washington University's Healthcare MBA


Abstract

Methods for treating conditions or disorders which can be alleviated by reducing food intake are disclosed which comprise administration of an effective amount of an exendin or an exendin agonist, alone or in conjunction with other compounds or compositions that affect satiety. The methods are useful for treating conditions or disorders, including obesity, Type II diabetes, eating disorders, and insulin-resistance syndrome. The methods are also useful for lowering the plasma glucose level, lowering the plasma lipid level, reducing the cardiac risk, reducing the appetite, and reducing the weight of subjects. Pharmaceutical compositions for use in the methods of the invention are also disclosed.

DETAILED DESCRIPTION OF THE INVENTION

Exendins and exendin agonists are useful as described herein in view of their pharmacological properties. Activity as exendin agonists can be indicated by activity in the assays described below. Effects of exendins or exendin agonists on reducing food intake can be identified, evaluated, or screened for, using the methods described in the Examples below, or other methods known in the art for determining effects on food intake or appetite.

Exendin Agonist Compounds

Preferred exendin agonist compounds include exendin analogs. Exendin analogs are functional variants having similar amino acid sequence and retaining, to some extent, the activities of the related exendin. By "functional variant" is meant an analog which has an activity that can be substituted for one or more activities of a particular exendin. Preferred functional variants retain all of the activities of a particular exendin, however, the functional variant may have an activity that, when measured quantitatively, is stronger or weaker, as measured in exendin functional assays, for example, such as those disclosed herein.

Preferred exendin analogs include exendin agonist compounds such as those described in U.S. Provisional Application No. 60/055,404, including compounds of the formula (I) [SEQ ID NO. 3]:

 1                  5                    10
Xaa1 Xaa2 Xaa3 Gly Thr Xaa4 Xaa5 Xaa6 Xaa7 Xaa8
                 15                  20
Ser Lys Gln Xaa9 Glu Glu Glu Ala Val Arg Leu
                 25                   30
Xaa10 Xaa11 Xaa12 Xaa13 Leu Lys Asn Gly Gly Xaa14
            35
Ser Ser Gly Ala Xaa15 Xaa16 Xaa17 Xaa18-Z


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

Preferred N-alkyl groups for N-alkylglycine, N-alkylpentylglycine and N-alkylalanine include lower alkyl groups preferably of 1 to about 6 carbon atoms, more preferably of 1 to 4 carbon atoms. Suitable compounds include those listed in FIG. 10 having amino acid sequences of SEQ. ID. NOS. 9 to 39.

Preferred exendin agonist compounds include those wherein Xaa1 is His or Tyr. More preferably Xaa1 is His.

Preferred are those compounds wherein Xaa2 is Gly.

Preferred are those compounds wherein Xaa9 is Leu, pentylglycine or Met.

Preferred compounds include those wherein Xaa13 is Trp or Phe.

Also preferred are compounds where Xaa4 is Phe or naphthylalanine; Xaa11 is Ile or Val and Xaa14, Xaa15, Xaa16 and Xaa17 are independently selected from Pro, homoproline, thioproline or N-alkylalanine. Preferably N-alkylalanine has a N-alkyl group of 1 to about 6 carbon atoms.

According to an especially preferred aspect, Xaa15, Xaa16 and Xaa17 are the same amino acid reside.

Preferred are compounds wherein Xaa18 is Ser or Tyr, more preferably Ser.

Preferably Z is —NH2.

According to one aspect, preferred are compounds of formula (II) wherein Xaa1 is His or Tyr, more preferably His; Xaa2 is Gly; Xaa4 is Phe or naphthylalanine; Xaa9 is Leu, pentylglycine or Met; Xaa10 is Phe or naphthylalanine; Xaa11 is Ile or Val; Xaa14, Xaa15, Xaa16 and Xaa17 are independently selected from Pro, homoproline, thioproline or N-alkylalanine; and Xaa18 is Ser or Tyr, more preferably Ser. More preferably Z is —NH2.

According to an especially preferred aspect, especially preferred compounds include those of formula (II) wherein: Xaa1 is His or Arg; Xaa2 is Gly; Xaa3 is Asp or Glu; Xaa4 is Phe or naphthylalaine; Xaa7 is Thr or Ser; Xaa6 is Ser or Thr; Xaa7 is Asp or Glu; Xaa8 is Leu or pentylglycine; Xaa9 is Leu or pentylglycine; Xaa10 is Phe or naphthylalanine; Xaa11 is Ile, Val or t-butyltylglycine; Xaa12 is Glu or Asp; Xaa13 is Trp or Phe; Xaa14, Xaa15, Xaa16, and Xaa17 are independently Pro, homoproline, thioproline, or N-methylalanine; Xaa18 is Ser or Tyr: and Z is —OH or —NH2; with the proviso that the compound does not have the formula of either SEQ. ID. NOS. 1 or 2. More preferably Z is —NH2. Especially preferred compounds include those having the amino acid sequence of SEQ. ID. NOS. 9, 10, 21, 22, 23, 26, 28, 34, 35 and 39.

According to an especially preferred aspect, provided are compounds where Xaa9 is Leu, Ile, Val or pentylglycine, more preferably Leu or pentylglycine, and Xaa13 is Phe, Tyr or naphthylalanine, more preferably Phe or naphthylalanine. These compounds will exhibit advantageous duration of action and be less subject to oxidative degration, both in vitro and in vivo, as well as during synthesis of the compound.

Exendin agonist compounds also include those described in U.S. Provisional Application No. 60/065,442, including compounds of the formula (II) [SEQ ID NO. 4]:

  • Xaa1 Xaa2 Xaa3 Gly Xaa5 Xaa6 Xaa7 Xaa8 Xaa9 Xaa10 Xaa11 Xaa12 Xaa13 Xaa14 Xaa15 Xaa16 Xaa17 Ala Xaa19 Xaa20 Xaa21 Xaa22 Xaa23 Xaa24 Xaa25 Xaa26 Xaa27 Xaa28-Z1; wherein
  • Xaa1 is His, Arg or Tyr;
  • Xaa2 is Ser, Gly, Ala or Thr;
  • Xaa3 is Asp or Glu;
  • Xaa5 is Ala or Thr;
  • Xaa6 is Ala, Phe, Tyr or naphthylalanine;
  • Xaa7 is Thr or Ser;
  • Xaa8 is Ala, Ser or Thr;
  • Xaa9 is Asp or Glu;
  • Xaa10 is Ala, Leu, Ile, Val, pentylglycine or Met;
  • Xaa11 is Ala or Ser;
  • Xaa12 is Ala or Lys;
  • Xaa13 is Ala or Gln;
  • Xaa14 is Ala, Leu, Ile, pentylglycine, Val or Met;
  • Xaa15 is Ala or Glu;
  • Xaa16 is Ala or Glu;
  • Xaa17 is Ala or Glu;
  • Xaa19 is Ala or Val;
  • Xaa20 is Ala or Arg;
  • Xaa21 is Ala or Leu;
  • Xaa22 is Ala, Phe, Tyr or naphthylalanine;
  • Xaa23 is Ile, Val, Leu, pentylglycine, tert-butylglycine or Met;
  • Xaa24 is Ala, Glu or Asp;
  • Xaa25 is Ala, Trp, Phe, Tyr or naphthylalanine;
  • Xaa26 is Ala or Leu;
  • Xaa27 is Ala or Lys;
  • Xaa28 is Ala or Asn;
  • Z1 is —OH,
    • —NH2
    • Gly-Z2,
    • Gly Gly-Z2,
    • Gly Gly Xaa31-Z2,
    • Gly Gly Xaa31 Ser-Z2,
    • Gly Gly Xaa31 Ser Ser-Z2,
    • Gly Gly Xaa31 Ser Ser Gly-Z2,
    • Gly Gly Xaa31 Ser Ser Gly Ala-Z2,
    • Gly Gly Xaa31 Ser Ser Gly Ala Xaa36-Z2,
    • Gly Gly Xaa31 Ser Ser Gly Ala Xaa36 Xaa37-Z2 or
    • Gly Gly Xaa31 Ser Ser Gly Ala Xaa36 Xaa37 Xaa38-Z2;
    • Xaa31, Xaa36, Xaa37 and Xaa38 are independently Pro,
    • homoproline, 3Hyp, 4Hyp, thioproline,
    • N-alkylglycine, N-alkylpentylglycine or
    • N-alkylalanine; and
    • Z2 is —OH or —NH2;
      provided that no more than three of Xaa3, Xaa5, Xaa6, Xaa8, Xaa10, Xaa11, Xaa12, Xaa13, Xaa14, Xaa15, Xaa16, Xaa17, Xaa19, Xaa20, Xaa21, Xaa24, Xaa25, Xaa26, Xaa27 and Xaa28 are Ala. Preferred N-alkyl groups for N-alkylglycine, N-alkylpentylglycine and N-alkylalanine include lower alkyl groups preferably of 1 to about 6 carbon atoms, more preferably of 1 to 4 carbon atoms.



    Preferred exendin agonist compounds include those wherein Xaa1 is His or Tyr. More preferably Xaa1 is His.

    Preferred are those compounds wherein Xaa2 is Gly.

    Preferred are those compounds wherein Xaa14 is Leu, pentylglycine or Met.

    Preferred compounds are those wherein Xaa25 is Trp or Phe.

    Preferred compounds are those where Xaa6 is Phe or naphthylalanine; Xaa22 is Phe or naphthylalanine and Xaa23 is Ile or Val.

    Preferred are compounds wherein Xaa31, Xaa36, Xaa37 and Xaa38 are independently selected from Pro, homoproline, thioproline and N-alkylalanine.

    Preferably Z1 is —NH2.

    Preferable Z2 is —NH2.

    According to one aspect, preferred are compounds of formula (I) wherein Xaa1 is His or Tyr, more preferably His; Xaa2 is Gly; Xaa6 is Phe or naphthylalanine; Xaa14 is Leu, pentylglycine or Met; Xaa22 is Phe or naphthylalanine; Xaa23 is Ile or Val; Xaa31, Xaa36, Xaa37 and Xaa38 are independently selected from Pro, homoproline, thioproline or N-alkylalanine. More preferably Z1 is —NH2.

    According to an especially preferred aspect, especially preferred compounds include those of formula (I) wherein: Xaa1 is His or Arg; Xaa2 is Gly or Ala; Xaa3 is Asp or Glu; Xaa5 is Ala or Thr; Xaa6 is Ala, Phe or naphthylalanine; Xaa7 is Thr or Ser; Xaa8 is Ala, Ser or Thr; Xaa9 is Asp or Glu; Xaa10 is Ala, Leu or pentylglycine; Xaa11 is Ala or Ser; Xaa12 is Ala or Lys; Xaa13 is Ala or Gln; Xaa14 is Ala, Leu or pentylglycine; Xaa15 is Ala or Glu; Xaa16 is Ala or Glu; Xaa17 is Ala or Glu; Xaa19 is Ala or Val; Xaa20 is Ala or Arg; Xaa21 is Ala or Leu; Xaa22 is Phe or naphthylalanine; Xaa23 is Ile, Val or tert-butylglycine; Xaa24 is Ala, Glu or Asp; Xaa25 is Ala, Trp or Phe; Xaa26 is Ala or Leu; Xaa27 is Ala or Lys; Xaa28 is Ala or Asn; Z1 is —OH, —NH2, Gly-Z2, Gly Gly-Z2, Gly Gly Xaa31-Z2, Gly Gly Xaa31 Ser-Z2, Gly Gly Xaa31 Ser Ser-Z2, Gly Gly Xaa31 Ser Ser Gly-Z2, Gly Gly Xaa31 Ser Ser Gly Ala-Z2, Gly Gly Xaa31 Ser Ser Gly Ala Xaa3-Z2, Gly Gly Xaa31 Ser Ser Gly Ala Xaa36 Xaa37-Z2, Gly Gly Xaa38 Ser Ser Gly Ala Xaa36 Xaa37 Xaa38-Z2; Xa31, Xaa36, Xaa37 and Xaa38 being independently Pro homoproline, thioproline or N-methylalanine; and Z2 being —OH or —NH2; provided that no more than three of Xaa3, Xaa6, Xaa6, Xaa8, Xaa10, Xaa11, Xaa12, Xaa13, Xaa14, Xaa15, Xaa6, Xaa17, Xaa19, Xaa20, Xaa21, Xaa24, Xaa21, Xaa26, Xaa27 and Xaa28 are Ala. Especially preferred compounds include those having the amino acid sequence of SEQ. ID. NOS. 40-61.

    According to an especially preferred aspect, provided are compounds where Xaa14 is Leu, Ile, Val or pentylglycine, more preferably Leu or pentylglycine, and Xaa25 is Phe, Tyr or naphthylalanine, more preferably Phe or naphthylalanine. These compounds will be less susceptive to oxidative degration, both in vitro and in vivo, as well as during synthesis of the compound.

    Exendin agonist compounds also include those described in U.S. Provisional Application No. 60/066,029, including compounds of the formula (III)[SEQ ID NO. 5]:

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



    In accordance with the present invention and as used herein, the following terms are defined to have the following meanings, unless explicitly stated otherwise.

    The term "amino acid" refers to natural amino acids, unnatural amino acids, and amino acid analogs, all in their D and L stereoisomers if their structure allow such stereoisomeric forms. Natural amino acids include alanine (Ala), arginine (Arg), asparagine (Asn), aspartic acid (Asp), cysteine (Cys), glutamine (Gln), glutamic acid (Glu), glycine (Gly), histidine (His), isoleucine (Ile), leucine (Leu), Lysine (Lys), methionine (Met), phenylalanine (Phe), proline (Pro), serine (Ser), threonine (Thr), typtophan (Trp), tyrosine (Tyr) and valine (Val). Unnatural amino acids include, but are not limited to azetidinecarboxylic acid, 2-aminoadipic acid, 3-aminoadipic acid, beta-alanine, aminopropionic acid, 2-aminobutyric acid, 4-aminobutyric acid, 6-aminocaproic acid, 2-aminoheptanoic acid, 2-aminoisobutyric acid, 3-aminoisbutyric acid, 2-aminopimelic acid, tertiarybutylglycine, 2,4-diaminoisobutyric acid, desmosine, 2,2′-diaminopimelic acid, 2,3-diaminopropionic acid, N-ethylglycine, N-ethylasparagine, homoproline, hydroxylysine, allo-hydroxylysine, 3-hydroxyproline, 4-hydroxyproline, isodesmosine, allo-isoleucine, N-methylalanine, N-methylglycine, N-methylisoleucine, N-methylpentylglycine, N-methylvaline, naphthalanine, norvaline, norleucine, ornithine, pentylglycine, pipecolic acid and thioproline. Amino acid analogs include the natural and unnatural amino acids which are chemically blocked, reversibly or irreversibly, or modified on their N-terminal amino group or their side-chain groups, as for example, methionine sulfoxide, methionine sulfone, S-(carboxymethyl)-cysteine, S-(carboxymethyl)-cysteine sulfoxide and S-(carboxymethyl)-cysteine sulfone.

    The term "amino acid analog" refers to an amino acid wherein either the C-terminal carboxy group, the N-terminal amino group or side-chain functional group has been chemically codified to another functional group. For example, aspartic acid-(beta-methyl ester) is an-amino acid analog of aspartic acid; N-ethylglycine is an amino acid analog of glycine; or alanine carboxamide is an amino acid analog of alanine.

    The term "amino acid residue" refers to radicals having the structure: (1) —C(O)—R—NH—, wherein R typically is —CH(R′)—, wherein R′ is an amino acid side chain, typically H or a carbon containing substitutent; or (2), ##STR1##
    wherein p is 1, 2 or 3 representing the azetidinecarboxylic acid, proline or pipecolic acid residues, respectively.

    The term "lower" referred to herein in connection with organic radicals such as alkyl groups defines such groups with up to and including about 6, preferably up to and including 4 and advantageously one or two carbon atoms. Such groups may be straight chain or branched chain.

    "Pharmaceutically acceptable salt" includes salts of the compounds described herein derived from the combination of such compounds and an organic or inorganic acid. In practice the use of the salt form amounts to use of the base form. The compounds are useful in both free base and salt form.

    In addition, the following abbreviations stand for the following:

  • "ACN" or "CH3CN" refers to acetonitrile.
  • "Boc", "tBoc" or "Tboc" refers to t-butoxy carbonyl.
  • "DCC" refers to N,N′-dicyclohexylcarbodiimide.
  • "Fmoc" refers to fluorenylmethoxycarbonyl.
  • "HBTU" refers to 2-(1H-benzotriazol-1-yl)-1,1,3,3,-tetramethyluronium hexaflurophosphate.
  • "HOBt" refers to 1-hydroxybenzotriazole monohydrate.
  • "homop" or hpro" refers to homoproline.
  • "MeAla" or "Nme" refers to N-methylalanine.
  • "naph" refers to naphthylalanine.
  • "pG" or pGly" refers to pentylglycine.
  • "tBuG" refers to tertiary-butylglycine.
  • "ThioP" or tpro" refers to thioproline.
  • 3Hyp" refers to 3-hydroxyproline
  • 4Hyp" refers to 4-hydroxyproline
  • NAG" refers to N-alkylglycine
  • NAPG" refers to N-alkylpentylglycine
  • "Norval" refers to norvaline
  • "Norleu" refers to norleucine
    Preparation of Compounds

    The exendins and exendin agonists described herein may be prepared using standard solid-phase peptide synthesis techniques and preferably an automated or semiautomated peptide synthesizer. Typically, using such techniques, an α-N-carbamoyl protected amino acid and an amino acid attached to the growing peptide chain on a resin are coupled at room temperature in an inert solvent such as dimethylformamide, N-methylpyrrolidinone or methylene chloride in the presence of coupling agents such as dicyclohexylcarbodiimide and 1-hydroxybenzotriazole in the presence of a base such as diisopropylethylamine. The α-N-carbamoyl protecting group is removed from the resulting peptide-resin using a reagent such as trifluoroacetic acid or piperidine, and the coupling reaction repeated with the next desired N-protected amino acid to be added to the peptide chain. Suitable N-protecting groups are well known in the art, with t-butyloxycarbonyl (tBoc) and fluorenylmethoxycarbonyl (Fmoc) being preferred herein.

    The solvents, amino acid derivatives and 4-methylbenzhydryl-amine resin used in the peptide synthesizer may be purchased from Applied Biosystems Inc. (Foster City, Calif.). The following side-chain protected amino acids may be purchased from Applied Biosystems, Inc.: Boc-Arg(Mts), Fmoc-Arg(Pmc), Boc-Thr(Bzl), Fmoc-Thr(t-Bu), Boc-Ser(Bzl), Fmoc-Ser(t-Bu), Boc-Tyr(BrZ), Fmoc-Tyr(t-Bu), Boc-Lys(Cl-Z), Fmoc-Lys(Boc), Boc-Glu(Bzl), Fmoc-Glu(t-Bu), Fmoc-His(Trt), Fmoc-Asn(Trt), and Fmoc-Gln(Trt). Boc-His(BOM) may be purchased from Applied Biosystems, Inc. or Bachem Inc. (Torrance, Calif.). Anisole, dimethylsulfide, phenol, ethanedithiol, and thioanisole may be obtained from Aldrich Chemical Company (Milwaukee, Wis.). Air Products and Chemicals (Allentown, Pa.) supplies HF. Ethyl ether, acetic ,acid and methanol may be purchased from Fisher Scientific (Pittsburgh, Pa.).

    Solid phase peptide synthesis may be carried out with an automatic peptide synthesizer (Model 430A, Applied Biosystems Inc., Foster City, Calif.) using the NMP/HOBt (Option 1) system and tBoc or Fmoc chemistry (see, Applied Biosystems User's Manual for the ABI 430A Peptide Synthesizer, Version 1.3B Jul. 1, 1988, section 6, pp. 49-70, Applied Biosystems, Inc., Foster City, Calif.) with capping. Boc-peptide-resins may be cleaved with HF (-5° C. to 0° C., 1 hour). The peptide may be extracted from the resin with alternating water and acetic acid, and the filtrates lyophilized. The Fmoc-peptide resins may be cleaved according to standard methods (Introduction to Cleavage Techniques, Applied Biosystems, Inc., 1990, pp. 6-12). Peptides may be also be assembled using an Advanced Chem Tech Synthesizer (Model MPS 350, Louisville, Ky.).

    Peptides may be purified by RP-HPLC (preparative and analytical) using a Waters Delta Prep 3000 system. A C4, C8 or C18 preparative column (10μ, 2.2×25 cm; Vydac, Hesperia, Calif.) may be used to isolate peptides, and purity may be determined using a C4, C8 or C18 analytical column (5μ, 0.46×25 cm; Vydac). Solvents (A=0.1% TFA/water and B=0.1% TFA/CH3CN) may be delivered to the analytical column at a flowrate of 1.0 ml/min and to the preparative column at 15 ml/min. Amino acid analyses may be performed on the Waters Pico Tag system and processed using the Maxima program. Peptides may be hydrolyzed by vapor-phase acid hydrolysis (1150° C., 20-24 h). Hydrolysates may be derivatized and analyzed by standard methods (Cohen, et al., The Pico Tag Method: A Manual of Advanced Techniques for Amino Acid Analysis, pp. 11-52, Millipore Corporation, Milford, Mass. (1989)). Fast atom bombardment analysis may be carried out by M-Scan, Incorporated (West Chester, Pa.). Mass calibration may be performed using cesium iodide or cesium iodide/glycerol. Plasma desorption ionization analysis using time of flight detection may be carried out on an Applied Biosystems Bio-Ion 20 mass spectrometer. Electrospray mass spectroscopy may be carried out on a VG-Trio machine.

    Peptide compounds useful in the invention may also be prepared using recombinant DNA techniques, using methods now known in the art. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, 2d Ed., Cold Spring Harbor (1989). Non-peptide compounds useful in the present invention may be prepared by art-known methods. For example, phosphate-containing amino acids and peptides containing such amino acids, may be prepared using methods known in the art. See, e.g., Bartlett and Landen, Biorg. Chem. 14:356-377 (1986).

    The compounds described above are useful in view of their pharmacological properties. In particular, the compounds of the invention possess activity as agents to reduce food intake. They can be used to treat conditions or diseases which can be alleviated by reducing food intake.

    Compositions useful in the invention may conveniently be provided in the form of formulations suitable for parenteral (including intravenous, intramuscular and subcutaneous) or nasal or oral administration. In some cases, it will be convenient to provide an exendin or exendin agonist and another food-intake-reducing, plasma glucose-lowering or plasma lipid-lowering agent, such as amylin, an amylin agonist, a CCK, or a leptin, in a single composition or solution for administration together. In other cases, it may be more advantageous to administer the additional agent separately from said exendin or exendin agonist. A suitable administration format may best be determined by a medical practitioner for each patient individually. Suitable pharmaceutically acceptable carriers and their formulation are described in standard formulation treatises, e.g., Remington's Pharmaceutical Sciences by E. W. Martin. See also Wang, Y. J. and Hanson, M. A. "Parenteral Formulations of Proteins and Peptides: Stability and Stabilizers," Journal of Parenteral Science and Technology, Technical Report No. 10, Supp. 42:2S (1988).

    Compounds useful in the invention can be provided as parenteral compositions for injection or infusion. They can, for example, be suspended in an inert oil, suitably a vegetable oil such as sesame, peanut, olive oil, or other acceptable carrier. Preferably, they are suspended in an aqueous carrier, for example, in an isotonic buffer solution at a pH of about 3.0 to 8.0, preferably at a pH of about 3.5 to 5.0. These compositions may be sterilized by conventional sterilization techniques, or may be sterile filtered. The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH buffering agents. Useful buffers include for example, sodium acetate/acetic acid buffers. A form of repository or "depot" slow release preparation may be used so that therapeutically effective amounts of the preparation are delivered into the bloodstream over many hours or days following transdermal injection or delivery.

    The desired isotonicity may be accomplished using sodium chloride or other pharmaceutically acceptable agents such as dextrose, boric acid, sodium tartrate, propylene glycol, polyols (such as mannitol and sorbitol), or other inorganic or organic solutes. Sodium chloride is preferred particularly for buffers containing sodium ions.

    The claimed compositions can also be formulated as pharmaceutically acceptable salts (e.g., acid addition salts) and/or complexes thereof. Pharmaceutically acceptable salts are non-toxic salts at the concentration at which they are administered. The preparation of such salts can facilitate the pharmacological use by altering the physical-chemical characteristics of the composition without preventing the composition from exerting its physiological effect. Examples of useful alterations in physical properties include lowering the melting point to facilitate transmucosal administration and increasing the solubility to facilitate the administration of higher concentrations of the drug.

    Pharmaceutically acceptable salts include acid addition salts such as those containing sulfate, hydrochloride, phosphate, sulfamate, acetate, citrate, lactate, tartrate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, cyclohexylsulfamate and quinate. Pharmaceutically acceptable salts can be obtained from acids such as hydrochloric acid, sulfuric acid, phosphoric acid, sulfamic acid, acetic acid, citric acid, lactic acid, tartaric acid, malonic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, cyclohexylsulfamic acid, and quinic acid. Such salts may be prepared by, for example, reacting the free acid or base forms of the product with one or more equivalents of the appropriate base or acid in a solvent or medium in which the salt is insoluble, or in a solvent such as water which is then removed in vacuo or by freeze-drying or by exchanging the ions of an existing salt for another ion on a suitable ion exchange resin.

    Carriers or excipients can also be used to facilitate administration of the compound. Examples of carriers and excipients include calcium carbonate, calcium phosphate, various sugars such as lactose, glucose, or sucrose, or types of starch, cellulose derivatives, gelatin, vegetable oils, polyethylene glycols and physiologically compatible solvents. The compositions or pharmaceutical composition can be administered by different routes including intravenously, intraperitoneal, subcutaneous, and intramuscular, orally, topically, transmucosally, or by pulmonary inhalation.

    If desired, solutions of the above compositions may be thickened with a thickening agent such as methyl cellulose. They may be prepared in emulsified form, either water in oil or oil in water. Any of a wide variety of pharmaceutically acceptable emulsifying agents may be employed including, for example, acacia powder, a non-ionic surfactant (such as a Tween), or an ionic surfactant (such as alkali polyether alcohol sulfates or sulfonates, e.g., a Triton).

    Compositions useful in the invention are prepared by mixing the ingredients following generally accepted procedures. For example, the selected components may be simply mixed in a blender or other standard device to produce a concentrated mixture which may then be adjusted to the final concentration and viscosity by the addition of water or thickening agent and possibly a buffer to control pH or an additional solute to control tonicity.

    For use by the physician, the compositions will be provided in dosage unit form containing an amount of an exendin or exendin agonist, for example, exendin-3, and/or exendin-4, with or without another food intake-reducing, plasma glucose-lowering or plasma lipid-lowering agent. Therapeutically effective amounts of an exendin or exendin agonist for use in reducing food intake are those that suppress appetite at a desired level. As will be recognized by those in the field, an effective amount of therapeutic agent will vary with many factors including the age and weight of the patient, the patient's physical condition, the blood sugar level and other factors.

    The effective daily appetite-suppressing dose of the compounds will typically be in the range of about 10 to 30 μg to about 5 mg/day, preferably about 10 to 30 μg to about 2 mg/day and more preferably about 10 to 100 μg to about 1 mg/day, most preferably about 30 μg to about 500 μg/day, for a 70 kg patient, administered in a single or divided doses. The exact dose to be administered is determined by the attending clinician and is dependent upon where the particular compound lies within the above quoted range, as well as upon the age, weight and condition of the individual. Administration should begin whenever the suppression of food intake, or weight lowering is desired, for example, at the first sign of symptoms or shortly after diagnosis of obesity, diabetes mellitus, or insulin-resistance syndrome. Administration may be by injection, preferably subcutaneous or intramuscular. orally active compounds may be taken orally, however dosages should be increased 5-10 fold.

    The optimal formulation and mode of administration of compounds of the present application to a patient depend on factors known in the art such as the particular disease or disorder, the desired effect, and the type of patient. While the compounds will typically be used to treat human subjects they may also be used to treat similar or identical diseases in other vertebrates such as other primates, farm animals such as swine, cattle and poultry, and sports animals and pets such as horses, dogs and cats.
     

    • Claim 1 of 42 Claims

    1. A method for reducing food intake in a subject desirous or in need of reducing food intake, comprising peripherally administering to said subject an amount of an exendin agonist effective to reduce food intake, wherein the exendin agonist is an exendin peptide analog.

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    If you want to learn more about this patent, please go directly to the U.S. Patent and Trademark Office Web site to access the full patent.

     

     
         
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