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US20080139481A1 - Non-Natural Amino Acids - Google Patents

Non-Natural Amino Acids Download PDF

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US20080139481A1
US20080139481A1 US11/629,806 US62980605A US2008139481A1 US 20080139481 A1 US20080139481 A1 US 20080139481A1 US 62980605 A US62980605 A US 62980605A US 2008139481 A1 US2008139481 A1 US 2008139481A1
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peptide
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amino acid
alkyl
compound
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Thomas A. Dix
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    • C07C279/00—Derivatives of guanidine, i.e. compounds containing the group, the singly-bound nitrogen atoms not being part of nitro or nitroso groups
    • C07C279/04—Derivatives of guanidine, i.e. compounds containing the group, the singly-bound nitrogen atoms not being part of nitro or nitroso groups having nitrogen atoms of guanidine groups bound to acyclic carbon atoms of a carbon skeleton
    • C07C279/14—Derivatives of guanidine, i.e. compounds containing the group, the singly-bound nitrogen atoms not being part of nitro or nitroso groups having nitrogen atoms of guanidine groups bound to acyclic carbon atoms of a carbon skeleton being further substituted by carboxyl groups
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    • A61P5/00—Drugs for disorders of the endocrine system
    • A61P5/02—Drugs for disorders of the endocrine system of the hypothalamic hormones, e.g. TRH, GnRH, CRH, GRH, somatostatin
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    • C07C229/00—Compounds containing amino and carboxyl groups bound to the same carbon skeleton
    • C07C229/02—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton
    • C07C229/04—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated
    • C07C229/06—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one amino and one carboxyl group bound to the carbon skeleton
    • C07C229/08—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one amino and one carboxyl group bound to the carbon skeleton the nitrogen atom of the amino group being further bound to hydrogen atoms
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    • C07C229/02—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton
    • C07C229/04—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated
    • C07C229/06—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one amino and one carboxyl group bound to the carbon skeleton
    • C07C229/10—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one amino and one carboxyl group bound to the carbon skeleton the nitrogen atom of the amino group being further bound to acyclic carbon atoms or to carbon atoms of rings other than six-membered aromatic rings
    • C07C229/12—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one amino and one carboxyl group bound to the carbon skeleton the nitrogen atom of the amino group being further bound to acyclic carbon atoms or to carbon atoms of rings other than six-membered aromatic rings to carbon atoms of acyclic carbon skeletons
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    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C257/00—Compounds containing carboxyl groups, the doubly-bound oxygen atom of a carboxyl group being replaced by a doubly-bound nitrogen atom, this nitrogen atom not being further bound to an oxygen atom, e.g. imino-ethers, amidines
    • C07C257/10—Compounds containing carboxyl groups, the doubly-bound oxygen atom of a carboxyl group being replaced by a doubly-bound nitrogen atom, this nitrogen atom not being further bound to an oxygen atom, e.g. imino-ethers, amidines with replacement of the other oxygen atom of the carboxyl group by nitrogen atoms, e.g. amidines
    • C07C257/14—Compounds containing carboxyl groups, the doubly-bound oxygen atom of a carboxyl group being replaced by a doubly-bound nitrogen atom, this nitrogen atom not being further bound to an oxygen atom, e.g. imino-ethers, amidines with replacement of the other oxygen atom of the carboxyl group by nitrogen atoms, e.g. amidines having carbon atoms of amidino groups bound to acyclic carbon atoms
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    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D231/00—Heterocyclic compounds containing 1,2-diazole or hydrogenated 1,2-diazole rings
    • C07D231/02—Heterocyclic compounds containing 1,2-diazole or hydrogenated 1,2-diazole rings not condensed with other rings
    • C07D231/06—Heterocyclic compounds containing 1,2-diazole or hydrogenated 1,2-diazole rings not condensed with other rings having one double bond between ring members or between a ring member and a non-ring member
    • C—CHEMISTRY; METALLURGY
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    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D233/00—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings
    • C07D233/04—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings having one double bond between ring members or between a ring member and a non-ring member
    • C07D233/28—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings having one double bond between ring members or between a ring member and a non-ring member with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D233/44—Nitrogen atoms not forming part of a nitro radical
    • C07D233/46—Nitrogen atoms not forming part of a nitro radical with only hydrogen atoms attached to said nitrogen atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D233/00—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings
    • C07D233/04—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings having one double bond between ring members or between a ring member and a non-ring member
    • C07D233/28—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings having one double bond between ring members or between a ring member and a non-ring member with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D233/44—Nitrogen atoms not forming part of a nitro radical
    • C07D233/48—Nitrogen atoms not forming part of a nitro radical with acyclic hydrocarbon or substituted acyclic hydrocarbon radicals, attached to said nitrogen atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D239/00—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings
    • C07D239/02—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings
    • C07D239/06—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings having one double bond between ring members or between a ring member and a non-ring member
    • C07D239/08—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings having one double bond between ring members or between a ring member and a non-ring member with hetero atoms directly attached in position 2
    • C07D239/12—Nitrogen atoms not forming part of a nitro radical
    • C07D239/14—Nitrogen atoms not forming part of a nitro radical with only hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, attached to said nitrogen atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • C07K7/083—Neurotensin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • This invention relates to non-natural desamino, alkyl amino acids, methods of making them, their utilization in peptides, and the therapeutic, diagnostic and screening use of those peptides.
  • Naturally occurring endogenous peptides are ideal drug candidate leads by virtue of their myriad activities in promoting and regulating biological processes. Inherent in the chemistry and biology of peptides, however, are several factors that also make them poor drug candidates. Peptides most often exert localized effects and are rapidly degraded within the body. In addition, most peptides are unable to cross biological membranes, including the small intestine and blood brain barrier (BBB). Finally, peptides often bind to more than one receptor or receptor subtype, thus rarely showing the selectivity required of a viable drug candidate. Therefore, for a peptide to become a viable drug candidate, improvements in blood stability, receptor selectivity, and barrier crossing should be made without eliminating inherent binding affinity.
  • BBB blood brain barrier
  • non-natural amino acids and for peptides incorporating such acids to achieve superior effects, such as, for example, improved diagnostic or disease fighting activity.
  • the non-natural amino acid concept could be applied to development of new peptide pharmaceuticals.
  • One example of such a development is the application to neuropeptides such as neurotensin.
  • alkyl refers to a branched or unbranched saturated hydrocarbon group of 1 to 24 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, pentyl, hexyl, heptyl, octyl, decyl, tetradecyl, hexadecyl, eicosyl, tetracosyl and the like.
  • Preferred alkyl groups herein contain from 1 to 6 carbon atoms.
  • alkenyl refers to a hydrocarbon group of 2 to 24 carbon atoms, with preferred groups within this class containing 2 to 6 carbon atoms, and structural formula containing a carbon-carbon double bond.
  • alkynyl refers to a hydrocarbon group of 2 to 24 carbon atoms, with preferred groups within this class containing 2 to 6 carbon atoms, and a structural formula containing a carbon-carbon triple bond.
  • the term “lower” refers to a moiety having from 1 to 6 carbon atoms, preferably 1 to 4 carbon atoms, more preferably 1 to 2 carbon atoms.
  • alkylating agent as provided herein is a compound with the structural formula RX, where R is an alkyl, alkenyl or alkynyl group as previously described, and X, which is preferably a halide such as chloride, bromide or iodide.
  • non-natural amino acid refers to an organic compound that is a congener of a natural amino acid in that it has a structure similar to a natural amino acid so that it mimics the structure and reactivity of a natural amino acid.
  • the non-natural amino acid as defined herein generally increases or enhances the properties of a peptide (e.g., selectivity, stability) when the non-natural amino acid is either substituted for a natural amino acid unit of a peptide or otherwise incorporated into a peptide.
  • peptide refers to a class of compounds composed of amino acids chemically bound together.
  • the amino acids are chemically bound together via amide linkages (—CONH—); however, the amino acids may be bound together by other chemical bonds known in the art.
  • the amino acids may be bound by amine linkages.
  • Peptide as used herein includes oligomers of amino acids and small and large peptides, including polypeptides.
  • the term “activity” refers to a biological activity.
  • pharmacological activity refers to the inherent physical properties of a peptide or polypeptide. These properties include but are not limited to half-life, solubility, and stability and other pharmacokinetic properties.
  • organic acid salt refers to the salt form of an amine group with an alkyl or aryl C 1 -C 9 carboxylic, sulfonic, or phosphoric acid.
  • inorganic acid salt refers to the salt form of an amine group with a mineral acid such as hydrochloric, sulfuric, sulfonic, phosphoric, nitric, nitrous, or hydrobromic acid.
  • aromatic of C 6 to C 18 refers to an aromatic hydrocarbon such as phenyl, naphthyl, anthracenyl, or an arylalkyl hydrocarbon such as benzyl, phenethyl or naphthylmethylenyl.
  • heteroaromatic of C 4 to C 18 and of one or two heteroatoms selected from oxygen, sulfur and nitrogen in any combination refers to a heteroaromatic hydrocarbon containing one or two heteroatoms or an alkyl heteroaromatic hydrocarbon such as thienyl, furyl, pyrrolyl, azathienyl, azafuryl, pyridinyl, thiapyridinyl, pyrazinyl, methylenylpyridinyl, ethylenylpyridinyl, methylenylpyrrolyl and the like.
  • the present invention concerns alpha-desamino, alpha-alkyl amino acid compounds (desamino, alkyl amino acid compounds) that are capable of carrying positively charged side chains, their synthesis, their application as substitutes for natural amino acid moieties of biologically active peptides and the resulting peptides as well.
  • alpha-alkyl, alpha-desamino arginine, lysine and ornithine as well as their substituted and derivatized side chain analogs constitute preferred embodiments of the invention.
  • desamino, alkyl amino acid compounds can be substituted for arginine and/or lysine moieties in any known, biologically active peptide such that the substituted peptide will be truncated at the substitution position.
  • these desamino, alkyl amino acid compounds can be coupled to the amino group of the N-terminus of any known biologically active peptide to produce an extended peptide.
  • the truncated and extended peptides have significant biological selectivity and biological half lives owing to their resistance toward amino peptidase degradation.
  • the invention relates to a non-natural desamino, alkyl amino acid compound having Formula I:
  • n is an integer of from 0 to 5, preferably, 2 to 5;
  • n is zero or an integer of 1;
  • R is a straight or branched chain alkyl group of C 1 -C 6 , or an aromatic group of C 6 -C 18 or a corresponding substituted aromatic group with one or two substituents selected from halogen, alkyloxy, carboxy, amide or alkyl in any combination, or a heteroaromatic group of C 4 -C 18 and one or two heteroatoms selected from oxygen, sulfur and nitrogen in any combination or a corresponding substituted heteraromatic group with one or two substituents selected from halogen, alkyloxy, carboxy, amide or alkyl in any combination;
  • R 1 , R 2 , and R 3 are, independently, hydrogen or branched or straight chain alkyl, alkenyl or alkynyl of C 1 -C 6 or an aromatic group of C 6 -C 18 or a corresponding substituted aromatic group with one or two substituents selected from halogen, alkyloxy, carboxy, amide or alkyl in any combination, or a heteroaromatic group of C 4 -C 18 and one or two heteroatoms selected from oxygen, sulfur and nitrogen in any combination or a corresponding substituted heteraromatic group with one or two substituents selected from halogen, alkyloxy, carboxy, amide or alkyl in any combination and with the proviso that a maximum of two of R 1 , R 2 , and R 3 may be selected to be the aromatic, substituted aromatic, heteroaromatic or substituted heteroaromatic group;
  • C ⁇ is a carbon atom having either R or S stereochemistry
  • the invention relates to a non-natural desamino, alkyl amino acid compound of the formula II:
  • n is an integer of from 0 to 6, preferably, 2 to 5;
  • X and Y are independently, hydrogen or lower branched or straight chain alkyl, alkenyl or alkynyl of C 1 -C 6 ;
  • X—Y is (CH 2 ) z , wherein z is an integer of from 1-8, preferably, 2 to 4;
  • R is a straight or branched chain alkyl group of C 1 -C 6 , or an aromatic group of C 6 -C 18 or a corresponding substituted aromatic group with one or two substituents selected from halogen, alkyloxy, carboxy, amide or alkyl in any combination, or a heteroaromatic group of C 4 -C 18 and one or two heteroatoms selected from oxygen, sulfur and nitrogen in any combination or a corresponding substituted heteraromatic group with one or two substituents selected from halogen, alkyloxy, carboxy, amide or alkyl in any combination;
  • R 4 is hydrogen or lower branched or straight chain alkyl, alkenyl or alkynyl of C 1 -C 6 , or an aromatic group of C 6 -C 18 or a corresponding substituted aromatic group with one or two substituents selected from halogen, alkyloxy, carboxy, amide or alkyl in any combination, or a heteroaromatic group of C 4 -C 18 and one or two heteroatoms selected from oxygen, sulfur and nitrogen in any combination or a corresponding substituted heteraromatic group with one or two substituents selected from halogen, alkyloxy, carboxy, amide or alkyl in any combination, and;
  • C ⁇ is a carbon atom and the stereochemistry at C ⁇ is either R or S;
  • a third aspect of the present invention relates to a non-natural desamino, alkyl amino acid compound of the formula III:
  • n is an integer of from 0 to 5, preferably, 2 to 5;
  • X—Y is (CH 2 ) z , wherein z is an integer of from 0 to 6, preferably, 2 to 4;
  • R is a straight or branched chain alkyl group of C 1 -C 6 , or an aromatic group of C 6 -C 18 or a corresponding substituted aromatic group with one or two substituents selected from halogen, alkyloxy, carboxy, amide or alkyl in any combination, or a heteroaromatic group of C 4 -C 18 and one or two heteroatoms selected from oxygen, sulfur and nitrogen in any combination or a corresponding substituted heteraromatic group with one or two substituents selected from halogen, alkyloxy, carboxy, amide or alkyl in any combination;
  • R 6 , and R 7 are, independently, hydrogen or lower branched or straight chain alkyl, alkenyl or alkynyl of C 1 -C 6 , or an aromatic group of C 6 -C 18 or a corresponding substituted aromatic group with one or two substituents selected from halogen, alkyloxy, carboxy, amide or alkyl in any combination, or a heteroaromatic group of C 4 -C 18 and one or two heteroatoms selected from oxygen, sulfur and nitrogen in any combination or a corresponding substituted heteraromatic group with one or two substituents selected from halogen, alkyloxy, carboxy, amide or alkyl in any combination; and
  • C ⁇ is a carbon atom and the stereochemistry at C ⁇ is either R or S;
  • a fourth aspect of the invention relates to a non-natural desamino, alkyl amino acid compound of the formula IV:
  • n is an integer of from 0 to 5, preferably, 2 to 4;
  • R is a straight or branched chain alkyl group of C 1 -C 6 , or an aromatic group of C 6 -C 18 or a corresponding substituted aromatic group with one or two substituents selected from halogen, alkyloxy, carboxy, amide or alkyl in any combination, or a heteroaromatic group of C 4 -C 18 and one or two heteroatoms selected from oxygen, sulfur and nitrogen in any combination or a corresponding substituted heteraromatic group with one or two substituents selected from halogen, alkyloxy, carboxy, amide or alkyl in any combination;
  • R 9 , R 10 , and R 11 are, independently, hydrogen or lower branched or straight chain alkyl, alkenyl or alkynyl of C 1 -C 6 , or an aromatic group of C 6 -C 18 or a corresponding substituted aromatic group with one or two substituents selected from halogen, alkyloxy, carboxy, amide or alkyl in any combination, or a heteroaromatic group of C 4 -C 18 and one or two heteroatoms selected from oxygen, sulfur and nitrogen in any combination or a corresponding substituted heteraromatic group with one or two substituents selected from halogen, alkyloxy, carboxy, amide or alkyl in any combination and with the proviso that a maximum of two of R 9 , R 10 , and R 11 may be selected to be the aromatic, substituted aromatic, heteroaromatic or substituted heteroaromatic group; and
  • C ⁇ is a carbon atom and the stereochemistry at C ⁇ is either R or S;
  • a further aspect of the invention relates to the addition of the non-natural desamino, alkyl amino acid compounds of the invention to the N-terminus amino group of biologically active peptides or their substitution for naturally occurring congener amino acid moieties of biologically active peptides.
  • Preferred congener moieties include arginine and/or lysine.
  • the addition to the N-terminus amino group of a known, biologically active peptide provides an extended peptide that has selective, long lasting biological activity of the same kind as the known, biologically active peptide.
  • the addition can be accomplished by known methods for coupling acid and amine groups together to form amide bonds, including use of acyl azide coupling, carbodiimide coupling, acid ion exchange resin, triaminoboranes and enzyme coupling.
  • a preferred method involves the use of an amino exopeptidase under conditions that promote the peptide bond formation.
  • the semisynthetic peptides are produced by substituting a non-natural amino acid compound for the N-terminal arginine residue of NT (8-13), e.g., ABS201.
  • Preferred embodiments of the peptides on which the extended peptides are based include biologically active peptides useful for treatment or prevention of malconditions.
  • a list of preferred categories and examples is included in the sections below. Some preferred categories include but are not limited to transcription factors, ligands for cellular receptors, hormones and extracellular binding peptides.
  • Some preferred examples include but are not limited to enkephlin, LHRH and analogs, neuropeptides, glycoincretins, integrin and analogs, glucagons and glucagon-like peptides, antithrombotic peptides, cytokines and interleukins, transferrins, interferons, endothelins, natriuretic hormones, extracellular kinase ligands, angiotensin enzyme inhibitors, peptide antiviral compounds, thrombin, substance P, substance G, somatotropin, somatostatin, GnRH and analogues, secretin, bradykinin, vasopressin and analogues, insulin and analogs thereof, growth factors, as well as others.
  • the extended peptide is formed by coupling the N-terminus amino group of a basis peptide to the carboxyl group of a desamino, alkyl amino acid compound of the invention.
  • substitution of desamino, alkyl amino acid moiety for an arginine or lysine moiety of a biologically active peptide provides a truncated peptide having selective, long-lasting biological activity.
  • Any known biologically active peptide having an arginine and/or lysine moiety within its amino acid sequence can serve as the basis for the corresponding truncated peptide. Beginning at that ARG or LYS moiety, the truncated peptide will have the same downstream sequence as the known, biologically active peptide but the upstream sequence will be absent.
  • ARG or LYS moiety will be exchanged for a desamino, alkyl amino acid moiety, thus providing the truncated peptide.
  • Several known biologically active peptides are penultimately formed as pro-peptides with an arginine or lysine moiety at the pro-peptide or precursor cleavage position, or are formed as final peptides containing an arginine or lysine moiety at a position that can be cleaved to provide an active truncated peptide. Trypsin is an enzyme specific for such cleavage points. Examples include glucagon-like peptide, neurotensin, proinsulin, and thrombin. The truncated versions of these examples with a desamino, alkyl amino acid compound substituted for the arginine or lysine moiety provide selective, long-lasting biological activity.
  • a further aspect of the invention includes pharmaceutical and cosmetic compositions of the desamino, alkyl amino acid compound, of the extended or truncated peptide, and combinations thereof.
  • Unit dosage forms and biologically effective formulations of the pharmaceutical compositions are included.
  • the cosmetic formulations include appropriate oil, creme, wax or aqueous base cosmetic carriers.
  • Yet another aspect of the invention includes methods of screening, diagnosis and treatment using the desamino, alkyl amino acid compounds of the invention and/or the addition or truncated peptides.
  • One embodiment of the invention is a truncated neurotensin peptide having a desamino, alkyl amino acid as its N-terminus amino acid moiety.
  • the invention also provides processes and intermediates disclosed herein that are useful for preparing compounds of the inventions, such as compounds of Formula I, II, III, and/or IV and peptides that contain such compounds.
  • One class of such intermediates includes the N-protected or carboxyl protected or N- and carboxyl protected compounds of Formulas I, II, III and IV. These protected intermediates are described in detail in the following sections of the application.
  • Another class of such intermediates includes the carboxylate salts of the compounds of Formulas I, II, III and IV, the organic or inorganic acid amine salts of those compounds and the double salts (carboxylate, amine salts).
  • FIG. 1 Structure comparisons of NT(8-13), ABS201, and peptide 30.
  • FIG. 2 Representative Examples of Compounds of Formulas I-IV.
  • FIG. 3A-3C Scheme for Synthesis of Compounds of Formulas I-IV.
  • FIG. 4 Asymmetric synthesis of ⁇ -bromo-2(S)-methyl acids.
  • FIG. 5 Synthesis of ethylene-bridged (N ⁇ to N ⁇ ) arginine analogues.
  • FIG. 6 Synthesis of cyclic and acyclic alkyl arginine analogues.
  • FIG. 7 Peptide synthesis of representative peptides of the invention.
  • FIG. 8A-8C Comparisons of induced hypothermia by ⁇ -methyl NT(8-13) analogues.
  • FIG. 9 Hypothermic effects of ABS201 after IP (solid symbols) and oral administration (open symbols).
  • FIG. 10A-10B Comparison of hypothermic effects after IP and oral administration for KH29 (10A) and KH30 (10B).
  • FIG. 11A-11B Dose-response curves for ABS201 after IP administration.
  • FIG. 12 Dose-dependent hypothermic response to ABS201 after oral administration.
  • FIG. 13 Attenuation of d-amphetamine induced hyperactivity after IP administration of ABS201.
  • FIG. 14 Attenuation of d-amphetamine induced hyperactivity after oral administration of ABS201.
  • FIG. 15 Effect of ABS201 and haloperidol on catalepsy.
  • FIG. 16 Hypothermic effects of chronic administration of ABS201 after daily dose of 5 mg/kg ABS201.
  • FIG. 17 Effect of repeated daily administration of ABS201 on d-amphetamine induced hyperlocomotion after daily doses of 5 mg/kg ABS201.
  • FIG. 18 Synthesis of Fmoc-Proline-OH*.
  • the present invention is directed to certain desamino, alkyl amino acid compounds, their incorporation as extenders or as congeners in known biologically active peptides, and the use of the compounds and peptides in medical diagnosis, treatment and screening.
  • Several aspects of the invention concern the mimicry of the alkyl desamino amino acid compounds for the natural amino acids arginine and/or lysine.
  • congeners for these natural amino acid moieties in known biologically active peptides By their use as congeners for these natural amino acid moieties in known biologically active peptides, truncated versions of the peptides can be prepared in which the biological activity is more selective and longer lasting than that of the known peptide.
  • By their use as extenders their position as the N-terminal moiety adduct of a known biologically active peptide will also provide longer lasting biological activity than that of the known peptide.
  • Neurotensin is a 13 amino acid peptide having neurological properties. Its cleavage at AA7 to produce truncated neurotensin (8-13) provides a peptide having selective biological activity. According to the invention, conversion of the AA8 arginine to a desamino, alkyl amino acid moiety results in a peptide also having significant and selective biological activity.
  • FIG. 1 The examples of NT and the converted versions are shown in FIG. 1 .
  • the biologically active peptides of the present invention have the desamino, alkyl amino acid moiety as their N-terminal moiety.
  • These peptides have known amino acid sequences of biologically active amino acids wherein the desamino alkyl amino acid is either covalently coupled through an amide bond with the N-terminus amine group of the known peptide (extended peptide) or is substituted for its corresponding congener moiety (analogous natural amino acid moiety) within the peptide (truncated version).
  • the peptide becomes truncated at the position of substitution so that the desamino, alkyl amino acid moiety becomes the new N-terminus and the amino acid residues upstream of this position are no longer part of the sequence (truncated peptide).
  • the extended and truncated peptides can have longer lifetimes in vivo and can have biological activities like those of the natural peptides except that the activities will be more selective.
  • R 1 , R 2 , and R 3 are independently, hydrogen or lower branched or straight chain alkyl of C 1 -C 5 , more preferably hydrogen or methyl.
  • n is 4.
  • R is methyl, ethyl or propyl. Additional preferred embodiments include those wherein R is methyl, ethyl, propyl or butyl and:
  • n 4, m is 0, R 1 is hydrogen, R 2 is methyl, the compound of formula I is an acid, and the stereochemistry at C ⁇ is R or S;
  • n 4, m is 1, R 1 and R 2 are methyl, R 3 is hydrogen or methyl, the compound of formula I is an acid, the stereochemistry at C ⁇ is R or S;
  • n 4, m is 1, R 1 is methyl, R 2 , and R 3 are hydrogen, the compound of formula I is an acid, the stereochemistry at C ⁇ is R or S;
  • n 4, m is 1, R 1 , R 2 , and R 3 are hydrogen, the compound of formula I is an acid, and the stereochemistry at C ⁇ is R or S;
  • n 3
  • m 0, R 1 and R 2 are methyl, the compound of formula I is an acid, the stereochemistry at C ⁇ is R or S;
  • n 3
  • m 0, R 1 and R 2 are ethyl, the compound of formula I is an acid, the stereochemistry at C ⁇ is R or S;
  • n 3
  • esters or salts of any of the foregoing preferred embodiments a-1 are also preferred.
  • Preferred embodiments of Formula II include those wherein when n is 3, dashed line a is not present. Additional preferred embodiments include those wherein X is hydrogen, and wherein Y and R 4 are the same lower branched or straight chain alkyl. In yet another preferred embodiment, R 4 and R 5 are, independently, hydrogen or methyl.
  • dashed line a is not present
  • X is hydrogen or lower branched or straight chain alkyl of C 1 -C 5 , preferably methyl or ethyl
  • Y is hydrogen or lower branched or straight chain alkyl of C 1 -C 5 , preferably methyl
  • dashed line a is present and z is 2, and preferably, n is 3.
  • Additional preferred embodiments include those wherein R is methyl, ethyl, propyl or butyl and:
  • n 3
  • dashed line a is not present, the compound of formula II is an acid, R 4 is methyl, X is hydrogen, Y is methyl, and the stereochemistry at C ⁇ is R or S;
  • n 3
  • dashed line a is present, the compound of formula II is an acid, z is 2, R 4 is hydrogen, and the stereochemistry at C ⁇ is R or S;
  • n 3
  • dashed line a is present, the compound of formula II is an acid, z is 2, R 4 is methyl, and the stereochemistry at C ⁇ is R or S;
  • n 3
  • dashed line a is not present, the compound of formula II is an acid, R 4 is hydrogen, X is methyl, Y is hydrogen, and the stereochemistry at C ⁇ is R or S;
  • n 3
  • dashed line a is not present, the compound of formula II is an acid, R 4 is hydrogen, X is ethyl, Y is hydrogen, and the stereochemistry at C ⁇ is R or S;
  • n 4
  • dashed line a is present, the compound of formula II is an acid, z is 2, R 4 is hydrogen, and the stereochemistry at C ⁇ is R or S;
  • n 3
  • dashed line a is present, the compound of formula II is an acid, z is 2, R 4 is methyl, and the stereochemistry at C ⁇ is R or S;
  • esters or salts of any of the forgoing preferred embodiments a-1 are also preferred.
  • a third aspect of the desamino, alkyl amino acid compounds of the invention is illustrated by Formula III.
  • Preferred embodiments of Formula III include those wherein R 6 and R 7 are independently, hydrogen or lower alkyl or straight chain alkyl of C 1 -C 5 , preferably hydrogen or methyl, even more preferably all are hydrogen.
  • z is 2 or 3, preferably 3.
  • n is 3.
  • Additional preferred embodiments include those wherein R is methyl, ethyl, propyl or butyl and:
  • n 4, z is 2, R 6 and R 7 are hydrogen, the compound of formula III is an acid, and the stereochemistry at C ⁇ is R or S;
  • n 4, z is 3, R 6 and R 7 are hydrogen, the compound of formula III is an acid, and the stereochemistry at C ⁇ is R or S;
  • n 4, z is 2, R 6 and R 7 are methyl, the compound of formula III is an acid, and the stereochemistry at C ⁇ is R or S;
  • n 4, z is 3, R 6 and R 7 are methyl, the compound of formula III is an acid, and the stereochemistry at C ⁇ is R or S.
  • esters or salts of the preferred foregoing embodiments a-j are also preferred.
  • a fourth aspect of the invention is provided by the desamino, alkyl amino acid compounds of Formula IV.
  • Preferred embodiments of the compounds of Formula IV include those wherein R 9 , R 10 , and R 11 are, independently, hydrogen or lower straight or branched chain alkyl of C 1 -C 5 , preferably hydrogen, methyl or ethyl.
  • R 10 is methyl.
  • R 9 is hydrogen
  • R 10 is methyl
  • R 12 is hydrogen
  • n is 3.
  • Additional preferred embodiments include those wherein R is methyl, ethyl, propyl or butyl and:
  • n 3
  • R 9 and R 11 are hydrogen
  • R 10 is methyl
  • the compound of formula IV is an acid
  • the stereochemistry at C ⁇ is R or S;
  • n 3
  • R 9 is hydrogen
  • R 10 and R 11 are methyl
  • the compound of formula IV is an acid
  • the stereochemistry at C ⁇ is R or S;
  • n 3
  • R 9 is hydrogen
  • R 10 is methyl
  • R 11 is ethyl
  • the compound of formula IV is an acid
  • the stereochemistry at C ⁇ is R or S;
  • n 2
  • R 9 and R 11 are hydrogen
  • R 10 is methyl
  • the compound of formula IV is an acid
  • the stereochemistry at C ⁇ is R or S;
  • n 2
  • R 9 is hydrogen
  • R 10 and R 11 are methyl
  • the compound of formula IV is an acid
  • the stereochemistry at C ⁇ is R or S;
  • n 4
  • R 9 is are hydrogen
  • R 10 is methyl
  • R 11 is ethyl
  • the compound of formula IV is an acid
  • the stereochemistry at C ⁇ is R or S.
  • esters or salts of the foregoing preferred embodiments a-f are also preferred.
  • Especially preferred non-natural desamino, alkyl amino acid compounds of the invention include the formulas provided in FIG. 2 wherein R is methyl or ethyl.
  • Certain embodiments of the invention provide protected intermediates and protected non-natural amino acids of the invention. Certain embodiments provide protected intermediates and protected non-natural amino acids of the invention, wherein the side chain amine group is protected by a protecting group that prevents undesired reaction of the amino group and is removable by a chemical method that does not also cause amide group cleavage. Certain embodiments provide protected intermediates and protected non-natural amino acids of the invention, wherein the side chain carboxyl group is protected by a protecting group that prevents undesired reaction of the carboxyl group and is removable by a chemical method that does not also cause carboxyl group cleavage.
  • the protecting group is t-butoxy carbonyl (BOC) or fluorenylmethoxycarbonyl (FMOC).
  • the protecting group is BOC, FMOC, Alloc (allyloxycarbonyl), CBZ (benzyloxycarbonyl), Pbf (2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl), NO2 (nitro), Pmc (2,2,5,7,8-pentamethylchroman-6-sulfonyl), Mtr (4-methoxy-2,3,6-trimethylbenzenesulfonyl), or Tos (tosyl).
  • the structures of the non-natural desamino, alkyl amino acids of formulas I-IV are similar to those of the naturally occurring amino acids lysine, arginine as well as the naturally occurring glutamate biosynthesis intermediate, ornithine.
  • the compounds of the invention differ from the corresponding natural amino acids due, inter alia, a longer or shorter methylene bridge between the (i) carboxyl terminus, which forms the N-terminus bond with the adjacent amino acid unit in a peptide, (ii) the presence of an alkyl group in place of the alpha amino group, and (iii) the organo group substitution of the amine side chain.
  • the extended bridge of the invention compared to the natural amino acid bridge is one carbon length longer or shorter (i.e., the homo- or des-forms).
  • the compounds of the invention have, inter alia, longer, shorter, or equivalent methylene bridge lengths and have substitutions at various moieties, form different moieties, or link moieties to form ring structures, compared to the comparable natural amino acid.
  • each of the compounds of the invention can be prepared as the acid, amide, salt or ester.
  • the non-natural amino acids of the present invention will be charged; however; in cell membranes and other non-polar regions of the cell, the non-natural amino acids may not be charged.
  • the ester group of the non-natural amino acids of the present invention is methyl, ethyl, t-butyl, benzyl or allyl.
  • the counter-ion for the salts of the non-natural amino acids is sodium, potassium, ammonium and tetra-alkyl ammonium.
  • Some embodiments of the invention provide semisynthetic peptides comprising a non-natural amino acid compound of the invention.
  • the semisynthetic peptide comprises a non-natural amino acid compound as its N-terminus moiety.
  • the semisynthetic peptide comprises a non-natural amino acid compound as the N-terminal moiety of a semisynthetic peptide of neurotensin (8-13).
  • the semisynthetic peptide is ABS201.
  • the semisynthetic peptide has an extended half-life in vivo as compared to a peptide having the same sequence as the semisynthetic peptide that does not comprise the non-natural amino acid compound substituted as its N-terminus moiety.
  • compositions comprising a peptide of the invention and a pharmaceutical carrier.
  • the peptide is present in unit dosage form.
  • Certain embodiments of the present invention provide cosmetic formulations comprising a non-natural amino acid compound of the invention and a cosmetic base formulation. Certain embodiments of the present invention provide cosmetic formulations comprising a semisynthetic peptide of the invention and a cosmetic base formulation. In certain embodiments, the cosmetic base formulation is an aqueous or oil base.
  • Certain embodiments of the present invention provide the non-natural amino acid compounds of the invention for use in medical therapy.
  • Certain embodiments of the present invention provide the use of the non-natural amino acid compounds of the invention for the manufacture of a medicament useful for treating psychosis in a mammal. Certain embodiments of the present invention provide the use of the semisynthetic peptides of the invention for the manufacture of a medicament useful for treating psychosis in a mammal. In certain embodiments, the psychosis is schizophrenia.
  • Certain embodiments of the present invention provide the use of a compound of the invention for the manufacture of a medicament useful for treating cancer in a mammal.
  • Certain embodiments of the present invention provide the use of a compound of the invention for the manufacture of a medicament useful for treating pain in a mammal.
  • Certain embodiments of the present invention provide the semisynthetic peptides of the invention for use in medical therapy.
  • Certain embodiments of the present invention provide a method to lower the body temperature of a patient, comprising administering to the patient an effective amount of a semisynthetic peptide of the invention so as to lower the body temperature of the patient.
  • Certain embodiments of the present invention provide a method to lower the body temperature of a patient, comprising administering to the patient an effective amount of a composition of the invention so as to lower the body temperature of the patient.
  • Certain embodiments of the present invention provide a method to treat a patient with psychosis, comprising administering to the patient an effective amount of a peptide of the invention so as to treat the psychosis.
  • Certain embodiments of the present invention provide a method to treat a patient with psychosis, comprising administering to the patient an effective amount of a composition of the invention so as to treat the psychosis.
  • Certain embodiments of the present invention provide a method to treat cancer, comprising administering to a patient an effective amount of a peptide of any of the invention so as to treat the cancer.
  • Certain embodiments of the present invention provide a method to treat cancer, comprising administering to a patient an effective amount of a composition of the invention so as to treat the cancer.
  • Certain embodiments of the present invention provide a method to treat pain, comprising administering to a patient an effective amount of a peptide of the invention so as to treat the pain.
  • Certain embodiments of the present invention provide a method to treat pain, comprising administering to a patient an effective amount of a composition of the invention so as to treat the pain.
  • Certain embodiments of the present invention provide a method for screening a peptide containing a non-natural amino acid compound for an activity, comprising the steps of: a) measuring a biological activity of a first peptide having a known amino acid sequence; and b) measuring the same biological activity of a semisynthetic peptide of any of the invention wherein the semisynthetic peptide has the same sequence as the first peptide except for the non-natural amino acid compound, or is a truncated version of the first peptide except for the non-natural amino acid compound.
  • the biological activity is poptosis, apoptosis, cell signaling, ligand binding, transcription, translation, metabolism, cell growth, cell differentiation, homeostasis, half-life, solubility, or stability.
  • the biological activity includes a direct or indirect assessment of the ability of the semisynthetic peptide to pass through a biological barrier.
  • the biological activity is selectivity.
  • Certain embodiments of the present invention provide a method of treating a patient with a disease that is affected by administration to the patient of a known first peptide, comprising administering to the patient a semisynthetic peptide of the invention wherein the semisynthetic peptide has the same sequence as the first peptide except for the non-natural amino acid compound, or is a truncated version of the first peptide except for the non-natural amino acid compound.
  • Certain embodiments of the present invention provide a method of increasing the ability of a known first peptide to cross a biological barrier of a subject, comprising substituting a semisynthetic peptide of the invention wherein the semisynthetic peptide has the same sequence as the first peptide except for the non-natural amino acid compound, or is a truncated version of the first peptide except for the non-natural amino acid compound.
  • the barrier comprises the blood brain barrier, a cell membrane, intestinal epithelium, skin, or blood-ocular.
  • Certain embodiments of the present invention provide a method of increasing the selectivity of a known peptide, comprising substituting for the known peptide a semisynthetic peptide of the invention wherein the semisynthetic peptide has the same sequence as the first peptide except for the non-natural amino acid compound, or is a truncated version of the first peptide except for the non-natural amino acid compound.
  • Certain embodiments of the present invention provide a method of increasing the resistance of a known peptide to digestion by a peptidase, comprising substituting for the known peptide a semisynthetic peptide of the invention wherein the semisynthetic peptide has the same sequence as the first peptide except for the non-natural amino acid compound, or is a truncated version of the first peptide except for the non-natural amino acid compound.
  • Certain embodiments of the present invention provide a method of treating a patient with a disease that is affected by administration to the patient of a known first peptide that crosses a body barrier, comprising administering to the patient a semisynthetic peptide of the invention wherein the semisynthetic peptide has the same sequence as the first peptide except for the non-natural amino acid compound, or is a truncated version of the first peptide except for the non-natural amino acid compound.
  • Certain embodiments of the present invention provide a method of treating a patient with a disease of the brain that is affected by administration to the patient of a known first peptide, comprising administering to the patient a semisynthetic peptide of any of the invention wherein the semisynthetic peptide has the same sequence as the first peptide except for the non-natural amino acid compound, or is a truncated version of the first peptide except for the non-natural amino acid compound.
  • Certain embodiments of the present invention provide a method for preparing a semisynthetic peptide with an extended half-life in vivo comprising substituting for a known peptide a semisynthetic peptide of the invention wherein the semisynthetic peptide has the same sequence as the first peptide except for the non-natural amino acid compound, or is a truncated version of the first peptide except for the non-natural amino acid compound.
  • the preparation of the desamino, alkyl amino acid compounds of the invention follows the overall synthetic scheme depicted in FIG. 3 .
  • the first step in this process is the production of alpha alkyl, omega halogen carboxylic acids having a methylene unit chain length corresponding to n of formulas I through IV.
  • this intermediate is designated as compound 27.
  • compound 27 Following the production of compound 27, its ⁇ -halo group can be easily displaced with excess nucleophilic agent to produce the desamino, alkyl amino acid compounds of Formulas I-IV.
  • the reaction conditions for production of compound 27 involve protection of the carboxyl group of an omega carboxylic acid by formation of an acyl oxazolone.
  • the acyl oxazolone is converted to an enolate and the enolate is combined with an alkylating agent such as alkyl iodide or alkyl mesylate to form compound 27.
  • an alkylating agent such as alkyl iodide or alkyl mesylate
  • the alpha alkyl omega halo carboxylic acid compounds 25 can be converted to any of the side chain modifications by coupling the appropriate side chain moiety and the omega halo group of compound 25. Appropriate protection of the carboxyl group is also advantageously employed.
  • the conditions for these reactions, and the appropriate alkylating and substituting agents follow the teaching set forth in “Advanced Organic Chemistry”, 4 th Edition, J. March, Wiley InterScience, New York, N.Y. 1992, the entire disclosure of which is incorporated herein by reference.
  • the omega halo carboxylic acid compound 27 can be combined with the appropriate amine nucleophile such as ammonia, a primary amine or a secondary amine.
  • the formulas of the amine nucleophiles correspond to the side chain moiety of Formula I.
  • the reaction conditions will follow those appropriate for amine nucleophilic substitution as are disclosed in “Advanced Organic Chemistry” cited above and incorporated herein as if fully repeated. These compounds can be used directly in the following peptide synthesis provided that the side chain amine group is appropriately protected or otherwise inhibited from carboxyl condensation.
  • the omega halo compound 27 may first be protected at the carboxyl position and then can be reacted sequentially with a diamine and cyanogen bromide. Deprotection and purification will afford the desamino, alkyl amino acid compounds of Formula II. These compounds can be used directly in peptide synthesis with appropriate side chain protection.
  • the compounds of Formulas III and IV can also be prepared by addition of the side chain moiety to the omega halo carboxylic acid compounds 27. In this instance, protection of the carboxyl group is unnecessary.
  • Preparation of the appropriate thiourea compound can be accomplished by addition of an alkylating agent such as an alkyl, alkenyl or alkynyl halide to thiourea, N-substituted thiourea or N,N-disubstituted thiourea (commercially available).
  • the nucleophilic substitution of the resulting appropriate thiourea compound at the omega halo position of compound 27 under basic conditions provides the desamino, alkyl amino acid compounds of Formula IV.
  • addition of the appropriate cyclic thiourea compounds to the omega halo compound 27 under basic conditions provides the desamino, alkyl amino acid compounds of Formula III.
  • the appropriate cyclic thiourea compounds can be prepared by combining an alkylating agent such as an alkyl, alkenyl or alkynyl halide with the corresponding unsubstituted, N-substituted or N,N-disubstituted cyclic diazathione (commercially available).
  • the crude reaction products can be purified by known methods such as ion-exchange chromatography to yield the alkyl desamino amino acid compounds of Formulas III and IV which can be used directly in peptide synthesis with appropriate side chain protection.
  • the side chains of the compounds of Formulas I-IV may either be appropriately protected or determined to be sufficiently hindered that they will not enter into a peptide condensation reaction.
  • the side chain of the compounds of Formula I is a primary amine group, it may be appropriately protected according to the teaching of the art associated with peptide synthesis. See for example, the review of amine protecting groups provided in “Compendium of Organic Synthetic Methods,” I&S Harrison, Wiley Interscience, New York, N.Y., 1971, the disclosure of which is incorporated herein by reference.
  • an appropriate protecting group can be t-butoxy carbonyl having the acronym BOC or fluorenylmethoxycarbonyl having the acronym FMOC.
  • the BOC and FMOC protecting groups can be removed by mild treatment with acid, such as aqueous trifluoroacetic acid, and base, such as piperidine, respectively.
  • the omega halo carboxylic acid compound 27 can be coupled with the penultimate peptide to form an omega halo acyl moiety at the N-terminus of the penultimate peptide. Because the omega halo carboxylic acid compound 27 does not contain an amino moiety on its side chain, protection and spurious peptide formation are of less concern.
  • the amino moiety can undergo nucleophilic reaction with the omega halo group of the acylated penultimate peptide as described above for formation of the compounds of Formulas I-IV. The desired peptide having a residue of a compound of Formula I-IV at its N-terminus is produced.
  • appropriate protection of carboxyl and amino side chains and appropriate protection of the C-terminus may be employed to prevent undesirable reactions of these groups.
  • the invention includes the truncated and extended peptides which contain as their N-terminus moiety the residue of the compound of Formula I, II, III or IV.
  • These peptides can be synthesized by the Merrifield solid phase method, which is an established method for preparing peptides to those skilled in the art. See R. B. Merrifield, Science, 232, 341-347 (1986), the disclosure of which is incorporated herein by reference for an explanation of, and conditions for the Merrifield solid phase peptide synthesis.
  • the peptide minus the N-terminal amino acid unit, or penultimate peptide can be expressed recombinantly by known biological methods and the desamino, alkyl amino acid compound of Formula I-IV can be added as the N-terminus by enzymatic condensation using an aminopeptidase. See “Enzyme Structure and Mechanism,” Alan Fersht, W.H. Freeman, New York, N.Y. (1985), the disclosure of which is incorporated herein by reference, for an explanation of, and conditions for, recombinant expression of peptides.
  • the Formulas I-IV compounds can be appropriately protected at the side chain amino group with standard protecting groups. In a preferred embodiment, the protecting groups are BOC and/or FMOC.
  • the penultimate peptide can be produced in bulk and then coupled to any of the Formula I-IV compounds using the protection and coupling techniques of the Merrifield solid phase synthesis.
  • an appropriate anchor resin designed for amino group exposure the carboxy terminus amino acid unit of the peptide having an amino protecting group such as an FMOC group is anchored to the resin through a selectively cleavable carboxyl coupling link.
  • the amino group of the anchored carboxy terminus unit is then deprotected and the additional amino protected amino acid units are then sequentially coupled in proper sequence.
  • Each coupling step will involve deprotection of the protected amino group of the anchored peptide chain followed by peptide condensation between that unprotected amino group and the carboxyl group of the next amino acid unit.
  • the condensation can be facilely obtained by carbodiimide coupling, by Schotten Bauman reaction or by activated acyl group condensation. These condensation reactions are described in “Advanced Organic Chemistry”, cited above. Protection of amine and carboxyl side chains using appropriate protecting groups that differ from the protecting groups of the alpha amino group entering into the peptide condensation will enable selective peptide condensation of the sequential amino acid units. Selection of appropriate protection groups and conditions for solid phase peptide synthesis are described in the Merrifield reference, cited above.
  • the penultimate peptides may also be produced by recombinant expression.
  • This biological method involves re-engineering a microbe to express the penultimate peptide.
  • a DNA segment encoding the penultimate peptide sequence can be inserted in proper reading from into a plasmid or other vector capable of causing microbial expression of the DNA.
  • the vector will also contain appropriate control, promoter and selection DNA segments.
  • the microbe mixture can be selected for appropriate transfection by treatment with the corresponding selection agent.
  • the agent will be an antibiotic and the vector will contain a sequence encoding the corresponding detoxifying enzyme for the antibiotic. Chloramphenacol and penicillin are two of such agents.
  • the penultimate peptide may be purified by known techniques such as lyophilization, chromatography and the like. These recombinant techniques for peptide expression are fully set forth in “Cold Spring Harbor—Current Protocols in Molecular Biology,” Wiley Interscience, Cold Spring Harbor (2003), the disclosure of which is incorporated herein by reference.
  • NT peptide An example of the solid phase peptide production is provided by the development of a set of neurotensin (8-13) compounds (NT peptide). These compounds incorporate the desamino, alkyl amino acid compounds of Formulas I-IV as their N-termini. They are a novel class of antipsychotic drugs, the biological study and background of which are described in the sections below.
  • the penultimate sequence of the peptide, NT(9-13), can be synthesized in bulk using p-alkoxybenzyl alcohol solid phase methodology (65) and stored in the fully protected form.
  • P-alkoxybenzyl alcohol resin-bound N ⁇ -Fmoc-leucine, N ⁇ -Fmoc-isoleucine, N ⁇ -Fmoc-tert-leucine, Noc-Fmoc-(But)-tyrosine, N ⁇ -Fmoc-(Boc)-tryptophan, N ⁇ -Fmoc-proline, and N ⁇ -Fmoc-(Pbf)-arginine were purchased from Advanced Chemtech (Louisville, Ky.). PyBOP® was purchased from Novabiochem (San Diego, Calif.).
  • N-hydroxybenzoriazole HABt
  • DIPEA N,N-diisopropylethylamine
  • TIS triisopropylsilane
  • TAS trifluoroacetic acid
  • Fmoc fluorenylmethoxycarbonyl
  • NH 3 ammonia
  • NH 2 CH 3 methylamine
  • NH(CH 3 ) 2 dimethylamine
  • N(CH 3 ) 3 trimethylamine
  • EtOH ethanol.
  • resin-bound N ⁇ -Fmoc-leucine can be swelled in DMF prior to Fmoc cleavage with piperidine (20% in DMF).
  • the piperidine solution can be removed with vacuum filtration and the resin-bound amino acid washed with DMF and CH 2 Cl 2 .
  • Amino acids (4 eq) can be activated in DMF with HOBt (4 eq), PyBOP (4 eq), and DIPEA (10 eq) and added directly to the peptide reaction vessel.
  • the amino acid couplings can be conducted for approximately 6 hr, the resin washed with DMF and CH 2 Cl 2 and monitored with the Kaiser test (66) for the presence of free amines. Residues can be recoupled when necessary.
  • Reverse phase high pressure liquid chromatography can be used to purify the foregoing crude peptides.
  • a Waters dual pump system in combination with a Waters C18 radial compression column can be used for this purpose.
  • Effluent can be monitored by UV absorbance at 280 mm.
  • the invention provides a method for screening a peptide for an activity or pharmacological activity.
  • the method includes the steps of: a) measuring an activity or pharmacological activity of a peptide having a selected natural amino acid sequence, and b) measuring the same activity or pharmacological activity of an extended or truncated peptide based upon the same amino acid sequence as the foregoing peptide wherein the N-terminus is a non-natural amino acid having the formula I-IV, described above; and c) comparing the measured activity or pharmacological activity of the peptides from steps a) and b) to determine whether the peptide of step b) has the desired activity or pharmacological activity.
  • the activities for which the present invention screens can include any activity associated with a biologically active peptide or peptidomimetic.
  • the following is a partial list of the many activities that can be determined in the present screening method:
  • Receptor agonist/antagonist activity A compendia of examples of specific screens for measuring these activities can be found in: “The RBI Handbook of Receptor Classification and Signal Transduction” K. J. Watling, J. W. Kebebian, J. L. Neumeyer, eds. Research Biochemicals International, Natick, Mass., 1995, and references therein. Methods of analysis can be found in: T. Kenakin “Pharmacologic Analysis of Drug-Receptor Interactions” 2 nd Ed. Raven Press, New York, 1993, and references therein.
  • Enzyme inhibition A compendia of examples of specific screens for measuring these activities can be found in: H. Zollner “Handbook of Enzyme Inhibitors”, 2 nd Ed. VCH Weinheim, FRG, 1989, and references therein.
  • Anticancer activities A compendia of examples of specific screens for measuring these activities can be found in: I. J. Fidler and R. J. White “Design of Models for Testing Cancer Therapeutic Agents”, Van Nostrand Reinhold Company, New York, 1982, and references therein.
  • Antibiotic and antiviral (especially anti-HIV) activities A compendia of examples of specific screens for measuring these activities can be found in: “antibiotics in Laboratory Medicine”, 3 rd Ed., V. Lorian, ed. Williams and Wilkens, Baltimore, 1991, and references therein. A compendia of anti-HIV screens for measuring these activities can be found in: “HIV Volume 2: Biochemistry, Molecular Biology and Drug Discovery”, J. Karn, ed., IRL Press, Oxford, 1995, and references therein.
  • Immunomodulatory activity A compendia of examples of specific screens for measuring these activities can be found in: V. St. Georgiev (1990) “Immunomodulatory Activity of Small Peptides” Trends Pharm. Sci. 11, 373-378.
  • Pharmacokinetic properties The pharmacological activities assayed in the screening method include half-life, solubility, or stability, among others.
  • methods of analysis and measurement of pharmacokinetic properties can be found in: J.-P. Labaune “Handbook of Pharmacokinetics: Toxicity Assessment of Chemicals”, Ellis Horwood Ltd., Chichester, 1989, and references therein.
  • the peptide of step a) can consist of natural amino acids.
  • the peptide of step a) can contain mostly natural amino acids, but also contain one or a small number of non-natural amino acids.
  • Such a peptide is considered to consist essentially of natural amino acids.
  • the peptide of step b) will be the truncated or extended peptide of the invention as described above.
  • the structures of the non-natural amino acids of formulas I-IV will be similar to those of the naturally occurring amino acids, lysine and arginine.
  • any extended or truncated peptide can be compared to any peptide having the same downstream sequence and having a known activity or pharmacological activity to determine whether or not the extended or truncated peptide has the same or similar activity or pharmacological activity at the same or different level.
  • the present screening method can also be used to detect an activity or pharmacological activity exhibited by the extended or truncated peptide. Also, the screening method can be used to detect and measure qualitative and quantitative differences in the same or similar activity or pharmacological activity.
  • the methods of the present invention provide evaluation of the alteration of activity of the extended or truncated peptide.
  • the hydrophobicity of the peptide is increased, which can result indirectly in increased binding activity when the desamino alkyl amino acid moiety is involved in binding (e.g., receptor-ligand binding, enzyme-cofactor binding, enzyme-substrate binding) and since binding strength is correlated with activity, a peptide higher potency (higher measured activity level) can result.
  • the desamino alkyl amino acids of the present invention also can enhance or increase the pharmacological activity of a peptide.
  • a peptide containing a non-natural amino acid is more able to pass a body barrier (e.g., blood brain, blood ocular, skin, intestinal epithelium).
  • a body barrier e.g., blood brain, blood ocular, skin, intestinal epithelium.
  • the desamino alkyl amino acids impart increased selectivity and stability to a peptide, the pharmacological activity can also be screened when compared to other peptides.
  • the invention further relates to a method of treating or preventing in a subject a malcondition comprising administering to the subject an extended or truncated peptide having as its N-terminus an amino acid having the formula I-IV.
  • the basis peptide from which the extended or truncated peptide is formed will have or will be believed to have a biochemical, physiological, pharmacological or biological relationship with the malcondition to be treated or prevented.
  • the malcondition may be a disease, biological or organic disfunction or an undesirable biological condition that is not ordinarily regarded as a disease or disfunction, such as but not limited to cosmetic malconditions such as skin blotches, acne, and the like.
  • the subject may be a medical or veterinary patient including mammals such as humans, and non-humans mammals such as dogs, cats, cows, sheep, pigs as well as avian.
  • the malconditions that can be treated or prevented and the peptides that can be used are numerous.
  • a partial list of peptides and malconditions is set out below.
  • Peptides for triggering B and T cell activity can be used to treat autoimmune disease, including uveitis, collagen-induced, adjuvant and rheumatoid arthritis, thyroiditis, myasthenia gravis, multiple sclerosis and diabetes.
  • autoimmune disease including uveitis, collagen-induced, adjuvant and rheumatoid arthritis, thyroiditis, myasthenia gravis, multiple sclerosis and diabetes.
  • these peptides are interleukins (referenced in Aulitzky, W E; Schuler, M; Peschel, C.; Huber, C.; Interleukins. Clinical pharmacology and therapeutic use. Drugs. 48(5):667-77, 1994 November) and cytokines (referenced in Peters, M.; Actions of cytokines on the immune response and viral interactions: an overview. Hepatology. 23(4):909-16, 1996 April).
  • Enkephlin and analogs, agonists and antagonists can be used to treat AIDS, ARC, and cancer, pain modulation, Huntington's, Parkinson's diseases.
  • LHRH and analogs, agonists and antagonists can be used to treat prostatic tumors and reproductive physiopathology, including breast cancer, and infertility.
  • Peptides and peptidomimetics that target crucial enzymes, oncogenes or oncogene products, tumor-suppressor genes and their products, growth factors and their corresponding receptors can be used to treat cancer. Examples of these peptides are described in Unger, C. Current concepts of treatment in medical oncology: new anticancer drugs. Journal of Cancer Research & Clinical Oncology. 122(4):189-98, 1996.
  • Neuropeptide Y and other pancreatic polypeptides, and analogs, agonists and antagonists can be used to treat stress, anxiety, depression and associated vasoconstrictive activities.
  • Gluco-incretins including gastric inhibitory polypeptide, glucose-dependent insulinotropic polypeptide, PACAP/Glucagon and glucagon-like polypeptide-1 and 2 and analogs, agonists and antagonists can be used to treat Type II diabetic hyperglycaemia.
  • Atrial natriuretic factor and analogs, agonists and antagonists can be used to treat congestive heart failure.
  • Integrin and analogs, agonists and antagonists can be used to treat osteoporosis, scar formation, bone synthesis, inhibition of vascular occlusion, and inhibition of tumor invasion and metastasis.
  • Glucagon, glucagon-like peptide 1, PACAP/Glucagon, and analogs, agonists and antagonists can be used to treat diabetes cardiovascular emergencies.
  • Antithrombotic peptides and analogs, agonists and antagonists can be used to treat cardiovascular and cerebrovascular diseases.
  • examples of these peptides RGD, D-Phe-Pro-Arg and others named are described in Ojima I.; Chakravarty S.; Dong Q. Antithrombotic agents: from RGD to peptide mimetics. Bioorganic & Medicinal Chemistry. 3(4):337-60, 1995.
  • Cytokines/interleukins and analogs, agonists and antagonists can be used to treat inflammatory disease, immune response dysfunction, hematopoiesis, mycosis fungoides, aplastic anemia, thrombocytopenia, and malignant melanoma.
  • Examples of these peptides are Interleukins, referenced in Aulitzky et al. and Peters et al.
  • Endothelin and analogs, agonists and antagonists can be used to treat arterial hypertension, myocardial infarction, congestive heart failure, atherosclerosis, shock conditions, renal failure, asthma and vasospasm
  • Natriuretic hormones and analogs, agonists and antagonists can be used to treat cardiovasicular disease and acute renal failure.
  • Examples of these peptides are named and described in Espiner, E. A; Richards, A. M.; Yandle, T. G.; Nicholls, M. G.; Natriuretic hormones. Endocrinology & Metabolism Clinics of North America. 24(3):481-509, 1995.
  • Peptides that activate or inhibit tyrosine kinase, or bind to TK-activating or inhibiting peptides and analogs, agonists and antagonists can be used to treat chronic myelogenous and acute lymphocytic leukemias, breast and ovarian cancers and other tyrosine kinase associated diseases. Examples of these peptides are described in Smithgall, T E.; SH2 and SH3 domains: potential targets for anti-cancer drug design. Journal of Pharmacological & Toxicological Methods. 34(3):125-32, 1995.
  • Renin inhibitors analogs, agonists and antagonists can be used to treat cardiovascular disease, including hypertension and congestive heart failure. Examples of these peptides are described in Rosenberg, S. H.; Renin inhibition. Cardiovascular Drugs & Therapy. 9(5):645-55, 1995.
  • Angiotensin-converting enzyme inhibitors, analogs, agonists and antagonists can be used to treat cardiovascular disease, including hypertension and congestive heart failure.
  • Peptides that activate or inhibit tyrosine phosphorylases can be used to treat cardiovascular diseases. Examples of these peptides are described in Srivastava, A. K.; Protein tyrosine phosphorylation in cardiovascular system. Molecular & Cellular Biochemistry. 149-150:87-94, 1995.
  • Peptide based antivirals can be used to treat viral diseases.
  • these peptides are described in Toes, R. E.; Feltkamp, M. C.; Ressing, M. E.; Vierboom, M. P.; Blom, R. J.; Brandt, R. M; Hartman, M.; Offringa, R.; Melief, C. J.; Kast, W. M.; Cellular immunity against DNA tumour viruses: possibilities for peptide-based vaccines and immune escape. Biochemical Society Transactions. 23(3):692-6, 1995.
  • Corticotropin releasing factor and peptide analogs, agonists and antagonists can be used to treat disease associated with high CRF, i.e Alzheimer's disease, anorexia nervosa, depressive disorders, arthritis, and multiple sclerosis.
  • Peptide agonists and antagonists of platelet-derived wound-healing formula can be used as a therapy for donor tissue limitations and wound-healing constraints in surgery. Examples of these peptides are described in Rudkin, G. H.; Miller, T. A.; Growth factors in surgery. Plastic & Reconstructive Surgery. 97(2):469-76, 1996.
  • Fibronectin, fibrinopeptide inhibitors and analogs, agonists and antagonists can be used to treat metastasis (i.e. enzyme inhibition, tumor cell migration, invasion, and metastasis).
  • metastasis i.e. enzyme inhibition, tumor cell migration, invasion, and metastasis.
  • Chemokine types of cytokine, including interleukin-8, RANTES, and monocyte chemotactic peptide
  • agonists and antagonists can be used to treat arthritis, hypersensitivity, angiogenesis, renal disease, glomerulonephritis, inflammation, and hematopoiesis.
  • Neutral endopeptidase inhibitors and analogs, agonists and antagonists can be used to treat hypertension and inflammation. Examples of these peptides are described in Gregoire, J. R; Sheps, S. G; Newer antihypertensive drugs. Current Opinion in Cardiology. 10(5):445-9, 1995.
  • Substance P and analogs, agonists and antagonists can be used to treat immune system dysfunction, pain transmission/perception and in autonomic reflexes and behaviors.
  • Alpha-melanocyte-stimulating hormone and analogs, agonists and antagonists can be used to treat AIDS, rheumatoid arthritis, and myocardial infarction.
  • Bradykinin (BK) and analogs, agonists and antagonists can be used to treat inflammatory diseases (edema, etc), asthma, allergic reactions (rhinitis, etc), anesthetic uses, and septic shock.
  • Secretin can be used to treat cardiovascular emergencies.
  • GnRH and analogs, agonists and antagonists can be used to treat hormone-dependent breast and prostate tumors.
  • Somatostatin and analogs, agonists and antagonists can be used to treat gut neuroendocrine tumors.
  • Gastrin, Gastrin Releasing Peptide and analogs, agonists and antagonists can be used as an adjuvant to chemotherapy or surgery in small cell lung cancer and other malignancies, or to treat allergic respiratory diseases, asthma and allergic rhinitis.
  • Laminin, the Laminin derivative antimetastatic drug YIGSR peptide, Laminin-derived synthetic peptides analogs, agonists and antagonists can be used to treat tumor cell growth, angiogenesis, regeneration studies, vascularization of the eye with diabetes, and ischemia.
  • the peptides of this category can inhibit the tumor growth and metastasis of leukemic cells and may be useful as a potential therapeutic reagent for leukaemic infiltrations. Peptides containing this sequence also inhibit experimental metastasis.
  • Exemplary references include McGowan I A. Marinkovich M P. Laminins and human disease. Microscopy Research & Technique. 51(3):262-79, 2000 Nov. 1; Yoshida N. Ishii E.
  • Defensins, corticostatins, dermaseptins, mangainins, and other antibiotic (antibacterial and antimicrobial) peptides and analogs, agonists and antagonists can be used to treat infections, tissue inflammation and endocrine regulation.
  • Vasopressin and analogs, agonists and antagonists can be used to treat neurological disorders, stress and Diabetes insipidus.
  • Oxytocin and analogs, agonists and antagonists can be used to treat neurological disorders and to induce labor.
  • ACTH-related peptides and analogs, agonists and antagonists can be used as neurotrophic, neuroprotective, and peripheral demyelinating neuropathy agents.
  • Amyloid-beta peptide and analogs, agonists and antagonists can be used to treat Alzheimer's disease.
  • Epidermal growth factor, receptor, and analogs, agonists and antagonists can be used to treat necrotizing enterocolitis, Zollinger-Ellison syndrome, gastrointestinal ulceration, colitis, and congenital microvillus atrophycarcinomas.
  • Leukocyte adhesion molecules and their ligands, and analogs, agonists and antagonists can be used to treat atherosclerosis, inflammation. Examples of these peptides are described in Barker, J. N.; Adhesion molecules in cutaneous inflammation. Ciba Foundation Symposium. 189:91-101.
  • Major histocompatibility complex (MHC) binding peptides and analogs, agonists and antagonists can be used to treat autoimmune, immunodysfunctional, immuno modulatory diseases and as well as used for their corresponding therapies.
  • MHC Major histocompatibility complex
  • Examples of these peptides are described in Appella, E.; Padlan, E. A.; Hunt, D. F; Analysis of the structure of naturally processed peptides bound by class I and class II major histocompatibility complex molecules. EXS. 73:105-19, 1995.
  • Corticotropin releasing factor can be used to treat neurological disorders.
  • Neurotrophins including brain-derived neurotrophic factor (BDNF), nerve growth factor, and neurotrophin 3 and analogs, agonists and antagonists can be used to treat neurological disorders.
  • BDNF brain-derived neurotrophic factor
  • nerve growth factor nerve growth factor
  • neurotrophin 3 analogs, agonists and antagonists can be used to treat neurological disorders.
  • Cytotoxic T-cell activating peptides can be used to treat infectious diseases and cancer. Examples of these peptides are described in: Chesnut R. W.; Sette, A.; Celis, E.; Wentworth, P.; Kubo, R. T.; Alexander, J.; Ishioka, G.; Vitiello, A.; Grey, H. M; Design and testing of peptide-based cytotoxic T-cell-mediated immunotherapeutics to treat infectious diseases and cancer. Pharmaceutical Biotechnology. 6:847-74, 1995.
  • Peptide immunogens for prevention of HIV-1 and HTLV-I retroviral infections can be used to treat AIDS. Examples of these peptides are described in Hart, M. K.; Palker, T. J.; Haynes, B F; Design of experimental synthetic peptide immunogens for prevention of HIV-1 and HTLV-I retroviral infections. Pharmaceutical Biotechnology. 6:821-45, 1995.
  • Galanin and analogs, agonists and antagonists can be used to treat Alzheimer's disease, depression, eating disorders, chronic pain, prevention of ischemic damage, and growth hormone modulation.
  • Tachykinins neurokinin A and neurokinin B
  • analogs, agonists and antagonists can be used to treat pain transmission/perception and in autonomic reflexes and behaviors.
  • RGD containing peptides can be used to treat various diseases involved with cell adhesion, antithrombotics, and acute renal failure.
  • Osteogenic growth peptide and analogs, agonists and antagonists can be used as treatment of systemic bone loss. Examples of these peptides are described in Bab IA. Regulatory role of osteogenic growth peptide in proliferation, osteogenesis, and hemopoiesis. Clinical Orthopaedics & Related Research. (313):64-8, 1995.
  • Parathyroid hormone, parathyroid hormone related-peptide and analogs, agonists and antagonists can be used to treat diseases affecting calcium homeostasis (hypercalcemia), bone metabolism, vascular disease, and atherosclerosis.
  • Kallidin and analogs, agonists and antagonists can be used to treat tissue injury or inflammation and pain signaling pathological conditions of the CNS.
  • T cell receptor peptide vaccines and analogs, agonists and antagonists can be used in immunotherapy. Examples of these peptides are described in Brostoff, S W; T cell receptor peptide vaccines as immunotherapy. Agents & Actions—Supplements. 47:53-8, 1995.
  • Platelet-derived growth factor (PDGF) and analogs, agonists and antagonists can be used to treat non-neoplastic hyperproliferative disorders, therapy for donor tissue limitations and wound-healing constraints in surgery.
  • PDGF Platelet-derived growth factor
  • Amylin, calcitonin gene related peptides (CGRP) and analogs, agonists and antagonists can be used to treat insulin-dependent diabetes.
  • Vasoactive intestinal polypeptide and analogs, agonists and antagonists can be used to treat allergic respiratory diseases, asthma and allergic rhinitis, and nervous control of reproductive functions.
  • Growth hormone-releasing hormone and analogs, agonists and antagonists can be used to treat growth hormone deficiency and immunomodulation.
  • HIV protease inhibiting peptides can be used to treat AIDS. Examples of these peptides are described in Bugelski, P. J.; Kirsh, R.; Hart, T. K; HIV protease inhibitors: effects on viral maturation and physiologic function in macrophages. Journal of Leukocyte Biology. 56(3):374-80, 1994.
  • Thymopoietin active fragment peptides and analogs, agonists and antagonists can be used to treat rheumatoid arthritis and virus infections.
  • Cecropins and analogs, agonists and antagonists can be used as antibacterials.
  • Thyroid releasing hormone and analogs, agonists and antagonists can be used to treat spinal cord injury and shock.
  • Erythropoietin and analogs, agonists and antagonists can be used to treat anemia.
  • Fibroblast growth factor FGF
  • receptor and analogs, agonists and antagonists can be as stimulation of bone formation, as well as used as a treatment for Kaposi's sarcoma, neuron regeneration, prostate growth, tumor growth inhibition, and angiogenesis.
  • Stem cell factor and analogs, agonists and antagonists can be used to treat anemias.
  • GP120, GP160, CD4 fragment peptides and analogs, agonists and antagonists can be used to treat AIDS.
  • Insulin-like growth factor, receptor, and analogs, agonists and antagonists can be used to treat breast and other cancers, noninsulin-dependen diabetest mellitus, cell proliferation, apoptosis, hematopoiesis, AIDS, growth disorders, osteoporosis, and insulin resistance.
  • Colony stimulating factors granulocyte-macrophage colony-stimulating factor, granulocyte colony-stimulating factor, and macrophage colony-stimulating factor and analogs, agonists and antagonists can be used to treat anemias.
  • Lymphocyte activating peptide and analogs, agonists and antagonists can be used for immunomodulation.
  • these peptides are described in Loleit, M.; Deres, K.; Wiesmuller, K. H.; Jung, G.; Eckert, M.; Bessler, W. G; Biological activity of the Escherichia coli lipoprotein: detection of novel lymphocyte activating peptide segments of the molecule and their conformational characterization.
  • Tuftsin and analogs, agonists and antagonists can be used for immunomodulation.
  • Prolactin and analogs, agonists and antagonists can be used to treat rheumatic diseases, systemic lupus erythematosus, and hyperprolactemia.
  • Angiotensin II and receptor(s) and analogs, agonists and antagonists can be used to treat hypertension, hemodynamic regulation, neurological disorders, diabetic nephropathies, aortoarterities induced RVH, hyperaldosteronism, heavy metal induced cardiovascular effects, diabetes mellitus and thyroid dysfunction.
  • Dynorphin and analogs, agonists and antagonists can be used to treat neurological disorders, pain management, algesia, spinal cord injury and epilepsy.
  • Calcitonin and analogs, agonists and antagonists can be used to treat neurological disorders, immune system dysfunction, calcium homeostasis, and osteoporosis.
  • Pituitary adenylate cyclase activating polypeptide can play a role in growth, signal transduction vasoactivity roles, exact role in diseases not determined yet.
  • Cholecystokinin and analogs, agonists and antagonists can be used to treat feeding disorders, panic disorders, and anti-opioid properties.
  • Pepstatin and analogs, agonists and antagonists can be used as pepsin and HIV protease inhibitors (AIDS).
  • Bestatin and analogs, agonists and antagonists can be used to treat muscular dystrophy, anticancer, antileukemia, immune response modulator, and acute non-lymphocytic leukemia.
  • Leupeptin and analogs, agonists and antagonists can be used as a protease inhibitor, exact role in diseases not determined yet.
  • Luteinizing hormone and releasing hormone and analogs, agonists and antagonists can be used as a infertility male contraceptive.
  • Neurotensin and analogs, agonists and antagonists can be used, e.g., as antipsychotic, analgesic, anti-cancer, and/or neuroprotective agents, e.g., for treating stroke victims, e.g., by inducing hypothermia so as to provide neuroprotection.
  • Motilin and analogs, agonists and antagonists can be used for the control of gastric emptying.
  • Insulin and analogs, agonists and antagonists can be used to treat diabetes.
  • TGF Transforming growth factor
  • analogs, agonists and antagonists can be used for cell proliferation and differentiation, cancer treatment, immunoregulation, therapy for donor tissue limitations, and wound-healing constraints in surgery.
  • Bone morphogenetic proteins and analogs, agonists and antagonists can be used as therapy for donor tissue limitations, osteogenesis, and wound-healing constraints in surgery.
  • Bombesin and Enterostatin as well as their analogs, agonists and antagonists can be used to prevent the proliferation of tumor cells, modulation of feeding, and neuroendocrine functions.
  • These peptides fall within a supercategory of the neuromedins described above. These peptides are described in such exemplary references as Yamada K. Wada E. Wada K.
  • Glucagon, glucagon-like peptide 1 and analogs, agonists and antagonists can be used to treat diabetes cardiovascular emergencies.
  • Endorphins and analogs, agonists and antagonists can be used to treat neurological disorders, alleviating pain, treatment of opioid abuse, obesity, and diabetes.
  • Examples of these peptides are named and described in Dalayeun, J. F.; Nores, J. M.; Bergal, S.; Physiology of beta-endorphins. A close-up view and a review of the literature. Biomedicine & Pharmacotherapy. 47(8):311-20, 1993.
  • Miscellaneous opioid peptides including (but not limited to) adrenal peptide E, alpha casein fragment, beta casomorphin, dermorphin, kyotorphin, metophamide neuropeptide FF (NPFF), melanocyte inhibiting factor, and analogues, agonists and antagonists can be used to treat neurological disorders, alleviating pain, as well as for the treatment of opioid abuse.
  • adrenal peptide E alpha casein fragment
  • beta casomorphin beta casomorphin
  • dermorphin dermorphin
  • kyotorphin metophamide neuropeptide FF (NPFF)
  • NPFF metophamide neuropeptide FF
  • melanocyte inhibiting factor melanocyte inhibiting factor
  • analogues melanocyte inhibiting factor
  • Vasotocin and analogues, agonists and antagonists can be used for clinical uses to be determined.
  • Protein kinase C and inhibitors and analogues, agonists and antagonists can be used to treat cancer, apoptosis, smooth muscle function, and Alzheimer's disease. Examples of these peptides are named and described in Philip, P. A.; Harris, A. L; Potential for protein kinase C inhibitors in cancer therapy. Cancer Treatment & Research. 78:3-27, 1995.
  • Amyloid, amyloid fibrin, fragments and analogues, agonists and antagonists can be used to treat neurodegenerative diseases and diabetes.
  • Calpain and other calmodulin-inhibitory proteins and analogues, agonists and antagonists can be used to treat neurodegenerative disorders, cerebral ischaemia, cataracts, myocardial ischaemia, muscular dystrophy and platelet aggregation.
  • Charybdotoxin, Apamin and analogues, agonists and antagonists can be used for treatment of neurodegenerative diseases and pain and cerebral ischemia.
  • Phospholipase A2 and receptor inhibiting/activating peptides and analogues, agonists and antagonists can be used to treat acute pancreatitis, pancreatic cancer, abdominal trauma, and inflammation, e.g., sepsis, infections, acute pancreatitis, various forms of arthritis, cancer, complications of pregnancy, and postoperative states.
  • Potassium channel activating and inhibiting proteins and analogues, agonists and antagonists can be used to treat various diseases. Examples of these peptides are described in Edwards, G.; Weston, A. H; Pharmacology of the potassium channel openers. Cardiovascular Drugs & Therapy. 9 Suppl 2:185-93, 1995 Mar.
  • IgG activators, inhibitors and analogues, agonists and antagonists can be used to treat autoimmune diseases and immune dysfunctions.
  • Examples of these peptides are described in Mouthon, L.; Kaveri, S. V.; Spalter, S. H.; Lacroix-Desmazes, S.; Lefranc, C.; Desai, R.; Kazatchkine, M. D; Mechanisms of action of intravenous immune globulin in immune-mediated diseases. Clinical & Experimental Immunology. 104 Suppl 1:3-9, 1996.
  • Endotoxin and inhibitors and analogues, agonists and antagonists can be used for decreasing cardiac output, systemic hypotension, decreased blood flow and O 2 delivery to tissues, intense pulmonary vasoconstriction and hypertension, bronchoconstriction, increased permeability, pulmonary oedema, ventilation-to-perfusion inequalities, hypoxaemia, and haemoconcentration.
  • Endotoxin and inhibitors and analogues, agonists and antagonists can be used for decreasing cardiac output, systemic hypotension, decreased blood flow and O 2 delivery to tissues, intense pulmonary vasoconstriction and hypertension, bronchoconstriction, increased permeability, pulmonary oedema, ventilation-to-perfusion inequalities, hypoxaemia, and haemoconcentration.
  • these peptides are named and described in Burrell, R; Human responses to bacterial endotoxin. Circulatory Shock. 43(3):137-53, 1994 Jul.
  • Orphan receptor ligands including but not limited to ADNF, Adrenomedullin, Apelin, Ghrelin, Mastoparan (MCD peptides), Melanin concentrating hormone, Nociceptin/Nocistatin, Orexin, Receptor activity modulating protein, Urotensin).
  • ADNF Adrenomedullin
  • Apelin Adrenomedullin
  • Ghrelin Ghrelin
  • Mastoparan MCD peptides
  • Melanin concentrating hormone Nociceptin/Nocistatin
  • Orexin Mastoparan
  • Receptor activity modulating protein Urotensin
  • Glycoprotein IIb/IIIa inhibitors The central role of platelet-rich thrombus in the pathogenesis of acute coronary syndromes (ACSs) is well-known. Glycoprotein IIb/IIIa (Gp IIb/IIIa) receptor antagonists are potent inhibitors of platelet function that may be expected to affect favorably the natural history of ACSs. Exemplary references for this category include Bhatt D L. Topol E J. Current role of platelet glycoprotein IIb/IIIa inhibitors in acute coronary syndromes. JAMA. 284(12):1549-58, 2000; Kereiakes D J. Oral blockade of the platelet glycoprotein IIb/IIIa receptor: fact or fancy? American Heart Journal.
  • the invention relates to a method of increasing the ability of a peptide to cross a body barrier of a subject by use of the extended or truncated peptide having as its N-terminus a residue of the compound formula I-IV.
  • the invention further relates to a method of treating or preventing in a subject a disease or condition treated or prevented by the administration of an extended or truncated peptide, whereby the extended or truncated peptide crosses the body barrier in higher amounts than the peptide having no non-natural amino acid.
  • the invention also relates to a method of treating or preventing in a subject a disease or condition of the brain treated or prevented by the administration of an extended or truncated peptide.
  • body barrier is defined herein as a cellular membrane or other structure that functions to prevent free (e.g., diffusional) passage of certain molecules.
  • body barriers include, but are not limited to, the blood brain barrier, a cell membrane, intestinal epithelium, skin cell, or the blood-ocular.
  • the body barrier is the blood brain barrier.
  • Certain embodiments of the invention relate to a method of increasing the selectivity of a chosen peptide through use of an extended or truncated peptide based upon the sequence of the chosen peptide as described above.
  • a peptide containing arginine and/or lysine can be converted according to the invention into an extended or truncated peptide in order to increase the selectivity of the peptide.
  • any of the non-natural amino acids disclosed herein can be used to increase the selectivity of a peptide.
  • the peptides of the invention can be used in any therapeutic procedure available to one of skill in the art to treat any disease or physiological problem with which the corresponding known peptide is associated.
  • the peptides of the invention can be formulated as pharmaceutical compositions and administered to a mammalian host, such as a human patient, in a variety of dosage forms adapted to the chosen route of administration, i.e., orally or parenterally, by intravenous, intramuscular, topical or subcutaneous routes.
  • the peptides may be systemically administered, for example, intravenously or intraperitoneally by infusion or injection.
  • Solutions of the peptide or peptide conjugate can be prepared in water, optionally mixed with a nontoxic surfactant.
  • Dispersions can also be prepared in glycerol, liquid polyethylene glycols, triacetin, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
  • the pharmaceutical dosage forms suitable for injection or infusion can include sterile aqueous solutions or dispersions or sterile powders comprising the active ingredient(s) that are adapted for the extemporaneous preparation of sterile injectable or infusible solutions or dispersions, optionally encapsulated in liposomes.
  • the liquid carrier or vehicle can be a solvent or liquid dispersion medium comprising, for example, water, ethanol, a polyol (for example, glycerol, propylene glycol, liquid polyethylene glycols, and the like), vegetable oils, nontoxic glyceryl esters, and suitable mixtures thereof.
  • the proper fluidity can be maintained, for example, by the formation of liposomes, by the maintenance of the required particle size in the case of dispersions or by the use of surfactants.
  • the prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, buffers or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.
  • Sterile injectable solutions are prepared by incorporating the peptide or peptide conjugate in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filter sterilization.
  • the preferred methods for preparation of such powders are vacuum drying and freeze drying techniques, which yield a powder of the active ingredient plus any additional desired ingredient present in the previously sterile-filtered solutions.
  • the peptides of the invention can also be administered orally, in combination with a pharmaceutically acceptable vehicle such as an inert diluent or an assimilable edible carrier.
  • a pharmaceutically acceptable vehicle such as an inert diluent or an assimilable edible carrier.
  • the peptides may be enclosed in hard or soft shell gelatin capsules, may be compressed into tablets, or may be incorporated directly with the food of the patient's diet.
  • the peptide or peptide conjugate may be combined with one or more excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like.
  • Such compositions and preparations should contain at least 0.1% of active compound.
  • compositions and preparations may, of course, be varied and may conveniently be between about 2 to about 60% to about 90% of the weight of a given unit dosage form.
  • amount of peptide in such therapeutically useful compositions is such that an effective dosage level will be obtained.
  • the tablets, troches, pills, capsules, and the like may also contain the following: binders such as gum tragacanth, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such as sucrose, fructose, lactose or aspartame or a flavoring agent such as peppermint, oil of wintergreen, or cherry flavoring may be added.
  • a liquid carrier such as a vegetable oil or a polyethylene glycol.
  • any material used in preparing any unit dosage form should be pharmaceutically acceptable and substantially non-toxic in the amounts employed.
  • the peptides of the invention may be incorporated into sustained-release preparations and devices.
  • Useful solid carriers include finely divided solids such as talc, clay, microcrystalline cellulose, silica, alumina and the like.
  • Useful liquid carriers include water, alcohols or glycols or water-alcohol/glycol blends, in which the present compounds can be dissolved or dispersed at effective levels, optionally with the aid of non-toxic surfactants.
  • Adjuvants such as fragrances and additional antimicrobial agents can be added to optimize the properties for a given use.
  • Thickeners such as synthetic polymers, fatty acids, fatty acid salts and esters, fatty alcohols, modified celluloses or modified mineral materials can also be employed with liquid carriers to form spreadable pastes, gels, ointments, soaps, and the like, for application directly to the skin of the user.
  • Useful dosages of the peptides of the invention can be determined by correlating their in vitro activity, and in vivo activity in animal models described herein.
  • the therapeutically effective amount of peptide of the invention necessarily varies with the subject and the disease or physiological problem to be treated and correlates with the effective amounts of the corresponding known peptide.
  • a therapeutic amount between 30 to 112,000 ⁇ g per kg of body weight can be effective for intravenous administration.
  • the amount can be varied depending on the method of administration.
  • the amount of the peptide of the invention, required for use in treatment will also vary with the route of administration, but also the nature of the condition being treated and the age and condition of the patient and will be ultimately at the discretion of the attendant physician or clinician.
  • the compound can conveniently be administered in unit dosage form; for example, containing 1 to 1000 mg, conveniently 10 to 750 mg, most conveniently, 20 to 500 mg of peptide per unit dosage form.
  • the peptide should be administered to achieve peak plasma concentrations of from about 0.1 to about 75 ⁇ M, preferably, about 1 to 50 ⁇ M, most preferably, about 2 to about 30 ⁇ M. This may be achieved, for example, by the intravenous injection of a 0.05 to 5% solution of the peptide, optionally in saline, or orally administered as a bolus containing about 1-100 mg of the peptide. Desirable blood levels may be maintained by continuous infusion to provide about 0.01-5.0 mg/kg/hr or by intermittent infusions containing about 0.4-15 mg/kg of the active ingredient(s).
  • the desired dose may conveniently be presented in a single dose, as divided doses, or as a continuous infusion.
  • the desired dose can also be administered at appropriate intervals, for example, as two, three, four or more sub-doses per day.
  • a cosmetic composition of the present invention contains the typical and common base carriers as well as a desamino alkyl amino acid compound of the invention.
  • the compound of the invention will be in the form of an ester, amide or salt for this purpose.
  • the cosmetic base will depend upon the kind of make-up being formulated: face creme, face powder, pancake make-up, skin creme, lip stick, rouge and the like.
  • These bases will contain appropriate, nontoxic colorants, emuliants, oils, waxes, solvents, emulsifiers, fatty acids, alcohols or esters, gums, inorganic inert builders and the like.
  • the gums may include various known polysaccharide compounds, for example, cellulose, hemicellulose, gum arabic, tragacanth gum, tamarind gum, pectin, starch, mannan, guar gum, locust bean gum, quince seed gum, alginic acid, carrageenan, agar, xanthane gum, dextran, pullulan, chitin, chitosan, hyaluronic acid, chondroitin sulfuric acid, etc., derivatives of polysaccharide compounds, for example, carboxymethylated derivatives, sulfate derivatives, phosphated derivatives, methylated derivatives, ethylated derivatives, addition derivatives of alkylene oxide such as ethylene oxide or propylene oxide, acylated derivatives, cationated derivatives, low molecular weight derivatives, and other polysaccharide derivatives may be mentioned.
  • polysaccharide compounds for example, carboxymethylated derivatives, sul
  • Powder components based on inorganic components such as talc, kaolin, mica, sericite, dolomite, phlogopite, synthetic mica, lepidolite, biotite, lithia mica, vermiculite, magnesium carbonate, calcium carbonate, aluminum silicate, barium silicate, calcium silicate, magnesium silicate, strontium silicate, metal salts of tungstenic acid, magnesium, silica, zeolite, barium sulfate, sintered calcium sulfate (sintered gypsum), calcium phosphate, fluorapatite, hydroxyapatite, ceramic powder, metal soap (zinc myristate, calcium palmitate, ammonium stearate), boronitride, etc.; and organic powder components such as polyamide resin powder (nylon powder), polyethylene powder, polymethyl methacrylate powder, polystyrene powder, copolymer resin powder of
  • powder components obtained by treating the surfaces of these powder components by a silicone compound, fluorine-modified silicone compound, fluorine compound, higher aliphatic acid, higher alcohol, aliphatic acid ester, metal soap, alkyl phosphate, etc. may be formulated into the external composition of the present invention depending upon the need.
  • inorganic white pigments such as titanium dioxide, zinc oxide, inorganic red pigments such as iron oxide (bengala), iron titanate, inorganic brown pigments such as .gamma.-iron oxide
  • inorganic yellow pigments such as yellow iron oxide, yellow earth
  • inorganic black pigments such as black iron oxide, carbon black, lower titanium oxide
  • inorganic violet pigments such as mango violet, cobalt violet
  • inorganic green pigments such as chromium oxide, chromium hydroxide, cobalt titanate
  • blue pigments such as prussian blue, ultramarine
  • pearl pigments such as titanium oxide coated mica, titanium oxide coated bismuth oxichloride, titanium oxide coated talc, colored titanium oxide coated mica, bismuth oxichloride, fish scales
  • metal powder pigments such as aluminum powder, copper powder
  • Lithol rubine B (Red No. 201), Lithol rubine BCA (Red No. 202), Lake red CBA (Red No. 204), Lithol red (Red No. 205), Deep maroon (Red No. 220), Helidone pink CN (Red No. 226), Permatone Red (Red No. 228), Permanent red F5R (Red No. 405), Permanent orange (Orange No. 203), Benzidine Orange (Orange No. 204), Benzidine yellow G (Yellow No. 205), Hanza Yellow (Yellow No. 401), Blue No. 404, and other organic pigments; Erythrosine (Red No. 3), Phloxine B (Red No.
  • Acid red (Red No. 106), Fast acid magenta (Red No. 227), Eosine YS (Red No. 230), Violamine R (Red No. 401), Oil red XO (Red No. 505), Orange II (Orange No. 205), Tartrazine (Yellow No. 4), Sunset yellow FCF (Yellow No. 5), Uranine (Yellow No. 202), Quinoline yellow (Yellow No. 203), Fast green FCF (Green No. 3), Brilliant blue FCF (Blue No. 1) may be mentioned.
  • the cosmetic composition of the present invention may be formulated with a liquid.
  • a volatile component ordinarily used in external compositions such as cosmetics.
  • volatile silicone oil for example, volatile silicone oil, water, or a lower alcohol (or mixtures of the same).
  • These volatile components may be suitably selected depending upon the specific form of the external composition of the present invention (for example, the later mentioned “roughness correcting composition” or “makeup composition” etc.) or type of carrier (for example, oil base or emulsion base etc.).
  • type of carrier for example, oil base or emulsion base etc.
  • the volatile silicone oil it is possible to use a volatile silicone oil which is used in the field of cosmetics and other external compositions. It is not particularly limited. Specifically, for example, a low boiling point linear silicone oil such as hexamethyl disiloxane, octamethyl trisiloxane, decamethyl cyclopentasiloxane, dodecamethyl cyclohexasiloxane, and tetradecamethyl cycloheptasiloxane; a low boiling point cyclic silicone oil such as octamethyl cyclotetrasiloxane, decamethyl cyclopentasiloxane, dodecamethyl cyclohexasiloxane, and tetradecamethyl cycloheptasiloxane; etc. may be mentioned.
  • a low boiling point linear silicone oil such as hexamethyl disiloxane, octamethyl trisiloxane, decamethyl cyclopentasi
  • the external composition of the present invention may contain, depending upon the need, the following other components as auxiliary components to an extent not detracting from the desired effect of the present invention.
  • hydrocarbon oils such as liquid paraffin, isoliquid paraffin, squalane, oils and fats such as olive oil, palm oil, coconut oil, macadamia nut oil, jojoba oil; higher alcohols such as isostearyl alcohol; ester oils such as higher aliphatic oils and isopropyl myristate, etc.
  • formulating a polar oil in the external composition of the present invention enables improvement of the stability with the elapse of time.
  • a benzophenon derivative, para-aminobenzoate derivative, para-methoxysuccinate derivative, salicylate derivative, and other UV absorbers may be blended in the external composition of the present invention.
  • the cosmetic formulation of the present invention may be produced in an appropriate medium including but not limited to a paste, a powder, a cake, a crème, an oil, a lotion, a grease, a wax or similar cosmetic bases.
  • the process to produce involves combining the cosmetic ingredients and desamino, alkyl amino acid compound of any of formulas I-IV preferably as the ester, amide or salt.
  • the combination is mixed, kneaded, rolled, ground, heated or otherwise treated to form a substantially homogeneous mass or mixture for use. These steps can be accomplished by use of a kneader, grind wheel, rollers, mixer, heat exchanger, extruder and the like.
  • the invention is exemplified by modification of the natural peptide neurotensin.
  • the background, modification and biological activity of neurotensin and the corresponding peptides of the invention are discussed.
  • NT Neurotensin
  • CNS central nervous system
  • NT neurotensin
  • hypothermia antinociception, attenuation of d-amphetamine-induced hyperlocomotion, and potentiation of barbiturate-induced sedation are promoted by direct injection of NT into the brain.
  • NT acts as a hormone to induce hypotension and decrease gastric acid secretion.
  • NT is a linear tridecapeptide with the following sequence: pGlu-Leu-Tyr-Glu-Asn-Lys-Pro-Arg-Arg-Pro-Tyr-Ile-Leu-OH.
  • pGlu-Leu-Tyr-Glu-Asn-Lys-Pro-Arg-Arg-Pro-Tyr-Ile-Leu-OH was shown that the C-terminal hexapeptide fragment, Arg 8 -Arg 9 -Pro 10 -Tyr 11 -Ile 12 -Leu 13 [NT(8-13)], was equipotent at producing the physiological effects of NT in vitro and in vivo.
  • NTR 1 NT receptor 1
  • human NTR 1 has been successfully cloned and expressed. Both are classic G-protein coupled receptors containing seven transmembrane (7TM) domains and share 84% homology.
  • Second messenger systems including cGMP production, calcium mobilization, and phosphatidylinositol turnover, are triggered upon NTR 1 activation.
  • the mRNA for NTR 1 is expressed in both rat and human brain and intestine.
  • NTR 2 is also a 7TM/G-protein coupled receptor, yet has a shorter N-terminal extracellular tail and a longer third intracytoplasmic loop compared to NTR 1 .
  • a third receptor (NTR 3 ) was cloned from a human brain cDNA library and found to be identical to the previously cloned gp95/sortilin.
  • NTR 3 is a non-G-protein coupled sorting protein having only a single transmembrane region.
  • NT neuropeptide derived neuropeptide
  • Advances in the dopamine theory of schizophrenia support that a flaw in the convergence of various neural circuits on the mesolimbic dopamine system is responsible for the development of the disorder.
  • the anatomical positioning of the NT system is such that it interacts with the glutaminergic, dopaminergic, GABAergic, and serotonergic systems within the brain.
  • the NT and dopamine systems are closely related within the nucleus accumbens, the area of the brain believed to be responsible for delusions and hallucinations.
  • NTR 1 receptors are dense in the ventral tegmental area, a brain region closely associated with the neuronal systems described above. Almost 90% of NT receptors are located on dopaminergic neurons and over 80% of dopamine neurons in the brain express NTR 1 .
  • Co-localization of the NT system with brain regions implicated in schizophrenia also imply its involvement.
  • NT was hypothesized as an “endogenous neuroleptic” and NT(8-13) was identified as its active fragment, efforts have been made to develop NT(8-13) derivatives as potential antipsychotics.
  • amino acid substitutions at Arg 8 , Arg 9 , Tyr 11 , and Ile 12 have yielded several analogues that are centrally active after peripheral administration.
  • An Eisai compound (the Eisai hexapeptide) was the first NT(8-13) analogue that elicited behavioral effects after peripheral administration.
  • the various modifications incorporated in this peptide resulted in a 700-fold loss of binding affinity at NTR 1 .
  • this analogue was not able to elicit central activity after oral administration.
  • NT69L also attenuates hyperactivity induced by both cocaine and d-amphetamine.
  • tolerance to its hypothermic effect and to its suppression of d-amphetamine induced hyperactivity was observed after chronic administration of the compound.
  • NT69L produced only a slight hypothermic response after oral administration.
  • N-terminal alpha methyl, alpha desamino homolysyl and orinthyl analogues of NT(8-13) prepared according to the invention were synthesized and screened for activity in numerous behavioral assays predictive of antipsychotic potential. These peptides induced hypothermia in a dose-dependent fashion after oral administration. In addition, oral administration of the peptides significantly reduced d-amphetamine induced hyperlocomotion, a measure of the therapeutic efficacy of current or potential APDs. The low dose of peptide (10 mg/kg) that elicits a significant response after oral administration in these assays is significant.
  • the peptides also demonstrate an ability to maintain efficacy after repeated administration. In fact they demonstrate an ability to increase maximal hypothermic response over time, implying that repeated administration may actually improve their CNS activity.
  • the NT peptides of the invention are shown to have biological activity like that of the known naturally occurring peptide NT and are more selective.
  • NT induces hypothermia when directly administered into the CNS.
  • induced hypothermia can be used to determine the ability of NT(8-13) peptides of the invention to cross the BBB after peripheral administration and indirectly to determine their in vivo CNS activity.
  • the hypothermic effect of NT can be attributed to its actions at NTR 1 , the NTR most often implicated in the pathophysiology of schizophrenia.
  • An NT(8-13) peptide that induced hypothermia after IP injection is thus shown to be an antipsychotic agent.
  • a significant hypothermic effect would demonstrate that the peptide showed marked improvements in blood stability and membrane crossing.
  • IP injection is the standard route of administration to determine the extent of BBB crossing of neurotensin analogues. The methods and protocols are provided in the Examples section. IV administration results in a dose that is completely available to the systemic circulation. By contrast, an IP injection is a more rigorous test of stability because the peptide is exposed to first pass metabolism in the liver.
  • hypothermic effects of peptides 28-30, after a 5 mg/kg IP injection are given in Table 2.
  • Each peptide exhibited a significant effect over a 5 hr time course.
  • the hypothermic results for these three peptides demonstrate that the substitution of an alpha alkyl group in place of the N-terminal amine group (i.e., the ⁇ -methyl group) does not abolish the in vivo activity of the NT(8-13) peptide.
  • the ability of these peptides to elicit CNS activity after oral administration was evaluated.
  • NT(8-13) peptides as antischizophrenic pharmaceuticals is to determine their ability to exhibit CNS activity after oral administration.
  • ABS201 an example of a peptide of the invention, demonstrated maximal hypothermic responses greater than 2° C. (Table 3) and its maximal hypothermic effect was equal to its hypothermic effect after IP dosing ( FIG. 9 ), resulting in an approximate oral bioavailability of 25%. While peptides 29 and 30 also were orally active, their ratio of oral activity to IP activity was not as balanced as that of ABS201. The oral activity of ABS201 was an important factor to support it as a lead NT(8-13) analogue for further evaluation of antipsychotic potential.
  • the blockade of locomotion caused by d-amphetamine, a “DA agonist”, has become the standard measure of therapeutic efficacy of current or potential drug candidates for treatment of schizophrenia.
  • This model operates on the assumption that the direct stimulation of DA receptors within the mesolimbic DA system is responsible for the locomotor response.
  • Catalepsy commonly defined as a state of tonic immobility in rodents, is regarded as analogous to (extrapyramidal side effects) EPSEs in humans. Consequently, catalepsy is a side effect to be avoided in a successful drug candidate. Concurrently, the degree to which a drug candidate causes catalepsy in rats may also be used as a predictor for the probable occurrence of EPSEs associated with that particular candidate.
  • ABS201 To examine the antipsychotic properties of ABS201, a dose-response curve for hypothermic induction after IP administration was generated. In addition, the hypothermic effects elicited by oral administration of ABS201 (10 mg/kg-30 mg/kg) were determined. The ability of ABS201 to reduce d-amphetamine induced hyperlocomotion after both IP and oral administration also was measured. To assess the effects on CNS activity caused by prolonged treatment of ABS201, hypothermia and attenuation of d-amphetamine induced hyperlocomotion were measured after repeated daily dosing. Finally, the bar test was utilized to measure catalepsy as a predictor of EPSEs in humans.
  • ABS201 also induced hypothermia in a dose-dependent fashion after oral administration ( FIG. 12 ). A significant hypothermic effect was demonstrated at 10 mg/kg, the lowest dose tested ( ⁇ 1.02 ⁇ 0.10° C. at 150 min PI). The generation of an ED 50 value for the oral administration of ABS201 was impractical due to the inordinate amount of peptide necessary to produce a complete dose-response curve.
  • Previous NT(8-13) analogues that have been under development as antipsychotic compounds have contained a Trp 11 substitution. Evidence from the studies presented herein supports the theory that this modification abolishes the oral activity of a NT analogue. Further studies are necessary to determine what specific role Tyr 11 plays in the oral bioavailability of NT(8-13) analogues.
  • d-amphetamine a “DA agonist”
  • NT(8-13) analogues currently under investigation as candidates have demonstrated the ability to decrease d-amphetamine induced hyperactivity in a dose-dependent fashion.
  • Sound- and light-attenuated locomotor cages are used to measure the ability of potential candidates to decrease d-amphetamine-induced hyperactivity.
  • ABS201 significantly reduced hyperlocomotion for all doses tested (doses of 3 mg/kg and 10 mg/kg not shown).
  • Another hallmark of current APDs is the ability to reduce spontaneous locomotor activity. All ABS201 dose groups responded significantly lower than saline during the drug phase, indicating the ability of ABS201 to reduce spontaneous activity.
  • ABS201 maintained a significant CNS effect after repeated daily dosing (Table 3) and over the 5-day period the absolute hypothermic response increased.
  • Table 3 A comparison of the induced hypothermia of ABS201 on days 1 and 5 was made. On day 5, the maximal hypothermic response was achieved faster (90 min) compared to day 1 (120 min). In contrast to day 1, on day 5 the maximal hypothermic effect was not maintained for an extended period, implying that while repeated dosing does not decrease the maximal effect, it may reduce the duration of the hypothermic effect.
  • Repeated daily dosing had no effect on the ability of ABS201 to attenuate d-amphetamine induced hyperlocomotion. Both the acute and chronic dosing groups produced a reduction in hyperactivity that was significant for almost two hours after amphetamine administration. Of note, chronic administration of ABS201 did abolish its inhibitory effect on spontaneous locomotor activity.
  • catalepsy In laboratory tests, catalepsy is characterized by the inability of an animal to correct its position after placement in an unnatural posture. Catalepsy tests can be greatly influenced by a number of variables. These include stress-induced inhibition of catalepsy caused by a new environment and the contribution of learned “pseudo-catalepsy” that can result upon repeated measures with the same animal. To circumvent these potential confounding factors, tests are performed on an animal only once in a quiet, controlled environment.
  • ABS201 did not induce catalepsy after peripheral administration, a hallmark of current clinically effective candidates.
  • Caco-2 cells derived from a human colorectal carcinoma, spontaneously differentiate into polarized cells that exhibit well-developed microvilli and brush-border enzymes. These features make the cells an excellent model of the human small intestine. A strong correlation between uptake of a compound in the Caco-2 cell model and oral bioavailability of the compound has been identified.
  • ABS201 is stable in rat serum for greater than 24 hours, however, its stability in cells has not been determined. Consequently, determination of the ability of intact peptide to enter the Caco-2 cells in the uptake experiments will show oral bioavailability and cellular stability.
  • Reverse phase HPLC is an ideal method to analyze the solubilized cell components for ABS201 and ABS201 degradation products. This analysis will show oral availability and cellular stability. Fractions can be collected at determined intervals and counted for radioactivity via LSC. By establishing the ABS201 elution time via a standard gradient, direct comparisons can be made to the contents of Caco-2 cells after uptake experiments.
  • Solvents are from Fisher Scientific (Pittsburgh, Pa.) and reagents from Aldrich (Milwaukee, Wis.) unless otherwise noted.
  • Trisyl-N 3 2,4,6-triisopropylbenzenesulfonyl azide; Et 3 N, triethylamine; t-BuCOCl, trimethylacetylchloride; n-BuLi, n-butyl lithium; H 2 , hydrogen gas; Pd—C, palladium on activated carbon; Xps, (S)-( ⁇ )-4-benzyl-2-oxazolidinone; KHMDS, potassium bis(trimethylsilyl) amide; CH 3 I, methyl iodide; H 2 O 2 , hydrogen peroxide; LiOH, lithium hydroxide; THF, tetrahydrofuran; CH 2 Cl 2 , dichloromethane; MgSO 4 , magnesium sulfate; Hex, hexane; EtOAc, ethyl acetate; NaHCO 3 , sodium bicarbonate; HCl, hydrochloric acid; N 2
  • Alpha methyl, alpha desamino omega N-substituted homo lysyl and orinthyl (8) neurotensin (8-13) were synthesized ( FIG. 7 ).
  • the solid state coupling was conducted as follows.
  • Resin bound N alpha Fmoc leucine was swelled in DMF prior to Fmoc cleavage with piperidine (20% in DMF). The piperidine solution was removed with vacuum filtration and the resin-bound amino acid washed with DMS and methylene chloride (5 ⁇ each). Amino acids (4 eq) were activated in DMF with HOBt (4 eq) PyBOP ((4 eq) and DIPEA (10 eq) and added directly to the peptide reaction vessel. Amino acids were coupled for 6 hours, the resins was washed with DMF and methylene chloride and monitored with a Kaiser test for the presence of free amines. Residues were recoupled when necessary. This procedure was repeated with subsequent amino acids to give the penultimate peptide sequence (pentamer).
  • Peptides were characterized and assessed for purity via MALDI-TOFMS on a Voyager DE-STR System 4117 mass spectrometer (Applied Biosystems, Foster City, Calif.). Peptides were used at greater than 95% purity in vivo.
  • Rats were restrained in Plas-Labs® plastic cages fitted with wooden dowels to restrict movement. Rectal temperature probes (Physitemp®, RET-2, Clifton, N.J.), lubricated with mineral oil, were inserted into the rectum of each animal. Probes were connected to a microprobe thermometer (Physitemp®, BAT-12) in conjunction with a thermocouple selector (Physitemp®, SWT-5). Rats were allowed to acclimate to the cages for 1 hr prior to IP injection.
  • IP Injection Peptides (5 mg/kg) were dissolved in saline (1 mL/kg). Following the equilibration period, rats were given an IP injection of peptide or saline. Initial temperature values were the average temperatures of the rats immediately before and after the injection. Subsequent measurements were taken every 30 min for 5 hr. One-way repeated measures ANOVAs followed by Tukey's post hoc test for multiple comparisons were performed for each peptide using GraphPad Prisms to measure significance. Results were considered significant for p ⁇ 0.05.
  • Dose-response curves for hypothermic induction All animal restraint and hypothermia protocols were as described previously. Variable slope dose-response curve and ED 50 value was generated using GraphPad Prism®.
  • rats were removed and given an IP injection of d-amphetamine (1 mg/kg) and returned to the chamber for a further 2 hr to assess the effect of the peptide on induced hyperlocomotion.
  • ABS201 (5 mg/kg) was dissolved in saline (1 ml/kg). A horizontal bar 5 mm in diameter was placed 7.5 cm above the cage floor. Rats were given an IP injection of peptide, saline, or haloperidol (1 mg/kg) and their front paws were placed directly on the bar. The rat was held in this position for 3 sec and then released. The time from release until the paws return to the cage floor was measured and recorded. A cut-off time of 30 sec was observed; this indicated a fully cataleptic animal. Measures were repeated every 30 min for 4 hr.
  • Dose-response curves The dose-response curve for ABS201 after IP injection over a concentration range of 0.1-10.0 mg/kg is shown in FIG. 11 .
  • the calculated ED 50 , value is 0.943 mg/kg.
  • the hypothermic response to the oral administration of ABS201 over a concentration range of 10.0-30.0 mg/kg is shown in FIG. 12 .
  • TIME TIME
  • DOSE DOSE
  • the main effect of TIME resulted from a decrease in activity for all doses relative to the first time point (70 min).
  • Tukey's post-hoc tests revealed that all ABS201 dose groups demonstrated reduced locomotor activity for time points 130-200 as compared to saline (p ⁇ 0.05).
  • ABS201 maintained a significant CNS effect after repeated daily dosing (Table 3) and over the 5-day period the absolute hypothermic response increased.
  • TIME TIME
  • F(5,90) 13.512 (p ⁇ 0.001)
  • GROUP GROUP
  • the main effect of TIME resulted from a decrease in activity for all doses relative to the first time point (70 min).
  • Tukey's post-hoc tests revealed that both the acute and chronic groups demonstrated reduced locomotor activity for time points 140-220 as compared to saline (p ⁇ 0.05).
  • Caco-2 cells derived from a human colorectal carcinoma, spontaneously differentiate into polarized cells that exhibit well-developed microvilli and brush-border enzymes (78). These features make the cells an excellent model of the human small intestine.
  • a strong correlation between uptake in the Caco-2 cell model and oral bioavailability has been identified (79).
  • Studies that focused on the transport of peptides across Caco-2 cells have identified solute-solvent hydrogen bonds as a major determining factor in the permeability of the peptide.
  • the non-natural amino acid technology is designed to reduce solute-solvent interactions, in particular, water solvation that occurs through hydrogen bonding, hence the current modifications should confer enhanced intestinal absorption in Caco-2 cells.
  • the studies described below are designed to evaluate the potential oral bioavailability of the NT(8-13) analogues and the mechanisms of transport responsible for their uptake.
  • ABS201 is a lead compound for the development of NT(8-13) analogues as novel APDs. ABS201 can therefore function as a prototype for evaluating the cellular uptake of the NT(8-13) analogues.
  • Liquid scintillation counting (LSC) is the preferred method of analysis for these assays, as extraction of the peptide from the cell monolayer is not required and dissolved cell components can be directly analyzed without an extraction protocol that can be inexact, resulting in inconsistent analysis.
  • L-[U- 14 C] ⁇ egrada was used as the radiolabel for these studies. Proline is easily protected at the ⁇ -amine for peptide synthesis with the base-labile Fmoc moiety.
  • Pro 10 has not been identified as a major site of cleavage of NT(8-13). NT(8-13) analogues that show antipsychotic potential have not included Pro 10 modifications.
  • NT(8-13) analogues elicit CNS activity after oral administration. They are the first analogues of NT(8-13) to exhibit oral activity, and these preliminary studies should provide information that aids in the development of future peptide analogues with enhanced oral activity.
  • the concentration of ABS201 used for these uptake studies can be chosen for two distinct reasons.
  • the concentration of a 20 mg/kg dose of peptide, delivered in saline (1 mL/kg), is 24 mM.
  • a concentration only slightly below 24 mM should be seen by the small intestine. Therefore, the concentration added to the Caco-2 cells is well below that theoretically seen in vivo.
  • the standard circulating blood volume in the rat is 64 mL/kg (82).
  • the concentration of a 20 mg/kg dose of peptide circulating throughout the entire rat is 377 ⁇ M.
  • 200 ⁇ M is determined to be a physiologically relevant concentration to study ABS201 uptake in vitro.
  • the compounds evaluated in Example 12 contain one non-natural amino acid (Scheme 1) or desaminoacid (Scheme 2).
  • hypothermia induction (NTR-1 receptor binding) activity was evaluated with both oral and IP dosing of each compound. As seen in Table 6, all of the compounds except for ABS201 exhibited ⁇ 10% oral activity. Interestingly, ABS201 had a 300% increase in oral activity over the previous most active compound. In addition, ABS201 achieved a faster response when administered orally versus IP. This is unique among the NT(8-13) derivatives.
  • IP Dose a in Oral Dose b App. Peptide t max c (min) BT d (° C.) t max c (min) ⁇ in BT d (° C.) Oral Saline 240 ⁇ 0.60 ⁇ 180 ⁇ 0.64 ⁇ NA ABS13 150 ⁇ 4.26 ⁇ 90 ⁇ 1.66 ⁇ ABS31 180 ⁇ 6.87 ⁇ 150 ⁇ 1.05 ⁇ NA ABS44 150 ⁇ 5.07 ⁇ 150 ⁇ 1.58 ⁇ ABS46 180 ⁇ 4.68 ⁇ 180 ⁇ 2.03 ⁇ ABS201 150 ⁇ 2.51 ⁇ 90 ⁇ 2.49 ⁇ ABS202 150 ⁇ 3.75 ⁇ 120 ⁇ 1.09 ⁇ NA ABS203 300 ⁇ 3.84 ⁇ 150 ⁇ 1.30 ⁇ NA a IP dose was 5 mg/kg for all peptides.
  • b Oral dose was 20 mg/kg for all peptides.
  • c t max (min) Time to maximal temperature decrease.
  • d ⁇ in BT (° C.) Decrease in body temperature measured at t max .
  • e Denotes a significant response (p ⁇ 0.05).
  • f Approximate oral bioavailability was calculated from the relative areas under the hypothermia curve for each dosing regimen, corrected for amount of compound administered.
  • g NA none apparent (as the oral dosing was not significant over baseline).
  • ABS201 is active in a dose-dependent fashion whether IP or orally injected ( FIGS. 13 and 14 ).
  • the action of ABS201 is apparent following both IV and PO administration; dose-dependent; and long acting (observable 1 hr post administration and apparent for at least one additional hour).
  • ABS201 does not induce a cataleptic state in rats ( FIG. 15 ), is not antinociceptive, and tolerance to multiple dosings does not occur either with monitoring hypothermia or inhibition of amphetamine-induced hyperlocomotion ( FIGS. 16 and 17 ).
  • ABS201 reliably induces hypothermia in rodents following both IV and PO administration.
  • the action of ABS201 is: dose-dependent; and long acting, being observable for a period of 3-4 hours following administration. The doses producing hypothermia are similar, if not identical, to those which reverse d-amphetamine responses.
  • ABS201 The following was observed during the period immediately during and following intravenous administration of ABS201: during the dosing period (slow push; >1 min ⁇ 2 min via the tail vein) the animals receiving ABS201 HCl became noticeably sedate in the body restraining cages; upon removal from the restraining cages, animals were obviously sedated, lacked spontaneous benchtop locomotor activity, assumed a curl position upon handling, and exhibited a greatly impaired, or loss of, the righting reflex; notwithstanding, ptosis was absent; there was no evidence of flaccid paralysis although muscle tone was substantially reduced; pupil reflex was present; the hind limb pinch response was impaired or absent; there was no evidence of parasympathetic responses, e.g., spontaneous urination, defecation, salivation and lacrimation were absent; there was no evidence of acute sympathetic responses, e.g., piloerection; there was no evidence of seizures, either tonic or clonic.
  • ABS201 Three separate concentrations of ABS201 (10 ⁇ 9 , 10 ⁇ 7 10 ⁇ 5 M) were screened individually against the following 16 receptors: adrenergic (alpha1, alpha 2, beta), dopamine, histamine (H1, H2, H3), muscarinic (central, peripheral), nicotinic, opioid (nonselective), orphanin, serotonin (transporter, nonselective), monoamine oxidase (A, B). No displacement of the receptors' endogenous substrate were observed. Hence ABS201 does not appear to bind with any of these receptors. In contrast, the ABS201 has nM affinity for the target receptor (NTR 1 ).
  • ABS201 was added to freshly isolated whole rat blood to a concentration of 100 ⁇ g/mL, allowed to partition, and the cellular fraction was removed by centrifugation. ABS201 at this concentration distributes almost evenly between the cellular and serum fractions. No metabolites of ABS201 were been detected, consistent with previous experiments in which a very long serum/plasma half-live was demonstrated.
  • ABS201 was administered to rats at IV doses up to 100 mg/kg and oral doses up to 500 mg/kg. No adverse effects of the compound (body weight loss, mortality, abnormal clinical evaluations panel) were seen out to 48 hr post administration. These experiments thus define lower limits for the MTD of ABS201 at 100 times the ED 50 for the compound in antipsychosis and other tests reflective of brain activity.
  • ABS201 was administered in one IV dose (5 mg/kg) or oral dose (50 mg/kg) to rats. At selected time points, blood was removed or brain harvested and the concentration of the compound determined. It was demonstrated that ABS201 could be detected in the blood and brain up to 120 minutes after both IV and oral administration. The amount in the brain was sufficient to saturate NTR-1 to produce the observed behavioral effects.
  • ABS201 The plasma and brain pharmacokinetics of ABS201 was studied following IV administration of 1 mg/kg and, oral administration of 30 mg/kg of ABS201 to non fasted rats. The results of this study indicated that: ABS201 was rapidly cleared from plasma following IV administration with a t 1/2 of about 5 minutes; the levels of the compound decreased below the LLOD by 45 min; the levels of ABS201 were below the LLOD at all times following oral administration; and the levels of ABS201 in brain were below the LLOD at all times following both IV and PO administration.
  • ABS201 In vitro metabolism and compartmentalization of ABS201 was also studied to evaluate the distribution of ABS201 in blood, the extent of protein binding in plasma, and to gain a preliminary assessment of the metabolism of ABS201 in blood and plasma.
  • ABS201 was cleared from whole blood in two phases with an initial phase and a second phase where compound was measurable at low levels up to 120 minutes following IV administration of 5 mg/kg; the levels of ABS201 in whole blood were below the LLOD at all times following oral administration of the compound (50 mg/kg); the levels of ABS201 in brain were below the LLOD at all times following IV administration; and measurable quantities of ABS201 were detected in brains of 2 of 3 animals 15 minutes post oral administration of 50 mg/kg.
  • ABS201 exhibited a 300% increase in central activity when administered orally, and achieved a more rapid response with oral versus IV injection.
  • ABS201 The pharmacokinetics, compartmentalization, and possible metabolism of ABS201 were evaluated in vitro and in vivo. The results suggest little or no metabolism, and complex pharmacokinetics which indicate that the compound initially undergoes a rapid clearance from blood, followed by a longer lasting, deep compartment phenomenon.
  • the pharmacodynamic response of ABS201 is long acting (2-4 hr) following both IV and PO administration; the acute effects of IV ABS201 are mediated via central nervous system; the compound does not appear to be metabolized upon co-incubation with plasma or whole blood; ABS201 partitions between the aqueous and cellular phases of blood in vitro; the PK profile of ABS201 in whole blood is consistent with a two phase clearance process; and the pharmacodynamic response which has been observed is likewise consistent with a two phase clearance process.
  • the compounds of the invention such as the semisynthetic peptide ABS201, could be administered on a once or twice daily basis.

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US20210388050A1 (en) * 2018-10-01 2021-12-16 University Of Houston System Engineered active single-polypeptide chain insulin analogs
US11760780B2 (en) * 2014-05-30 2023-09-19 Albert Einstein College Of Medicine Targeting dimerization of BAX to modulate BAX activity

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AU2009210570B2 (en) * 2008-01-30 2014-11-20 Indiana University Research And Technology Corporation Ester-based insulin prodrugs
JP2012505637A (ja) 2008-10-15 2012-03-08 アンジオケム，インコーポレーテッド Ｇｌｐ−１アゴニストのコンジュゲート及びその使用
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EP2585102B1 (fr) 2010-06-24 2015-05-06 Indiana University Research and Technology Corporation Promédicaments insuliniques à base d'amide
UY35144A (es) 2012-11-20 2014-06-30 Novartis Ag Miméticos lineales sintéticos de apelina para el tratamiento de insuficiencia cardiaca
US8921307B2 (en) 2012-11-20 2014-12-30 Novartis Ag Synthetic linear apelin mimetics for the treatment of heart failure
TN2016000031A1 (en) 2013-07-25 2017-07-05 Novartis Ag Cyclic polypeptides for the treatment of heart failure
KR20160031552A (ko) 2013-07-25 2016-03-22 노파르티스 아게 합성 아펠린 폴리펩티드의 생체접합체
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US20210388050A1 (en) * 2018-10-01 2021-12-16 University Of Houston System Engineered active single-polypeptide chain insulin analogs
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