WO2010004535A2 - Therapeutic methods and compositions employing peptide compounds - Google Patents

Abstract

Methods for alleviating pain in a mammal, methods for alleviating a symptom associated with cessation or withdrawal from an addiction to a habit-forming drug in a mammal, methods for alleviating inflammation in a mammal, and methods for alleviating a symptom associated with a neuropsychiatric disorder, wherein the neuropsychiatric disorder is a cognitive deficiency or anxiety, in a mammal, comprise administering to the mammal a therapeutically effective amount of a peptide compound of Formula (I), Formula (II), or Formula (III), or a pharmaceutically acceptable salt thereof: Formula (I), Formula (II), Formula (III) as described herein. Pharmaceutical compositions which may be employed, inter alia, in such methods comprise a peptide compound of Formula (I), Formula (II), or Formula (III) and a pharmaceutically acceptable carrier.

Description

THERAPEUTIC METHODS AND COMPOSITIONS EMPLOYING PEPTIDE

COMPOUNDS

FIELD OF THE INVENTION

[0001] The present invention is directed to methods for alleviating pain in a mammal, methods for alleviating a symptom associated with cessation of or withdrawal from an addiction to a habit-forming drug in a mammal, methods for alleviating inflammation in a mammal, methods for alleviating a symptom associated with a neuropsychiatric disorder, wherein the neuropsychiatric disorder is a cognitive deficiency or anxiety, in a mammal, and pharmaceutical compositions which may be employed, inter alia, in such methods. In additional embodiments, the invention is directed to peptide compounds which are suitable, inter alia, for use in such methods and compositions. As described in detail below, the term "peptide compounds" as used in the present disclosure to refers to both peptides and peptide-based analogues thereof.

BACKGROUND OF THE INVENTION

[0002] The undecapeptide Substance P (SP), discovered in 1931, was the first mammalian neuropeptide to be identified. SP is a neurotransmitter and neuromodulator at G-protein coupled neurokinin- 1 (NK-I) receptors and is involved in a variety of effects in different tissues. In the past, its role in pain transmission in the central nervous system (CNS) has attracted most attention. However, interest has also recently been directed to the involvement of SP in mental disorders including depression and anxiety. Despite many efforts worldwide to develop SP- related drugs, aprepitant (MK-869, approved as an anti-emetic agent) remains the only NK-I receptor antagonist on the market.

[0003] Neuropeptides are in many cases degraded into bioactive fragments with similar or modified biological activity. Additionally, degradation products often exert a modulating effect on the response of the parent peptide. SP is cleaved enzymatically into at least five metabolites, of which the N-terminal heptapeptide SP₁-₇ (H-Arg-Pro-Lys-Pro-Gln-Gln-Phe-OH) is the major fragment. SP₁-₇ has been shown to not only modulate several actions of SP but also opposes several effects of SP, e.g. the nociceptive effect, inflammatory effect, and the potentiating effect on opioid withdrawal symptoms. In addition, SPi-₇has also been shown to promote memory function.

[0004] Recent research suggests that SPi_₇ does not mediate its effects through any of the known tachykinin receptors or through activation of any opioid receptor. The SPi_₇ fragment rather appears to produce its effects through specific sites for this heptapeptide, which are not recognized by the parent peptide SP. Specific sites for SPi_₇ have been identified and characterized in the rat and mouse spinal cord (see Botros et al, "Endomorphin-1 and endomorphin-2 differentially interact with specific binding sites for substance P (SP) aminoterminal SPi_₇ in the rat spinal cord," Peptides, 27(4):753-759 (April 2006)) and in the rat brain (see Botros et al, "Endomorphins interact with the substance P (SP) aminoterminal SP₁-₇ binding in the ventral tegmental area of the rat brain," Peptides, 29(10):1820-1824 (October 2008, available online May 25, 2008)). The affinity of SPi_₇ for these sites highly exceeds those of the other N-terminal fragments of SP. During the characterization of the specific binding sites for SP i_₇, several potent analogues of SPi_₇ with high affinity have been discovered (see Fransson et al, "Small peptides mimicking substance Pi_₇ and encompassing a C-terminal amide functionality," Neuropeptides, 42(l):31-37 (February 2008, available online December 19, 2007)). Although the actions of SPi_₇ are well known, no explicit receptor for this peptide fragment has yet been identified. The affinity of SPi_₇ to the rat spinal cord site was much greater than the affinity of SP and other degradation fragments thereof, and established ligands of the neurokinin receptors NK-I, NK-2, and NK-3 showed no affinity at all towards this binding site. Additionally, negligible binding of SP₁-₇ to any of the known tachykinin and μ-opioid receptors indicates the occurrence of a specific binding site for SPi_₇.

[0005] Historically, the identification of receptors (mostly G-protein coupled receptors,

GPCRs), combined with the development of non-peptide antagonists and agonists, have been crucial for the elucidation of the functional role of neuropeptides. Since they are encoded in the genome, the function of a neuropeptide and its role in the mechanism behind diseases can be studied by knock-out techniques in transgenic animals. By nature, the specific biological effects of metabolites of genome-encoded peptides cannot be studied by such a technique. Thus, metabolically stable and selective analogues and non-peptide ligands of metabolites are essential pharmacological tools for such studies.

SUMMARY OF THE INVENTION

[0006] The present invention is directed to improved therapeutic methods, compositions and peptide compounds.

[0007] In one embodiment, the invention is directed to a method for alleviating pain in a mammal, comprising administering to the mammal a therapeutically effective amount of a compound of Formula I, Formula II, or Formula III, or a pharmaceutically acceptable salt thereof:

Formula I:

wherein:

1 9

R and R are individually H or alkyl; R³ and R⁴ are individually H or alkyl, or together form a ring having 3-5 carbon atoms;

R⁵ is H or alkyl; n is 0-3;

A is a 5-membered aromatic ring or a 6-membered aromatic ring, either ring optionally containing from 1 to 3 heteroatoms selected from the group consisting of N, S and O, optionally substituted with 1 to 3 substituents of halogen, alkyl, alkoxy, CF₃, nitro, or nitrile;

10

R

\— X

R⁶ and R⁷ are individually H, alkyl, aromatic, heteroaromatic, of the formula R⁹>" wherein

R and R are individually H or alkyl, or R forms a ring having 4 or 5 carbon atoms with R , or R forms a ring having 4 or 5 carbon atoms with R , and X is a 5-membered aromatic ring or a 6-membered aromatic ring, either ring optionally containing from 1 to 3 heteroatoms selected from the group consisting of N, S and O, optionally substituted with 1 to 3 substituents of

halogen, alkyl, alkoxy, CF₃, nitro, or nitrile, or of the formula

wherein R and R are individually H or alkyl, m is 1 to 4, and Y is CONH₂, CONH-alkyl, aromatic or heteroaromatic, or R or R forms a ring having 4 or 5 carbon atoms with R ; and

R⁸ is H, alkyl, acyl or aromatic, or R⁸ forms a ring having 4 or 5 carbon atoms with R⁶, R⁷ or R⁹; Formula II:

wherein:

R¹ and R² are individually H or alkyl;

R³ and R⁴ are individually H or alkyl, or together form a ring having 3-5 carbon atoms;

R⁵ is H or alkyl; n is 0-3;

A is a 5-membered aromatic ring or a 6-membered aromatic ring, either ring optionally containing from 1 to 3 heteroatoms selected from the group consisting of N, S and O, optionally substituted with 1 to 3 substituents of halogen, alkyl, alkoxy, CF₃, nitro, or nitrile; and

R¹⁹ is alkyl, O-alkyl, O-benzyl, O-9-fluorenylmethyl, N-alkyl, N-benzyl, or N-9-fluorenylmethyl;

Formula III:

wherein:

Z is NH₂, OH or NH-alkyl;

R² is H or alkyl;

R³ and R⁴ are individually H or alkyl, or together form a ring having 3-5 carbon atoms;

R⁵ is H or alkyl; n is 0-3;

A is a 5-membered aromatic ring or a 6-membered aromatic ring, either ring optionally containing from 1 to 3 heteroatoms selected from the group consisting of N, S and O, optionally substituted with 1 to 3 substituents of halogen, alkyl, alkoxy, CF₃, nitro, or nitrile; and R ,2 ^z0^υ is of the formula (i)

wherein p is 1-6,

wherein p is 1-5,

, wherein p is 1-4,

wherein p is 1-5, or

, wherein R' and R" are amino acid side chains, and R²¹ is H or CH₃C(O)-.

[0008] In another embodiment, the invention is directed to a method for alleviating a symptom associated with cessation of or withdrawal from an addiction to a habit-forming drug in a mammal, comprising administering to the mammal a therapeutically effective amount of a compound of Formula I, Formula II, or Formula III, as described above, or a pharmaceutically acceptable salt thereof.

[0009] In a further embodiment, the invention is directed to a method for alleviating inflammation in a mammal, comprising administering to the mammal a therapeutically effective amount of a compound of Formula I, Formula II, or Formula III, as described above, or a pharmaceutically acceptable salt thereof.

[0010] In another embodiment, the invention is directed to a method for alleviating a symptom associated with a neuropsychiatric disorder, wherein the neuropsychiatric disorder is a cognitive deficiency or anxiety, in a mammal, comprising administering to the mammal a therapeutically effective amount of a compound of Formula I, Formula II, or Formula III, as described above, or a pharmaceutically acceptable salt thereof

[0011] In additional embodiments, the invention is directed to pharmaceutical compositions which may be employed, inter alia, in such methods. The pharmaceutical compositions comprise a therapeutically effective dose of a peptide compound of Formula I, Formula II, or Formula III, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier. [0012] In yet further embodiments, the invention is directed to peptide compounds of Formula I or Formula II, or pharmaceutically acceptable salts thereof. In other embodiments, the invention is directed to a compound selected from the group consisting of Ala-Pro-Phe-Phe-NH₂, Tyr-Ala-Phe-Phe-NH₂, Tyr-Pro-Ala-Phe-NH₂, Tyr-Pro-Phe-Phe-OH, and Pro-Phe-Phe- NH₂. [0013] The methods, compositions and peptide compounds of the invention are advantageous in providing smaller, drug-like molecules exhibiting stable SPi_₇-mimetic properties. Additional advantages and aspects of the various embodiments of the invention will be more fully apparent in view of the following detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

[0014] The detailed description and the examples will be more fully understood in view of the drawings in which:

[0015] Figs. IA and IB show the anti-nociceptive effects of intrathecal (i.t.) injection of SPi_ 7-NH₂, SP_1-7 and Phe-Phe-NH₂ in non-diabetic and diabetic mice, respectively, as described in the Examples;

[0016] Figs. 2A and 2B show the effect of various opioid receptor antagonists on the antinociceptive effects of i.t. injected SP_1-7-NH₂ in non-diabetic and diabetic mice, respectively, as described in the Examples; and

[0017] Figs. 3 A-3C show the effect of intracerebroventricular (i.c.v.) injected SPi_₇-NH₂ on teeth chattering, ptosis and writhing, respectively, during nalox one-precipitated morphine withdrawal in rats, as described in the Examples.

[0018] Further details of the drawings are provided in the Examples. DETAILED DESCRIPTION [0019] The present invention is directed to peptide compounds, and to compositions and methods employing peptide compounds. As noted above, the term "peptide compounds" is used in the present disclosure to refer to both peptides and peptide-based analogues thereof. The peptide compounds of the invention and those employed in the inventive compositions and methods have been found to exhibit good affinity to a SPi_₇ binding site. In one embodiment, the compounds of the invention exhibit an affinity K₁ of less than 1000 nM in a competitive binding assay using spinal cord membrane as described in the Examples below. The compounds are metabolically stable SPi_₇ mimetics and, as lower molecular weight analogs, may therefore be conveniently used as research tools, for example, for the identification of the SPi_₇ receptor and for functional studies in complex animal models. According to additional embodiments of the invention, the compounds have pharmaceutical use, i.e., as analgesics in methods of alleviating pain; as actives in methods of alleviating one or more symptoms associated with cessation or withdrawal from an addiction to a habit-forming drug; as anti-inflammatory actives in methods of alleviating inflammation, as active agents in methods for alleviating a symptom associated with a neuropsychiatric disorder, for example, neuropsychiatric disorders such as a cognitive deficiency, wherein the methods can, for example, reverse and/or delay onset of such symptoms, or anxiety, wherein the methods can alleviate or reduce onset of anxiety, and the like. [0020] In a first embodiment, compounds according to the invention are of Formula I, or are a pharmaceutically acceptable salt thereof: Formula I:

wherein:

R¹ and R² are individually H or alkyl;

R³ and R⁴ are individually H or alkyl, or together form a ring having 3-5 carbon atoms;

R⁵ is H or alkyl; n is 0-3;

A is a 5-membered aromatic ring (i.e., a 5-membered ring having unsaturation) or a 6-membered aromatic ring, either ring optionally containing from 1 to 3 heteroatoms selected from the group consisting of N, S and O, optionally substituted with 1 to 3 substituents of halogen, alkyl, alkoxy,

CF₃, nitro, or nitrile;

R "> 6 and j - Rr> 7 are individually H, alkyl, aromatic, heteroaromatic, of the formula

wherein R and R are individually H or alkyl, or R forms a ring having 4 or 5 carbon atoms with R , or R forms a ring having 4 or 5 carbon atoms with R , and X is a 5-membered aromatic ring or a 6-membered aromatic ring, either ring optionally containing from 1 to 3 heteroatoms selected from the group consisting of N, S and O, optionally substituted with 1 to 3 substituents of

halogen, alkyl, alkoxy, CF₃, nitro, or nitrile, or of the formula

wherein R and R are individually H or alkyl, m is 1 to 4, and Y is CONH₂, CONH-alkyl, aromatic or heteroaromatic, or R⁶ or R⁷ forms a ring having 4 or 5 carbon atoms with R⁸; and

R⁸ is H, alkyl, acyl or aromatic, or R⁸ forms a ring having 4 or 5 carbon atoms with R⁶, R⁷ or R⁹. [0021] Within the present disclosure, the term "alkyl" refers to alkyl groups of from 1 to 8 carbon atoms, optionally substituted with one or more halogen atoms. In a specific embodiment, the alkyl group substituents are from 1 to 3 carbon atoms, i.e., methyl, ethyl, propyl or isopropyl, optionally substituted with one or more halogen atoms.

[0022] In specific embodiments, the compounds are according to Formula I wherein n is 0 or

1 , A is

wherein R¹³, R¹⁴ and R¹⁵ are individually H, halogen or alkyl, R⁶ is H

or alkyl or forms a ring having 4 or 5 carbon atoms with R 8 , R 7 is

wherein R 9 is H or

forms a ring having 4 or 5 carbon atoms with R⁸, R¹⁰ is H, and X is

wherein

R¹⁶, R¹⁷ and R¹⁸ are individually H, halogen or alkyl. In more specific embodiments, R¹ and R⁸ are each H and R³ is H or alkyl, more specifically methyl, in combination with the preceding defined variables and therefore the compounds of this embodiment are of the formula

wherein R³ is H or alkyl, and R¹³, R¹⁴, R¹⁵, R¹⁶, R¹⁷ and R¹⁸ are individually H, halogen or alkyl, or a pharmaceutically acceptable salt thereof. Specific examples of these compounds are set forth in the Examples. It should be specifically noted that the inventive compounds of Formula I encompass all racemates and enantiomers of the compounds of the disclosed formulae. Further, the compounds of the invention may be prepared, inter alia, using known techniques, for example solid-phase peptide synthesis, examples of which are also set forth below in the Examples.

[0023] In another embodiment, the compounds of the invention are of Formula II, or are a pharmaceutically acceptable salt thereof: Formula II:

wherein:

R¹ and R² are individually H or alkyl;

R³ and R⁴ are individually H or alkyl, or together form a ring having 3-5 carbon atoms;

R⁵ is H or alkyl; n is 0-3;

A is a 5-membered aromatic ring or a 6-membered aromatic ring, either ring optionally containing from 1 to 3 heteroatoms selected from the group consisting of N, S and O, optionally substituted with 1 to 3 substituents of halogen, alkyl, alkoxy, CF₃, nitro, or nitrile; and

R¹⁹ is alkyl, O-alkyl, O-benzyl, O-9-fluorenylmethyl, N-alkyl, N-benzyl, or N-9-fluorenylmethyl. [0024] In specific embodiments, R and R are H, n is 0 or 1, and A is

wherein R , R and R are individually H, halogen or alkyl. Specific examples of these compounds are set forth in the Examples. It should be specifically noted that the inventive compounds of Formula II encompass all racemates and enantiomers of the compounds of the disclosed formulae. Further, the compounds of the invention may be prepared, inter alia, using known techniques, for example solid-phase peptide synthesis, examples of which are also set forth below in the Examples.

[0025] Additional compounds suitable for use in the present invention include those of

Formula III, or a pharmaceutically acceptable salt thereof:

Formula III:

wherein:

Z is NH₂, OH or NH-alkyl;

R² is H or alkyl;

R³ and R⁴ are individually H or alkyl, or together form a ring having 3-5 carbon atoms;

R⁵ is H or alkyl; n is 0-3; A is a 5-membered aromatic ring or a 6-membered aromatic ring, either ring optionally containing from 1 to 3 heteroatoms selected from the group consisting of N, S and O, optionally substituted with 1 to 3 substituents of halogen, alkyl, alkoxy, CF₃, nitro, or nitrile; and

R²⁰ is of the formula (i)

wherein p is 1-6,

2 (corresponding to amino acids-Gin-) wherein p is 1-5,

, (corresponding to amino acids-Gln-Gln-)

wherein p is 1-4,

(corresponding to amino acids- Phe-) wherein p is 1-5, or

(corresponding to

91

Arg-Pro-Lys-Pro-Gln-Gln-), wherein R' and R" are amino acid side chains, and R is H or

CH₃C(O)-. In another specific embodiment, A is

R wherein R¹³, R¹⁴ and R¹⁵ are individually H, halogen or alkyl. Specific examples of these compounds include Arg-Pro- Lys-Pro-Gln-Gln-Phe-NH₂, Gln-Gln-Phe-NH₂, Ala-Pro-Phe-Phe-NH₂, Tyr-Ala-Phe-Phe-NH₂, Tyr-Pro-Ala-Phe-NH₂, Tyr-Pro-Phe-Phe-OH, and Pro-Phe-Phe- NH₂. Additional specific examples of these compounds are set forth in the Examples. It should be specifically noted that the inventive compounds of Formula III encompass all racemates and enantiomers of the compounds of the disclosed formulae. Further, the compounds of the invention may be prepared, inter alia, using known techniques, for example solid-phase peptide synthesis, examples of which are also set forth below in the Examples.

[0026] The pharmaceutically acceptable salts of the subject compounds include the conventional non-toxic salts or quaternary ammonium salts known for pharmaceutical use, suitably prepared, e.g., from non-toxic organic or inorganic acids. For example, such conventional non-toxic salts include those derived from inorganic acids such as hydrochloric, hydrobromic, sulphuric, sulfamic, phosphoric, nitric, and the like; and the salts prepared from organic acids such as acetic, valeric, oleic, lauric, naphthoic, glucoheptonic, laurylsulphonic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, palmitic, maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicyclic, sulfanilic, 2-acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, isothionic, and the like. [0027] The compositions according to the invention comprise a therapeutically-effective amount of one or more of the peptide compounds described above, together with one or more pharmaceutically acceptable carriers. Any pharmaceutical carrier known in the art may be employed, including, but not limited to, saline, sugar, lactose, mannitol, starch, celluloses, including microcrystalline cellulose, and the like. Conventional additives and/or diluents employed in pharmaceutical formulations, including, but not limited to, glidants, lubricants, wetting agents, emulsifying agents, pH buffering agents, stabilizers, and the like, may be employed as well. The pharmaceutical compositions of the present invention may be specially formulated for administration in solid or liquid form, including those adapted for the following

(1) oral administration, for example, drenches (aqueous or non-aqueous solutions or suspensions), tablets, capsules, boluses, powders, granules, pastes and the like, in immediate release form, sustained or controlled release form, delayed release form, or combinations thereof;

(2) parenteral administration, for example, by subcutaneous, intramuscular or intravenous injection as, for example, a sterile solution or suspension; (3) topical application, for example, as a cream, ointment or spray applied to the skin; or (4) intravaginally or intrarectally, for example, as a pessary, cream or foam. The compositions of the invention may also include one or more additional active agents as desired.

[0028] The phrase "therapeutically-effective amount" as used herein means that amount of a compound of the present invention which is effective for producing the desired therapeutic effect in at least a sub-population of cells in an animal at a reasonable benefit/risk ratio applicable to the therapeutic treatment. In general, a suitable amount of a peptide compound of the invention will be that amount of the compound which is the lowest dose effective to produce the therapeutic effect. Such an effective dose will typically depend upon various factors. Generally, doses will range from about 0.0001 to about 100 mg peptide compound per kilogram of body weight per day. In more specific embodiments, doses will range from about 0.001 to about 100 mg peptide compound per kilogram of body weight per day. In another embodiment, doses will range from about 0.001 to about 10 mg peptide compound per kilogram of body weight per day, or, alternatively, from about 0.1 to about 100 mg peptide compound per kilogram of body weight per day. If desired, the effective daily dose of the active compound may be administered as a single dose or as two, three, four, five, six or more sub-doses administered separately at appropriate intervals throughout the day, optionally, in unit dosage forms. [0029] The above compounds exhibit anti-nociceptive and anti-inflammatory properties and are suitable for use in methods for alleviating pain and in methods for alleviating inflammation, particularly in mammals, and, more specifically, in humans. The methods comprise administering a therapeutically effective amount of a peptide compound of Formula I, Formula II or Formula III as described, or a pharmaceutically acceptable salt thereof. In particular, the methods for alleviating pain may comprise alleviating, inter alia, acute or chronic pain, or, more specifically, chronic neuropathic pain. Further, the methods for alleviating inflammation may comprise alleviating acute, late phase acute, recurrent or chronic injury-related inflammation. In a specific embodiment, the methods comprise alleviating inflammation related to acute or recurrent injury. In one embodiment, the methods comprise alleviating peripheral neurogenic inflammation. The methods of alleviating inflammation result in alleviating one or more symptoms of inflammation, for example, heat release, vasodilation, erythema, edema and/or pain. In a specific embodiment, the methods of alleviating inflammation result in alleviating vasodilation.

[0030] The compounds also exhibit an attenuating effect on symptoms associated with cessation of or withdrawal from an addiction, i.e., an addictive use, of a habit-forming drug, such as opioids, and therefore are suitable for use in methods for alleviating one or more symptoms associated with cessation of or withdrawal from an addition of a habit-forming drug, particularly in mammals, and, more specifically, in humans. The methods comprise administering a therapeutically effective amount of a peptide compound of Formula I, Formula II or Formula III as described, or a pharmaceutically acceptable salt thereof. In a specific embodiment, the methods are directed to alleviating a symptom associated with cessation of or withdrawal from opioid use. In an additional embodiment, the methods are directed to alleviating one or more symptoms including, but not limited to, dysphoric mood, nausea, vomiting, muscle aches, lacrimation, rhinorrhoea, papillary dilation, piloerection, sweating, diarrhea, yawning, fever, insomnia, and opioid craving.

[0031] Various aspects and embodiments of the invention are illustrated in the following Examples. In these Examples, the following general procedures were employed. Preparative RP-HPLC was performed on a system equipped with a Zorbax SB-C8 column (150 x 21.2 mm) or a 10 μm Vydac C18 column (250 x 22 mm), in both cases with UV detection at 230 nm. Analytical RP-HPLC-MS was preformed on a Gilson-Finnigan ThermoQuest AQA system (Onyx monolithic Cl 8 column, 50 x 4.6 mm; MeCN/H₂O gradient with 0.05% HCOOH) in ESI mode, using UV (214 and 254 nm) and MS detection. The purity of each of the peptides was determined by RP-HPLC using the columns: ACE 5 Cl 8 (50 x 4.6 mm) and ACE 5 Phenyl (50 x 4.6 mm) or Thermo Hypersil Fluophase RP (50 x 4.6 mm) with a H₂OZMeCN gradient with 0.1% TFA and UV detection at 220 nm. NMR spectra were recorded on a Varian Mercury plus spectrometer (¹H at 399.8 MHz and ¹³C at 100.5 MHz or ¹H at 399.9 MHz and ¹³C at 100.6 MHz) at ambient temperature. Chemical shifts (δ) are reported in ppm referenced indirectly to TMS via the solvent residual signal. Exact molecular masses were determined on a Micromass Q-Tof2 mass spectrometer equipped with an electrospray ion source at the Department of Pharmaceutical Biosciences, Uppsala University, Sweden. Amino acid analyses were performed at the Department of Biochemistry and Organic Chemistry, Uppsala University, Sweden. All other chemicals and solvents were of analytical grade from commercial sources. Example 1

[0032] This example describes the general synthesis of Peptides 1 -6. The Peptides 1 -6 were synthesized with a Symphony instrument (Protein Technologies, INC., Tucson, AZ, USA) using standard Fmoc chemistry techniques known in the art, for example as earlier described by Fransson et al (2008), cited herein. Cleavage and purification were carried out as described below. The reported yields are based on the loading of the starting resin and with correction for peptide content according to amino acid analysis.

[0033] Peptide 1 (H-Tyr-Pro-Phe-Phe-NH₂). An aliquot of the crude cleavage product was purified to yield 17.5 mg (53%). HPLC purity: Cl 8 column 99.8%, Phenyl column > 99.9%. LC/MS (M: 571.3): 572.3 (M + H⁺). Amino acid analysis: Tyr, 1.00; Pro, 1.02; Phe, 1.98 (78% peptide).

[0034] Peptide 2 (H-Ala-Pro-Phe-Phe-NH₂). An aliquot of the crude cleavage product was purified to yield 15.9 mg (46%). HPLC purity: Cl 8 column 98.5%, Phenyl column > 99.9%. LC/MS (M: 479.3): 480.3 (M + H⁺). Amino acid analysis: Ala, 1.00; Pro, 1.02; Phe, 1.99 (71% peptide).

[0035] Peptide 3 (H-Tyr-Ala-Phe-Phe-NEb). An aliquot of the crude cleavage product was purified to yield 22.2 mg (66%). HPLC purity: Cl 8 column 99.8%, Phenyl column > 99.9%.

LC/MS (M: 545.3): 546.3 (M + H⁺). Amino acid analysis: Tyr, 0.99; Ala, 1.00; Phe, 2.01 (85% peptide).

[0036] Peptide 4 (H-Tyr- PrO-AIa-PhC-NH₂). An aliquot of the crude cleavage product was purified to yield 15.7 mg (57%). HPLC purity: Cl 8 column 99.6%, Phenyl column > 99.9%.

LC/MS (M: 495.3): 496.3 (M + H⁺). Amino acid analysis: Tyr, 0.99; Pro, 1.01; Ala, 1.02; Phe,

0.98 (76% peptide).

[0037] Peptide 5 (H-Tyr- PrO-PhC-AIa-NH₂). An aliquot of the crude cleavage product was purified to yield 17.9 mg (75%). HPLC purity: Cl 8 column 99.7%, Phenyl column > 99.9%.

LC/MS (M: 495.3): 496.3 (M + H⁺). Amino acid analysis: Tyr, 1.00; Pro, 1.00; Phe, 0.99 Ala,

1.02; (76% peptide).

[0038] Peptide 6 (H-Tyr- Pro-Phe-Phe-OH). An aliquot of the crude cleavage product was purified to yield 14.9 mg (42%). HPLC purity: Cl 8 column > 99.9%, Phenyl column > 99.9%.

LC/MS (M: 572.3): 573.3 (M + H⁺). Amino acid analysis: Tyr, 1.01; Pro, 1.01; Phe, 1.99 (81% peptide).

Example 2

[0039] This example describes the general synthesis of Peptide 8. Peptide 8 (H-Phe-Phe-

NH₂). To a solution of Z-Phe-OSu (200 mg, 0.505 mmol) and the hydrochloride salt of H-Phe-

NH₂ (IIl mg, 0.555 mmol) in EtOAc (15 ml) on ice, was added DIEA (97 μl, 0.555 mmol) and the mixture was stirred at room temperature. After 1.5 h, an additional portion of DIEA (97 μl, 0.555 mmol) was added and the mixture was stirred over night. The precipitate was filtered and dried under vacuum to yield Z-Phe-Phe-NH₂ (131 mg) as a white solid. The crude product was analysed by LC-MS and found to be adequate for further reaction.

[0040] The product was solved in MeOHiCHCl₃ (1 : 1 , 10 ml) in a round-bottom flask and

Pd/C (10%, 33 mg, 0.032 mmol) was added. The flask was sealed with a septum and purged, first with nitrogen and then with hydrogen gas. The reaction mixture was stirred under hydrogen atmosphere over night and was then filtered and evaporated. The product was purified by RP-

HPLC as described below to yield H-Phe-Phe-NH₂ in 19 mg (20%). HPLC purity: Cl 8 column >

99.9%, Phenyl column > 99.9%. LC/MS (M: 311.2): 312.1 (M + H⁺). ¹H NMR (MeOD) δ 2.96

(dd, / = 8.5, 13.8 Hz, IH), 3.01 (dd, J = 8.6, 14.3 Hz, IH), 3.14(dd, J = 6.2, 13.8 Hz, IH), 3.26

(dd, J = 5.5, 14.3 Hz, IH), 4.08 (J = 5.5, 8.6 Hz, IH), 4.66 (dd, J = 6.2, 8.5 Hz, IH), 7.17-7.39

(m, 10H). ¹³C NMR (MeOD) δ 38.6, 39.1, 55.5, 56.0, 127.9, 128.9, 129.5, 130.2, 130.3, 130.5,

135.5, 138.2, 169.5, 175.3. Amino acid analysis: Phe, 2.00 (70% peptide).

[0041] For comparison, as described below, Peptide 9 (H-PhC-NH₂) of analytical grade was obtained from a commercial source and tested without any further purification.

Example 3

[0042] This example describes the general synthesis of Peptides 7 and 10-20.

[0043] Coupling: The Peptides 7 and 10-20 were synthesized manually from Rink Amide

MBHA resin (0.66 mmol/g) or Phe-2-chlorotrityl resin (0.85mmol/g) in 2 mL disposable syringes fitted with porous polyethylene filter. Standard Fmoc conditions were used as described above. The Fmoc protecting group was removed by treatment with 20% piperidine in DMF (2 x

1.5 mL, 2 + 10 min) and the polymer was washed with DMF (6 x 1.5 mL, 6 x 1 min). Coupling of the appropriate amino acid, Fmoc-AA-OH (4 equiv) was performed in DMF (1.5 mL) using HBTU (4 equiv.) in the presence of DIEA (8 equiv). The resin was washed with DMF (5 x 1.5 mL, 5 x 1 min) and subsequently deprotected and washed as described above. After completion of the coupling cycle the resin was also washed with several portions of DMF, CH₂Cl₂ and

MeOH before it was dried in in vacuo.

[0044] Cleavage. The final peptide was cleaved from the resin by treatment with triethylsilane (100 μL) and 95% aqueous TFA (1.5 mL) followed by agitation for 2 h in room temperature. The resin was filtered off and washed with TFA (2 x 0.3-0.5 mL). The filtrate was collected in a centrifuge tube and concentrated in a stream of nitrogen. Cold diethyl ether (< 7 mL) was used to precipitate the product, which was collected by centrifugation, washed with cold diethyl ether (< 3 x 7 mL) and dried.

[0045] Purification. The crude peptide was dissolved in MeCN/0.1% aqueous TFA, filtered through a 0.45 μm nylon membrane and purified in 1-2 runs by RP-HPLC. Selected fractions were analyzed by RP-HPLC and RP-HPLC-MS, and those containing pure product were pooled and lyophilized. The reported yields are based on the loading of the starting resin and with correction for peptide content according to amino acid analysis.

[0046] Peptide 7 (H-Pro-Phe-Phe-NH₂). The crude peptide was purified to yield 19.7 mg

(48%). HPLC purity: Cl 8 column > 99.9%, Fluophase > 99.9%. LC/MS (M: 408.2): (M + H⁺).

Amino acid analysis: Pro, 1.00; Phe, 2.00 (78% peptide).

[0047] Peptide 10 (H-Phe-Phe-OH). The crude peptide was purified to yield 15.1 mg (97%).

HPLC purity: Cl 8 column > 99.9%, Fluophase 96.2% (Ring closure in water). ¹H NMR

(MeOD) δ 2.49 (dd, J = 8.8, 14.4 Hz, IH), 3.04 (dd, J = 8.80 14.1 Hz, IH), 3.25 (dd, J = 5.2,

14.1 Hz, IH), 3.28 (dd, J = 5.0, 14.3 Hz, IH), 4.07 (dd, J = 5.0, 8.8 Hz, IH), 4.70 (dd, J = 5.2,

8.8 Hz, IH), 7.18-7.40 (m, 10H). ¹³C NMR (MeOD) δ 38.3, 38.5, 55.5, 55.6, 127.9, 128.9, 129.6, 130.1, 130.3, 130.6, 135.5, 138.3, 169.7, 174.2. LC/MS (M: 312.2): 313.1 (M + H⁺). Amino acid analysis: Phe, 2.00 (73% peptide).

[0048] Peptide 11 (Ac-Phe-Phe-NEb). The synthesis was conducted as described above. Acetylation was performed by allowing the resin to react with acetic anhydride (5 equiv.) and DIEA (6 equiv.) in DMF (1.5 mL) for 2 h directly after the last Fmoc deprotection. The resin was washed and dried, and the final peptide was subjected to the standard cleavage and purification procedures to yield 16 mg (43%). HPLC purity: Cl 8 column > 99.9%, Fluophase > 99.9%. ¹H NMR (CDCl₃: MeOD 2:1) δ 1.85 (s, 3H), 2.80 (dd, J = 8.1, 13.8 Hz, IH), 2.87 (dd, J = 8.3, 13.9 Hz, IH), 2.98 (dd, J = 6.7, 13.8 Hz, IH), 3.11 (dd, J= 5.8, 13.9 Hz, IH), 4.51 (dd, J = 6.7, 8.1 Hz, IH), 4.54 (dd, J= 5.8, 8.3 Hz, IH), 7.12-7.26 (m, 10H). ¹³C NMR (CDCl₃: MeOD 2:1) 522.5, 38.0 (two signals) 54.6, 55.4, 127.3, 127.4, 128.9, 129.0, 129.6, 129.8, 137.2, 137.3,

172.2, 172.3, 174.6. LC/MS (M: 353.2): 354.1 (M + H⁺). Amino acid analysis: Phe, 2.00 (95% peptide).

[0049] Peptide 12 (L-Phe-D-Phe-NEb). The crude peptide was purified to yield 7.3 mg (35%). HPLC purity: C18 column 98.4%, Fluophase > 99.996.¹H NMR (MeOD) δ 2.74 (dd, J = 8.0, 14.2 Hz, IH), 2.81 (dd, J = 9.8, 13.9 Hz, IH), 2.92 (dd, J= 5.8, 14.2 Hz, IH), 3.14 (dd, J = 5.3, 13.9 Hz, IH), 4.05 (dd, J = 5.8, 8.0 Hz, IH), 4.65 (dd, J = 5.3, 9.8 Hz, IH), 6.98-7.06 (m, 2H), 7.15-7.35 (m, 8H). ¹³C NMR (MeOD) δ 38.4, 39.1, 55.5, 55.7, 128.0, 128.8, 129.6, 130.1,

130.3, 130.4, 135.3, 138.3, 169.4, 175.8. LC/MS (M: 311.2): 312.1 (M + H⁺). Amino acid analysis: Phe, 2.00 (68% peptide).

[0050] Peptide 13 (D-Phe-D-Phe-NH₂). The crude peptide was purified to yield 7.8 mg (38%). HPLC purity: C18 column > 99.9%, Fluophase > 99.9%. ¹H NMR (MeOD) δ 2.97 (dd, J = 8.5, 13.8 Hz, IH) 3.00 (dd, J = 8.5, 14.3 Hz, IH), 3.14 (dd, J = 6.3, 13.8 Hz, IH), 3.26 (dd, J = 5.4, 14.3 Hz, IH) 4.08 (dd, J = 5.54, 8.5 Hz, IH), 4.65 (dd, J = 6.3, 8.5 Hz, IH) 7.19-7.40 (m, 10H). ¹³C NMR (MeOD) δ 38.6, 39.1, 55.5, 56.0, 127.9, 128.9, 129.5, 130.2, 130.3, 130.5, 138.2, 169.5, 175.2. LC/MS (M: 311.2): 312.1 (M + H⁺). Amino acid analysis: Phe, 2.00 (68% peptide).

[0051] Peptide 14 (D-Phe-L-Phe-NH₂). The crude peptide was purified to yield 13.6 mg (42%). HPLC purity: C18 column 99.3%, Fluophase 99.4%. ¹U NMR (MeOD) δ 2.75 (dd, J = 8.0, 14.1 Hz, IH), 2.81 (dd, J = 9.8, 13.9 Hz, IH), 2.93 (dd, J= 5.9, 14.1 Hz, IH), 3.15 (dd, J = 5.4, 13.9 Hz, IH), 4.08 (dd, J = 5.9, 8.0 Hz, IH), 4.67 (dd, J = 5.4, 9.8 Hz, IH), 7.01-7.05 (m, 2H), 7.18-7.32 (m, 8H). LC/MS (M: 311.2): 312.1 (M + H⁺). Amino acid analysis: Phe, 2.00 (64% peptide).

[0052] Peptide 15 (H-Phe-Phg-NH₂). During the synthesis of Peptide 15, racemisation of the phenylglycine moiety occurred. The crude peptide was purified to only give one of the two diastereomers in a yield of 9.3 mg (a total yield of 31%). HPLC purity: Cl 8 column > 99.9%, Fluophase 99.8%. ¹H NMR (MeOD) δ 3.07 (dd, J = 8.5, 14.2 Hz, IH), 3.32 (dd, J= 5.8, 14.2 Hz, IH), 4.20 (dd, J= 5.8, 8.5 Hz, IH), 5.46 (s, IH), 7.29-7.41 (m, 8H), 7.44-7.48 (m, 2H). ¹³C NMR (MeOD) δ 38.7, 55.6, 58.5, 128.8, 128.9, 129.5, 129.8, 130.2, 130.6, 135.6, 138.6, 169.1, 174.1. HRMS (M + H⁺): 298.1559, Ci₇H₂₀N₃O₂ requires 298.1556. [0053] Peptide 16 (H-LeU-PhC-NH₂). The crude peptide was purified to yield 16.6 mg (60%). HPLC purity: C18 column > 99.9%, Fluophase 99.7%. ¹H NMR (MeOD) δ 0.95-0.99 (m, 6H), 1.59-1.76 (m, 3H), 2.99 (dd, J = 8.8, 13.8 Hz, IH), 3.14 (dd, J= 6.3, 13.8 Hz, IH), 3.84 (m, IH), 4.65 (dd, J = 6.3, 8.8 Hz, IH), 7.19-7.35 (m,5H). ¹³C NMR (MeOD) δ 20.7, 22.0, 24.1, 37.7, 40.5, 51.6, 54.9, 126.7, 128.3, 129.1, 137.1, 169.4, 174.3. HRMS (M + H⁺): 278.1871, Ci₅H₂₄N₃O₂ requires 278.1869. [0054] Peptide 17 (H-Omt-2MP-NH₂). The crude peptide was purified to yield 15.5 mg (44%). HPLC purity: C18 column > 99.9%, Fluophase > 99.9%. ¹U NMR (MeOD) δ 2.37 (s, 3H), 2.97 (dd, J = 8.1, 14.4 Hz, IH), 3.00 (dd, J = 7.7, 14.0 Hz, IH) 3.18 (dd, J = 5.6, 14.4 Hz, IH) 3.12 (dd, J = 7.5, 14.0 Hz, IH), 3.77 (s, 3H), 4.04 (dd, J = 5.6, 8.1 Hz, IH), 4.65 (t, J= 7.6 Hz, IH), 6.87-6.92 (m, 2H), 7.06-7.22 (m, 6H). ¹³C NMR (MeOD) δ 19.5, 36.5, 37.7, 54.7, 55.6, 55.7, 115.5, 127.0, 127.1, 128.0, 130.8, 131.4, 131.6, 136.1, 137.9, 160.8, 169.4, 175.3. HRMS (M + H⁺): 356.1972, C₂₀H₂₆N₃O₃ requires 356.1974.

[0055] Peptide 18 (H-Phe-Thi-NH₂). The crude peptide was purified to yield 22.7 mg (72%). HPLC purity: C18 column > 99.9%, Fluophase > 99.9%. ¹H NMR (MeOD) δ 3.03 (dd, J = 8.3, 14.3, IH), 3.21(dd, J = 8.1, 15.0 Hz, IH), 3.26 (dd, J = 5.6, 14.3 Hz, IH) 3.36 (dd, J = 6.0, 15.0 Hz, IH), 4.13 (dd, J = 5.6, 8.3 Hz, IH), 4.64 (d, J = 6.0, 8.1 Hz, IH), 6.90-6.96 (m, 2H), 7.22-7.25 (m, IH), 7.26-7.39 (m, 5H). ¹³C NMR (MeOD) δ 33.1, 38.5, 55.5, 56.1, 125.5, 127.6, 127.9, 128.9, 130.2, 130.5, 135.4, 139.8, 169.5, 174.7. HRMS (M + H⁺): 318.1278, Ci₆H₂₀N₃O₂S requires 318.1276.

[0056] Peptides 19(1) (D-Phg-Phe-NH₂) and 19(11) (L-Phg-Phe-NH₂). During the synthesis of Peptide 19, some racemisation (L to D) of the phenylglycine moiety occurred. The crude peptide was purified to yield the two diastereomers 19(1) (2.4 mg) and 19(11) (3.9 mg), in a total yield of 21%. Peptide 19(1). HPLC purity: C18 column > 99.9%, Fluophase > 99.9%. ¹U NMR (MeOD) δ 2.95 (dd, J= 8.5, 13.8 Hz, IH), 3.12 (dd, J = 6.2, 13.8 Hz, IH), 4.65 (dd, J = 6.2, 8.5 Hz, IH), 4.93 (s, IH), 7.18-7.30 (m, 5H), 7.44- 7.51 (m, 5H). ¹³C NMR (MeOD) δ 38.8, 56.2, 57.7, 127.9, 129.4, 129.5, 130.3, 130.5, 131.1, 134.2, 138.3, 168.6, 175.0. HRMS (M + H⁺): 298.1552, Ci₇H₂₀N₃O₂ requires 298.1556. Peptide 19(11). 2.76 (dd, J = 9.9, 14.1 Hz, IH), 3.10 (dd, J= 4.7, 14.1 Hz, IH), 4.72 (dd, J = 4.6, 9.9 Hz, IH), 4.94 (s, IH), 6.94 (d, J= 7.2 Hz, 2H), 7.02-7.13 (m, J = 7.2 Hz, 3H), 7.25 (d, J = 7.6 Hz, 2H), 7.37 (t, J= 7.6 Hz, 2H), 7.44 (t, J = 7.6

Hz, IH). ¹³C NMR (MeOD) δ 38.8, 55.6, 57.9, 127.7, 129.1, 129.4, 130.0, 130.5, 130.9, 134.1,

137.9, 168.7, 175.7. HRMS (M + H⁺): 298.1549, Ci₇H₂₀N₃O₂ requires 298.1556.

[0057] Peptide 20 (H-Cha-3Fp-NH₂). The crude peptide was purified to yield 11 mg (33%).

HPLC purity: C18 column > 99.9%, Fluophase > 99.9%. ¹H NMR (MeOD) δ 0.9-1.02 (m, 2H),

1.12-1.42 (m, 4H), 1.59-1.81 (m, 7H), 2.95 (dd, J = 8.7, 13.8 Hz, IH), 3.15 (dd, J = 6.3, 13.9 Hz,

IH), 3.88 (dd, J = 5.7, 8.9 Hz, IH), 4.65 (dd, J = 6.3, 8.7 Hz, IH), 6.95 (dddd, J = 1.0, 2.7, 8.2,

9.9 Hz, IH), 7.05 (dddd, J = 0.5, 1.6, 2.7, 10.0 Hz, IH), 7.10 (ddd, J = 1.0, 1.6, 7.6 Hz, IH), 7.30

(dddd, J = 0.5, 6.0, 7.6, 8.2 Hz, IH). ¹³C NMR (MeOD) δ 26.9, 27.1, 27.3, 33.3, 34.4, 34.6, 38.5,

40.4, 52.2, 55.8, 114.6 (d, J = 21.2 Hz, 1C), 117.0 (d, J= 21.6 Hz, 1C), 126.2 (d, J= 2.8 Hz,

1C), 131.2 (d, J = 8.5 Hz, 1C), 141.1 (d, J = 7.4 Hz, 1C), 164.3 (d, J= 244 Hz, 1C), 170.8,

175.1. HRMS (M + H⁺): 336.2084, Ci₈H₂₇FN₃O₂ requires 336.2087.

Example 4

[0058] In this example, Peptides 21-33, 50 and 54 were prepared using methods as described above.

[0059] Peptide 21 (N-Me-Phe-Phe-NH₂). The crude peptide was purified to yield 14.75 mg

(45,4%). HPLC purity: C18-kolonn > 99%, Fluophase > 98%. ¹H-NMR (MeOD) δ 2.11 (s 3H),

2.79 (dd, J= 10.4, 13.9 Hz, IH), 3.05-3.15 (m, 2H), 3.21 (dd, J = 5.04, 13.9 Hz, IH), 3.86-3.93

(m, IH), 4.77 (dd, J = 5.04, 10.4 Hz, IH), 7.18-7.36 (m, 10H). ¹³C-NMR (MeOD) δ 32.1, 37.9,

39.3, 55.5, 64.1, 127.9, 129.0, 129.5, 130.2, 130.5, 130.51, 135.0, 138.4, 167.7, 174.8. LC/MS

(M: 325.18): 326.2 (M + H⁺).

[0060] Peptide 22 (α-Me-Phe-Phe-NH₂). The crude peptide was purified to yield 3.63 mg

(11.2%). HPLC purity: C18-column > 98%, Fluophase > 99%. ¹H-NMR (MeOD) δ 1.38 (s, 3H), 2.98 (dd, J = 10.1, 13.9 Hz, IH), 3.30 (d, J= 14.4 Hz, 1 H), 3.23 (dd, J = 5.2, 13.9 Hz, IH), 3.31 (d, J = 14.4 Hz, IH), 4.78 (dd, J = 5.2, 10.1 Hz, IH), 7.2-7.36 (m, 10H). ¹³C-NMR (MeOD) δ 22.8, 39.2, 43.7, 55.7, 61.8, 127..9, 129.1, 129.5, 130.0, 130.4, 131.4, 134.2, 138.4, 171.5, 175.5. LC/MS (M: 325.18): 326.2 (M + H⁺).

[0061] Peptide 23 (Phe-N-Me-Phe-NH₂). The crude peptide was purified to yield 9.80 mg (30.1%). ¹H-NMR (MeOD) δ 2.82 (s, 3H), 2.95-3.15 (m, 4H), 4.51 (dd, J = 6.5, 7.6 Hz, IH), 5.21 (dd, J= 7.4, 8.6 Hz, IH), 7.13-7.39 (m, 10H).LC/MS (M: 325.18): 326.2 (M + H⁺). [0062] Peptide 24 (Phe-α-Me-Phe-NH₂). The crude peptide was purified to yield 17.3 mg (53.2%). HPLC purity: C18-column > 99%, Fluophase > 99%. ¹H-NMR (MeOD) δ 1.46 (s, 3H), 2.97 (dd, J= 9.5, 14.2 Hz, IH), 3.14 (dd, J= 5.6, 14.2 Hz, IH), 3.25 (d, J = 13.4 Hz, IH), 3.37 (d, J = 13.4 Hz, IH), 4.10 (dd, J = 5.6, 9.5 Hz, IH), 7.1-7.4 (m, 10H). ¹³C-NMR (MeOD) δ 23.9, 38.6, 41.5, 55.9, 61.8, 127.99, 128.9, 129.2, 130.2, 130.4, 131.7, 135.9, 137.5, 168.9, 178.2. LC/MS (M: 325.18): 326.2 (M + H⁺).

[0063] Peptide 25 (Phe-Phe-N-Me-NH). The crude peptide was purified to yield 25.7 mg (79.1%). HPLC purity: C18-column > 99%, Fluophase > 99%. ¹H-NMR (MeOD) δ 2.65 (bs, 3H), 2.95 (dd, J = 7.73, 13.6 Hz, IH), 3.03 (dd, J = 8.17, 14.21 Hz, IH), 3.07 (dd, J = 7.32, 13.66 Hz, IH), 3.23 (dd, J = 5.79, 14.2 Hz, IH), 4.09 (dd, J = 5.81, 8.10 Hz, IH), 4.55 (t, J = 7.50, IH), 7.16-7.38 (m, 10H). ¹³C-NMR (MeOD) δ 26.2, 38.5, 39.2, 55.5, 56.6, 127.9, 128.8, 129.5, 130.1, 130.2, 130.5, 135.5, 138.1, 169.4, 173.1. LC/MS (M: 325.18): 326.2 (M + H⁺). [0064] Peptide 26a (See Table 1). The crude peptide was purified to yield 5.4 mg (32%). ¹H- NMR (MeOD) δ 2.35-2.50 (m, 2H), 2.77 (dd, J = 7.5, 13.7 Hz, IH), 3.00 (dd, J = 6.5, 13.7 Hz IH), 3.38 (ddd, J = 7.4, 9.7, 11.4 Hz, IH), 3.73 (ddd, J = 3.6, 7.8, 11.4 Hz IH), 3.88 (ddd, J = 7.4, 9.3 Hz IH) 4.33 (dd, J = 6.5, 7.5 Hz, IH), 4.48 (d, J = 9.3 Hz, IH) 7.15-7.40 (m, 10H).

LC/MS (M: 337.18): 338.2 (M + H⁺).

[0065] Peptide 26b (See Table 1). The crude peptide was purified to yield 9.3 mg (55%). ¹H-

NMR (MeOD) δ 2.20 (dd, J= 5.9, 13.6 Hz, IH), 2.41-2.49 (m, 2H), 3.50 (dd, J = 8.6 13.6 Hz

IH), 3.42 (ddd, J = 8.7, 11.2 Hz, IH), 3.75 (ddd, J = 5.4, 11.2 Hz IH), 3.89 (ddd, J= 8.8 Hz IH)

4.11 (dd, J= 5.9, 8.6 Hz, IH), 4.55 (d, J = 9.3 Hz, IH) 6.94-6.99 (m, 2H), 7.16-7.36 (m, 8H).

LC/MS (M: 337.18): 338.2 (M + H⁺).

[0066] Peptide 27 (See Table 1). The crude peptide was purified to yield 4.6 mg (27%). ¹H-

NMR (MeOD) δ 1.92 (m, J= 6.2, 13.2 Hz, IH), 2.58 (m, J= 8.5, 10.7, 11.8, 13.2 Hz, IH), 2.73

(m, J= 6.2, 9.6, 10.9 Hz IH), 3.15-3.18 (d, J = 7.6 Hz, 2H), 3.31 (m, IH), 3.84 (m, J = 8.5, 9.7

Hz, IH) 4.42 (dd, J= 7.6 Hz, IH), 4.53 (d, J= 8.6 Hz, IH) 7.21-7.41 (m, 10H). ¹³C-NMR

(MeOD) 29.56, 38.30, 47.75, 47.95, 54.06, 65.61, 128.59, 129.17, 129.43, 129.44, 130.13,

130.71, 135.51, 137.49, 168.40, 174.46. LC/MS (M: 337.18): 338.2 (M + H⁺).

[0067] Peptide 28a (See Table 1). The crude peptide was purified to yield 4.7 mg (29%). ¹H-

NMR (MeOD) δ 1.31 (dd, J= 1.6, 7.1, Hz, 3H), 2.95 (dd, J = 8.4, 14.3 Hz, IH), 3.13-3.21 (m,

2H), 3.93 (ddd, J = 1.6, 5.0, 8.4 Hz, IH), 4.65 (dd, J= 1.6, 9.7 Hz, IH) 7.16-7.40 (m, 10H).

LC/MS (M: 325.4): 326.2 (M + H⁺).

[0068] Peptide 28b (See Table 1). The crude peptide was purified to yield 4.5 mg (28%). ¹H-

NMR (MeOD) δ 1.29 (dd, J= 0.9, 7.0, Hz, 3H), 2.42 (dd, J = 8.9, 14.5 Hz, IH), 2.57 (dd, J =

5.0, 14.5 Hz, IH), 3.13 (dddd, J= 6.7, 9.9 Hz, IH), 3.95 (dd, J = 5.0, 8.9 Hz, IH) 4.66 (dd, J =

0.9, 9.9 Hz, IH), 6.96-7.01 (m, 2H), 7.17-7.33 (m, 18H). LC/MS (M: 325.4): 326.2 (M + H⁺).

[0069] Peptide 29 (See Table 1). The crude peptide was purified to yield 21.8 mg (67%). ¹H-

NMR (MeOD) δ 1.91-2.15 (m, 2H), 2.60-2.77 (m, 2H), 3.06 (dd, J = 8.5, 14.2 Hz, IH), 3.31 (dd, J = 5.9, 14.3 Hz, IH), 3.19 (dd, J = 5.9, 8.5 Hz, IH) 4.41 (dd, J= 5.4, 8.5 Hz, IH), 7.14-7.40 (m,

10H). ¹³C-NMR (MeOD) 33.04, 35.40, 38.59, 54.42, 55.59, 127.17, 128.92, 129.41, 129.54,

130.19, 130.55, 135.58, 142.35, 169.63, 175.79. LC/MS (M: 325.2): 326.2 (M + H⁺).

[0070] Peptide 30 (See Table 1). The crude peptide was purified to yield 12.9 mg (40%). ¹H-

NMR (MeOD) δ 2.98 (s, 3H), 3.00 (dd, J = 7.6, 14.0 Hz, IH), 3.02 (dd, J = 8.4, 14.3 Hz, IH),

3.12 (dd, J= 7.6, 14.0 Hz, IH), 3.25 (dd, J= 5.7, 14.3 Hz, IH), 4.09 (dd, J = 5.7, 8.4 Hz, IH)

4.66 (t, J= 7.6 Hz, IH), 7.07-7.21 (m, 4H), 7.26-7.41 (m, 5H). ¹³C-NMR (MeOD) 19.55, 36.53,

38.57, 54.73, 55.52, 127.01, 128.04, 128.90, 130.16, 130.53, 130.78, 131.42, 135.47, 136.13,

137.89, 169.32, 175.29. LC/MS (M: 325.2): 326.2 (M + H⁺).

[0071] Peptide 31 (See Table 1). The crude peptide was purified to yield 12.4 mg (38%). ¹H-

NMR (MeOD) δ 2.98 (dd, J= 8.4, 13.9 Hz, IH), 3.01 (dd, J = 8.5, 14.3 Hz, IH), 3.15 (dd, J =

6.3, 13.9 Hz, IH), 3.26 (dd, J = 5.4, 14.3 Hz, IH) 4.08 (dd, J= 5.4, 8.5 Hz, IH) 4.66 (dd, J = 6.3,

8.4 Hz, IH), 6.92-7.00 (m, IH), 7.01-7.13 (m, 2H), 7.26-7.40 (m, 6H). ¹³C-NMR (MeOD) 38.57,

38.73, 55.47, 55.73, 114.64 (7= 21.4), 117.01 (/ = 21.7), 126.24 (7 = 2.77), 128.92, 130.17,

130.53, 131.21 (/= 8.29), 135.44, 141.00 (7 = 7.56), 164.27 (7= 244.4), 169.51, 174.89 .

LC/MS (M: 329.2): 330.2 (M + H⁺).

[0072] Peptide 32 (GIn-PhC-NH₂). An aliquot of the crude cleavage product was purified to yield 13.7 mg (29%). HPLC purity: C18 column 95%. LC/MS (M: 292.2): 293.2 (M + H⁺).

Amino acid analysis: GIn, 1.00; Phe, 1.00 (74% peptide).

[0073] Peptide 33 (See Table 1). ¹H NMR (Acetonitril, CD₃CN, 400 MHz) δ 2.83 (IH, dd,

CH₂-C₆H_5, J = 9.36 Hz, J= 14.04 Hz ), 3.14 (IH, dd, CH₂-C₆H_5, J = 4.88 Hz, J = 14.04 Hz) 4.31

(IH, ddd , NH-CH J = 4.88 Hz /=8.39 Hz, J= 9.36 Hz ), 4.97 (IH, d (AB spinsystem), O-CH₂-

C₆H₅J = 12.67 Hz ), 5.03 (IH, d (AB spinsystem), Q-CH₂-C₆H₅J= 12.68 Hz) 5.84 (IH, br s, 1 x CONH₂) 5.89 (IH, d, CONH, J = 8.39 Hz) 6.42 (IH, br s, 1 x CONH₂) 7.29 (1OH, unresolved m, 2 x C₆H₅), ¹³C NMR (Acetonitril, CD₃CN, 400 MHz) δ 38.65, 56.98, 67.01, 127.60, 128.55, 128.85, 129.33, 129.43, 130.26, 138.15, 138.65, 156.94, 174.30.

[0074] Peptide 50 (Arg-Pro-Lys-Pro-Gln-Gln-Phe-NH₂). An aliquot of the crude cleavage product was purified to yield 15.9 mg (46%). HPLC purity: C18 column 98.5%, Phenyl column > 99.9%. LC/MS (M: 479.3): 480.3 (M + H⁺). Amino acid analysis: Ala, 1.00; Pro, 1.02; Phe, 1.99 (71% peptide).

[0075] Peptide 54 (GIn-GIn-PhC-NH₂). An aliquot of the crude cleavage product was purified to yield 7.7 mg (37%). HPLC purity: C18 column >94%, Phenyl column > 94%. LC/MS (M: 420.21): 421.1 (M + H⁺). Amino acid analysis: GIu, 2.02; Phe, 0.98 (70% peptide). Example 5

[0076] This example describes evaluation and results of radioligand binding assays conducted with Peptides 1-20 as described above and Peptides 21-55 as described in Table 1 using spinal cord membrane from Sprangue-Dawley rats and the analogue [ H]SPi_₇ as tracer. The precursor peptide for tritium-labeling [2,4-DehydroPro]SPi_₇ was prepared by standard solid-phase peptide synthesis techniques using FmocΛ-butyl protection and purified as described above. Tritium labeling of the precursor was performed by Amersham Biosciences (Cardiff, UK) and resulted in 370 MBq (10 mCi) of [³H]-SPi_₇ with a specific activity of 3.11 TBq/mmol (84 Ci/mmol). The procedure as described by Fransson et al (2008) and Botros (2006), both cited herein, was employed. Competition experiments were performed at six different concentrations and corrected for non-specific binding. The results are set forth in Table 1.

Table 1: Binding Affinity (Kj)

[0077] Compounds having a K₁ of less than 1000 nM for the SPi_₇ binding site are preferred. Example 6

[0078] This example describes a method of alleviating pain according to the invention. SP₁-₇ and a shorter fragment thereof, the dipeptide Phe-Phe-NH₂ (Peptide 8), are shown to induce a dose-dependent antihyperalgesic effects in streptozotocin (STZ)-induced diabetic mice, i.e. the peptides may relieve a condition considered as neuropathic pain.

[0079] Male ICR 4-week-old mice were used. Animals were rendered diabetic by an injection of streptozotocin (200 mg/kg, i.v.) prepared in 0.1 N citrate buffer at pH 4.5. Age-matched non- diabetic mice were injected with vehicle alone. The experiments were carried out 2 weeks after injection of streptozotocin or vehicle. Mice with serum glucose levels above 400 mg/dl were considered diabetic. The anti-nociceptive effect was determined by the tail-flick test (Hylden et al, Eur. J. Pharmacol, 67:313-316 (1980)). The peptides SP_1-7-NH₂, SPi_₇, and Phe-Phe-NH₂ were injected i.t. 30 min before the testing. Tail-flick latency was measured 30, 60 and 90 min after the injection (Ohsawa et al, Eur. J. Pharmacol, 372:221-228 (1999)). [0080] Figs. IA and IB show the anti-nociceptive effects of i.t. injected SP1-7-NH2, SP_1-7 and Phe-Phe-NH₂ in non-diabetic and diabetic mice, respectively. The anti-nociceptive effect was evaluated by the AUC calculated from the time-response curve of tail-flick latency. Each point represents the mean with S.E.M. (n=8-10). *P<0.05 vs. respective saline-treated mice (Bonferroni-Dunn test). Figs. 2A and 2B show the effect of various opioid receptor antagonists on the anti-nociceptive effects of i.t. injected SP_1-7-NH₂ in non-diabetic and diabetic mice, respectively. The anti-nociceptive effect was evaluated by the AUC calculated from the time- response curve of tail-flick latency. Each point represents the mean with S.E.M. (n=8-10). *P<0.05 vs. respective saline-treated mice (Bonferroni-Dunn test). FNA represents β- funaltrexamine and BNI represents nor-binaltorphimine.

[0081] As shown in Figs. IA and IB, all peptides showed significant anti-nociception in both non-diabetic and diabetic mice. The anti-nociceptive effect induced by i.t. injection of each peptide was significantly greater in diabetic mice than in non-diabetic mice (more than 2 times). Additionally, the anti-nociceptive effect induced by i.t. injection of SPi_₇ was significantly less than the anti-nociceptive effect induced by other peptides in diabetic mice. However, there were no such differences on anti-nociception observed in non-diabetic mice. The relative strength of the anti-nociceptive effect of each peptide in diabetic mice was SP1-7-NH2 > Phe-Phe-NF^ > SP_{1- 7}. The anti-nociceptive effect of each peptide was not mediated by μ-opioid receptor or k-opioid receptor. Example 7

[0082] This example describes a method of alleviating symptoms associated with opioid use withdrawal. It has previously been shown that intracerebroventricular (i.c.v.) administration of SPi_₇ attenuates the expression of morphine withdrawal in the male rat. The present example used a synthetic analogue of this peptide, i.e. the SPi_₇ -NH_2, having higher binding affinity to spinal cord receptor membranes than the native heptapeptide, in a similar experimental set-up. [0083] Sprague-Daley male rats were rendered tolerant to morphine by daily injections of the opiate during 8 days. After peptide administration (i.c.v.) and a subsequent naloxone challenge, a variety of physical syndromes of withdrawal were recorded. The SP ₁-₇ -NH₂ potently and dose- dependently reduced several signs of morphine withdrawal, showing that SP₁-₇ -NH₂ mimics the effect of the native SP fragment but with higher potency.

[0084] The procedure for assessing opiate withdrawal was described previously (Zhou et al., Peptides, 24:147-153 (2003)). Briefly, animals were anaesthetized with chloral hydrate- pentobarbital sodium and surgically implanted with a guide cannulae in the right lateral ventricle (coordinates: A - 0.8 mm; L -1.4 mm, V -4 mm). After a period of recovery, morphine was injected (s.c.) twice daily for 8 days. The morphine dose was progressively increased from 2.5 mg to 5 mg and then to 10 mg/kg over a period of 3 days. Thereafter, the dose of 10 mg/kg twice daily was maintained until the rats became tolerant to the drug. The development of tolerance was followed by assessment of the tail flick latency every second day. On day 9, rats were challenged with a single dose of naloxone (2 mg/kg, s.c.) to precipitate somatic signs of withdrawal. Prior to naloxone (30 minutes), rats received i.c.v. administration of SP₁-₇ amide, SP_1-7 or artificial CSF (as a control). Immediately after naloxone injection, withdrawal signs (diarrhea, digging, escape jumping, face washing, grooming, rearing, teeth chattering, wet-dog shakes, writhing, yawn) were recorded during a period of 30 min. Statistical evaluation of data was performed using Student's t-test and Factorial ANOVA analysis computer program. [0085] Figs. 3 A-3C show the effect of i.c.v. injected SPi_₇ -NH₂ on teeth chattering, ptosis and writhing, respectively, on the naloxone-precipitated morphine withdrawal in rats. ** p < 0.01 versus artificial CSF-treated group, *** p < 0.001 versus artificial CSF-treated group. (Artificial CSF, SPi_₇ amide 18.7 and 55.5 nmol: n = 8; SPi_₇ amide 165.5 nmol: n = 7). These results indicate that the amidated analogue of SPi_₇ mimicked the previous shown attenuating effect of the native heptapeptide on naloxone-precipitated morphine withdrawal. A clear dose-dependent effect was seen on signs as teeth chattering, ptosis and writhing. Some signs, for example, escape jumping, digging, diarrhea and wet dog shake were not significantly affected in this study, although a clear tendency to attenuate, e.g. wet dog shake, was observed. However, the total withdrawal scores recorded following peptide infusion indicated a significant attenuating effect of the SPi-₇ -NH₂. The effect of the SP₁-₇ -NH₂ was significantly more potent compared to that obtained by native SPi_₇.

[0086] The specific examples and embodiments described herein are exemplary only in nature and are not intended to be limiting of the invention defined by the claims. Further embodiments and examples, and advantages thereof, will be apparent to one of ordinary skill in the art in view of this specification and are within the scope of the claimed invention.

Claims

WHAT IS CLAIMED IS:

1. A method for alleviating pain in a mammal, comprising administering to the mammal a therapeutically effective amount of a compound of Formula I, Formula II, or Formula III, or a pharmaceutically acceptable salt thereof:

Formula I:

wherein:

R¹ and R² are individually H or alkyl;

R³ and R⁴ are individually H or alkyl, or together form a ring having 3-5 carbon atoms;

R⁵ is H or alkyl; n is 0-3;

A is a 5-membered aromatic ring or a 6-membered aromatic ring, either ring optionally containing from 1 to 3 heteroatoms selected from the group consisting of N, S and O, optionally substituted with 1 to 3 substituents of halogen, alkyl, alkoxy, CF₃, nitro, or nitrile;

R⁶ and R⁷ are individually H, alkyl, aromatic, heteroaromatic, of the formula

wherein R⁹ and R are individually H or alkyl, or R forms a ring having 4 or 5 carbon atoms with R , or R forms a ring having 4 or 5 carbon atoms with R , and X is a 5-membered aromatic ring or a 6- membered aromatic ring, either ring optionally containing from 1 to 3 heteroatoms selected from the group consisting of N, S and O, optionally substituted with 1 to 3 substituents of halogen, alkyl, alkoxy, CF₃, nitro, or nitrile, or of the formula

wherein R and R are individually H or alkyl, m is 1 to 4, and Y is CONH₂, CONH-alkyl, aromatic or heteroaromatic, or R or R forms a ring having 4 or 5 carbon atoms with R ; and

R⁸ is H, alkyl, acyl or aromatic, or R⁸ forms a ring having 4 or 5 carbon atoms with R⁶, R⁷ or R⁹; Formula II:

wherein:

R¹ and R² are individually H or alkyl;

R³ and R⁴ are individually H or alkyl, or together form a ring having 3-5 carbon atoms;

R⁵ is H or alkyl; n is 0-3;

A is a 5-membered aromatic ring or a 6-membered aromatic ring, either ring optionally containing from 1 to 3 heteroatoms selected from the group consisting of N, S and O, optionally substituted with 1 to 3 substituents of halogen, alkyl, alkoxy, CF₃, nitro, or nitrile; and

R¹⁹ is alkyl, O-alkyl, O-benzyl, O-9-fluorenylmethyl, N-alkyl, N-benzyl, or N-9-fluorenylmethyl;

Formula III:

wherein:

Z is NH₂, OH or NH-alkyl; R² is H or alkyl;

R³ and R⁴ are individually H or alkyl, or together form a ring having 3-5 carbon atoms; R⁵ is H or alkyl; n is 0-3;

A is a 5-membered aromatic ring or a 6-membered aromatic ring, either ring optionally containing from 1 to 3 heteroatoms selected from the group consisting of N, S and O, optionally substituted with 1 to 3 substituents of halogen, alkyl, alkoxy, CF₃, nitro, or nitrile; and

R²⁰ is of the formula (i)

wherein p is 1-6,

'2 wherein p is 1-5, , wherein p is 1 -4,

wherein p is 1-5, or

, wherein R' and R" are amino acid side chains, and R ,2^Z1¹ is H or CH₃C(O)-.

2. A method for alleviating a symptom associated with cessation or withdrawal from an addiction to a habit-forming drug in a mammal, comprising administering to the mammal a therapeutically effective amount of a compound of Formula I, Formula II, or Formula III, or a pharmaceutically acceptable salt thereof:

Formula I:

wherein:

R¹ and R² are individually H or alkyl;

R³ and R⁴ are individually H or alkyl, or together form a ring having 3-5 carbon atoms;

R⁵ is H or alkyl; n is 0-3;

A is a 5-membered aromatic ring or a 6-membered aromatic ring, either ring optionally containing from 1 to 3 heteroatoms selected from the group consisting of N, S and O, optionally substituted with 1 to 3 substituents of halogen, alkyl, alkoxy, CF₃, nitro, or nitrile;

10

R

\— X

R and R are individually H, alkyl, aromatic, heteroaromatic, of the formula R⁹>" wherein R and R are individually H or alkyl, or R forms a ring having 4 or 5 carbon atoms with R , or R forms a ring having 4 or 5 carbon atoms with R , and X is a 5-membered aromatic ring or a 6- membered aromatic ring, either ring optionally containing from 1 to 3 heteroatoms selected from the group consisting of N, S and O, optionally substituted with 1 to 3 substituents of halogen,

1 1

R

m

12 alkyl, alkoxy, CF₃, nitro, or nitrile, or of the formula R wherein R and R are individually H or alkyl, m is 1 to 4, and Y is CONH₂, CONH-alkyl, aromatic or heteroaromatic, or R⁶ or R⁷ forms a ring having 4 or 5 carbon atoms with R⁸; and R⁸ is H, alkyl, acyl or aromatic, or R⁸ forms a ring having 4 or 5 carbon atoms with R⁶, R⁷ or R⁹; Formula II:

wherein:

R¹ and R² are individually H or alkyl;

R³ and R⁴ are individually H or alkyl, or together form a ring having 3-5 carbon atoms;

R⁵ is H or alkyl; n is 0-3;

A is a 5-membered aromatic ring or a 6-membered aromatic ring, either ring optionally containing from 1 to 3 heteroatoms selected from the group consisting of N, S and O, optionally substituted with 1 to 3 substituents of halogen, alkyl, alkoxy, CF₃, nitro, or nitrile; and

R¹⁹ is alkyl, O-alkyl, O-benzyl, O-9-fluorenylmethyl, N-alkyl, N-benzyl, or N-9-fluorenylmethyl;

Formula III:

wherein

Z is NH₂, OH or NH-alkyl;

R² is H or alkyl;

R and R are individually H or alkyl, or together form a ring having 3-5 carbon atoms; R⁵ is H or alkyl; n is 0-3;

A is a 5-membered aromatic ring or a 6-membered aromatic ring, either ring optionally containing from 1 to 3 heteroatoms selected from the group consisting of N, S and O, optionally substituted with 1 to 3 substituents of halogen, alkyl, alkoxy, CF₃, nitro, or nitrile; and

wherein p is 1-6,

'2 wherein p is 1-5,

wherein p is 1 -4,

wherein p is 1-5, or

, wherein R' and R" are amino acid side chains, and R²¹ is H or CH₃C(O)-.

3. A method for alleviating inflammation in a mammal, comprising administering to the mammal a therapeutically effective amount of a compound of Formula I, Formula II, or Formula III, or a pharmaceutically acceptable salt thereof:

Formula I:

wherein:

R¹ and R² are individually H or alkyl;

R³ and R⁴ are individually H or alkyl, or together form a ring having 3-5 carbon atoms;

R⁵ is H or alkyl; n is 0-3;

A is a 5-membered aromatic ring or a 6-membered aromatic ring, either ring optionally containing from 1 to 3 heteroatoms selected from the group consisting of N, S and O, optionally substituted with 1 to 3 substituents of halogen, alkyl, alkoxy, CF₃, nitro, or nitrile; R 6 and R 7 are individually H, alkyl, aromatic, heteroaromatic, of the formula

wherein R 9

and R are individually H or alkyl, or R forms a ring having 4 or 5 carbon atoms with R , or R forms a ring having 4 or 5 carbon atoms with R³, and X is a 5-membered aromatic ring or a 6- membered aromatic ring, either ring optionally containing from 1 to 3 heteroatoms selected from the group consisting of N, S and O, optionally substituted with 1 to 3 substituents of halogen,

are individually H or alkyl, m is 1 to 4, and Y is CONH₂, CONH-alkyl, aromatic or heteroaromatic, or R⁶ or R⁷ forms a ring having 4 or 5 carbon atoms with R⁸; and

R⁸ is H, alkyl, acyl or aromatic, or R⁸ forms a ring having 4 or 5 carbon atoms with R⁶, R⁷ or R⁹;

Formula II:

wherein:

R and R are individually H or alkyl;

R and R are individually H or alkyl, or together form a ring having 3-5 carbon atoms;

R⁵ is H or alkyl; n is 0-3; A is a 5-membered aromatic ring or a 6-membered aromatic ring, either ring optionally containing from 1 to 3 heteroatoms selected from the group consisting of N, S and O, optionally substituted with 1 to 3 substituents of halogen, alkyl, alkoxy, CF₃, nitro, or nitrile; and R¹⁹ is alkyl, O-alkyl, O-benzyl, O-9-fluorenylmethyl, N-alkyl, N-benzyl, or N-9-fluorenylmethyl; Formula III:

wherein:

Z is NH₂, OH or NH-alkyl; R² is H or alkyl;

R and R are individually H or alkyl, or together form a ring having 3-5 carbon atoms; R⁵ is H or alkyl; n is 0-3;

A is a 5-membered aromatic ring or a 6-membered aromatic ring, either ring optionally containing from 1 to 3 heteroatoms selected from the group consisting of N, S and O, optionally substituted with 1 to 3 substituents of halogen, alkyl, alkoxy, CF₃, nitro, or nitrile; and

R ,2 ^z0^υ is of the formula (i)

wherein p is 1-6,

wherein p is 1-5,

, wherein p is 1-4,

wherein p is 1-5, or

, wherein R' and R" are amino acid side chains, and R²¹ is H or CH₃C(O)-.

4. A method for alleviating a symptom associated with a neuropsychiatric disorder, wherein the neuropsychiatric disorder is a cognitive deficiency or anxiety, in a mammal, comprising administering to the mammal a therapeutically effective amount of a compound of Formula I, Formula II, or Formula III, or a pharmaceutically acceptable salt thereof:

Formula I:

wherein:

R¹ and R² are individually H or alkyl;

R³ and R⁴ are individually H or alkyl, or together form a ring having 3-5 carbon atoms;

R⁵ is H or alkyl; n is 0-3; A is a 5-membered aromatic ring or a 6-membered aromatic ring, either ring optionally containing from 1 to 3 heteroatoms selected from the group consisting of N, S and O, optionally substituted with 1 to 3 substituents of halogen, alkyl, alkoxy, CF₃, nitro, or nitrile;

R⁶ and R⁷ are individually H, alkyl, aromatic, heteroaromatic, of the formula

wherein R⁹ and R¹⁰ are individually H or alkyl, or R⁹ forms a ring having 4 or 5 carbon atoms with R⁸, or R¹⁰ forms a ring having 4 or 5 carbon atoms with R³, and X is a 5-membered aromatic ring or a 6- membered aromatic ring, either ring optionally containing from 1 to 3 heteroatoms selected from the group consisting of N, S and O, optionally substituted with 1 to 3 substituents of halogen,

R^{1 1}

m

12 alkyl, alkoxy, CF₃, nitro, or nitrile, or of the formula R wherein R¹¹ and R¹² are individually H or alkyl, m is 1 to 4, and Y is CONH₂, CONH-alkyl, aromatic or heteroaromatic, or R⁶ or R⁷ forms a ring having 4 or 5 carbon atoms with R⁸; and

R is H, alkyl, acyl or aromatic, or R forms a ring having 4 or 5 carbon atoms with R , R or R ; Formula II:

wherein:

1 9

R and R are individually H or alkyl;

R³ and R⁴ are individually H or alkyl, or together form a ring having 3-5 carbon atoms; R⁵ is H or alkyl; n is 0-3;

A is a 5-membered aromatic ring or a 6-membered aromatic ring, either ring optionally containing from 1 to 3 heteroatoms selected from the group consisting of N, S and O, optionally substituted with 1 to 3 substituents of halogen, alkyl, alkoxy, CF₃, nitro, or nitrile; and

R¹⁹ is alkyl, O-alkyl, O-benzyl, O-9-fluorenylmethyl, N-alkyl, N-benzyl, or N-9-fluorenylmethyl;

Formula III:

wherein:

Z is NH₂, OH or NH-alkyl;

R² is H or alkyl;

R³ and R⁴ are individually H or alkyl, or together form a ring having 3-5 carbon atoms;

R⁵ is H or alkyl; n is 0-3;

A is a 5-membered aromatic ring or a 6-membered aromatic ring, either ring optionally containing from 1 to 3 heteroatoms selected from the group consisting of N, S and O, optionally substituted with 1 to 3 substituents of halogen, alkyl, alkoxy, CF₃, nitro, or nitrile; and R ,2 ^z0^υ is of the formula (i)

wherein p is 1-6, wherein p is 1-5,

, wherein p is 1-4,

wherein p is 1-5, or

, wherein R' and R" are amino acid side chains, and R ,2^Z1¹ is H or CH₃C(O)-.

5. The method of any one of claims 1-4, wherein the compound is of Formula I or a pharmaceutically acceptable salt thereof.

6. The method of claim 5, wherein in the compound of Formula I,

n is 0 or 1,

A is

wherein R¹³, R¹⁴ and R¹⁵ are individually H, halogen or alkyl,

R is H or alkyl or forms a ring having 4 or 5 carbon atoms with R

R⁷ is

wherein R⁹ is H or forms a ring having 4 or 5 carbon atoms with R⁸, R¹⁰ is H,

and X is

wherein R , R and R are individually H, halogen or alkyl.

7. The method of claim 5, wherein the compound of Formula I is of the formula

wherein R³ is H or alkyl, and R¹³, R¹⁴, R¹⁵, R¹⁶, R¹⁷ and R¹⁸ are individually H, halogen or alkyl, or a pharmaceutically acceptable salt thereof.

8. The method of claim 5, wherein the compound of Formula I is selected from the group consisting of:

and enantiomers, racemates and pharmaceutically acceptable salts thereof.

9. The method of any one of claims 1-4, wherein the compound is of Formula II or a pharmaceutically acceptable salt thereof.

10. The method of claim 9, wherein in the compound of Formula II, R⁴ and R⁵ are H,

n is 0 or 1 , and A is

R wherein R , R and R are individually H, halogen or alkyl.

11. The method of claim 9, wherein the compound of Formula II is selected from the group consisting of

and , and enantiomers, racemates and pharmaceutically acceptable salts thereof.

12. The method of any one of claims 1-4, wherein the compound is of Formula III, or a pharmaceutically acceptable salt thereof.

13. The method of claim 12, wherein A is

wherein R , R and R are individually H, halogen or alkyl.

14. The method of claim 12, wherein the compound is Arg-Pro-Lys-Pro-Gln-Gln- Phe-NH₂ or Gln-Gln-Phe-NH₂.

15. The method of any one of claims 1-4, wherein the therapeutically effective amount is in the range of from about 0.001 to about 100 mg of the compound per kilogram of body weight per day.

16. The method of claim 1 , wherein the pain is chronic neuropathic pain.

17. The method of claim 2, wherein the symptom is associated with cessation or withdrawal from an opioid.

18. The method of claim 2 wherein the symptom is one or more of dysphoric mood, nausea, vomiting, muscle aches, lacrimation, rhinorrhoea, papillary dilation, piloerection, sweating, diarrhea, yawning, fever, insomnia, and opioid craving.

19. The method of claim 3, wherein the inflammation is acute or recurrent injury related.

20. The method of claim 3, wherein one or more of inflammation-related heat release, vasodilation, erythema, edema and/or pain is alleviated.

21. A compound of Formula I:

Formula I :

wherein:

1 9

R and R are individually H or alkyl;

R and R are individually H or alkyl, or together form a ring having 3-5 carbon atoms;

R⁵ is H or alkyl; n is 0-3;

A is a 5-membered aromatic ring or a 6-membered aromatic ring, either ring optionally containing from 1 to 3 heteroatoms selected from the group consisting of N, S and O, optionally substituted with 1 to 3 substituents of halogen, alkyl, alkoxy, CF₃, nitro, or nitrile;

and R are individually H or alkyl, or R forms a ring having 4 or 5 carbon atoms with R , or R forms a ring having 4 or 5 carbon atoms with R³, and X is a 5-membered aromatic ring or a 6- membered aromatic ring, either ring optionally containing from 1 to 3 heteroatoms selected from the group consisting of N, S and O, optionally substituted with 1 to 3 substituents of halogen,

are individually H or alkyl, m is 1 to 4, and Y is CONH₂, CONH-alkyl, aromatic or heteroaromatic, or R⁶ or R⁷ forms a ring having 4 or 5 carbon atoms with R⁸; and

R⁸ is H, alkyl, acyl or aromatic, or R⁸ forms a ring having 4 or 5 carbon atoms with R⁶, R⁷ or R⁹, or a pharmaceutically acceptable salt thereof.

22. A compound according to claim 21, wherein n is 0 or 1,

A is

wherein R¹³, R¹⁴ and R¹⁵ are individually H, halogen or alkyl,

R is H or alkyl or forms a ring having 4 or 5 carbon atoms with R , R¹⁰

R⁷ Is R^{β %} wherein R is H or forms a ring having 4 or 5 carbon atoms with R , R is H,

and X is

wherein R , R and R are individually H, halogen or alkyl.

23. A compound according to claim 21, of the formula

wherein R³ is H or alkyl, and R¹³, R¹⁴, R¹⁵, R¹⁶, R¹⁷ and R¹⁸ are individually H, halogen or alkyl, or a pharmaceutically acceptable salt thereof.

24. A compound according to claim 21, selected from the group consisting of

and enantiomers, racemates and pharmaceutically acceptable salts thereof.

25. A compound of the Formula II:

Formula II:

wherein:

R¹ and R² are individually H or alkyl;

R³ and R⁴ are individually H or alkyl, or together form a ring having 3-5 carbon atoms;

R⁵ is H or alkyl; n is 0-3;

A is a 5-membered aromatic ring or a 6-membered aromatic ring, either ring optionally containing from 1 to 3 heteroatoms selected from the group consisting of N, S and O, optionally substituted with 1 to 3 substituents of halogen, alkyl, alkoxy, CF₃, nitro, or nitrile; and

R¹⁹ is alkyl, O-alkyl, O-benzyl, O-9-fluorenylmethyl, N-alkyl, N-benzyl, or N-9-fluorenylmethyl, or a pharmaceutically acceptable salt thereof.

26. A compound according to claim 25, wherein R⁴ and R⁵ are H, n is 0 or 1, and A is

wherein R , R and R are individually H, halogen or alkyl.

27. A compound according to claim 26 selected from the group consisting of

and enantiomers, racemates and pharmaceutically acceptable salts thereof.

28. A compound selected from the group consisting of Ala-Pro-Phe-Phe-NH₂, Tyr- Ala-Phe-Phe-NH₂, Tyr-Pro-Ala-Phe-NH₂, Tyr-Pro-Phe-Phe-OH, and Pro-Phe-Phe- NH₂.

29. A pharmaceutical composition comprising a therapeutically effective dose of a compound according to any one of claims 21-28, and at least one pharmaceutically acceptable carrier.

30. A pharmaceutical composition comprising a therapeutically effective dose of a compound of Formula III, and at least one pharmaceutically acceptable carrier:

Formula III:

wherein:

Z is NH₂, OH or NH-alkyl;

R² is H or alkyl;

R³ and R⁴ are individually H or alkyl, or together form a ring having 3-5 carbon atoms;

R⁵ is H or alkyl; n is 0-3;

A is a 5-membered aromatic ring or a 6-membered aromatic ring, either ring optionally containing from 1 to 3 heteroatoms selected from the group consisting of N, S and O, optionally substituted with 1 to 3 substituents of halogen, alkyl, alkoxy, CF₃, nitro, or nitrile; and R ,2 ^z0^υ is of the formula (i)

wherein p is 1-6,

wherein p is 1-5,

, wherein p is 1-4,

wherein p is 1-5, or

, wherein R' and R" are amino acid side chains, and R²¹ is H or CH₃C(O)-, or a pharmaceutically acceptable salt thereof.