WO2023247999A1

WO2023247999A1 - Peptidic pain modulators and methods for producing and using the same

Info

Publication number: WO2023247999A1
Application number: PCT/IB2022/055898
Authority: WO
Inventors: Liang Zeng Yan; Junge ZHANG
Original assignee: Mainline Biosciences (Shanghai) Co., Ltd.
Priority date: 2022-06-24
Filing date: 2022-06-24
Publication date: 2023-12-28

Abstract

Provided is a peptide of the formula: X1-X2-Gly-X4–X5-X6-X7-Trp-L-R(SEQ ID NO:1) or a pharmaceutically acceptable salt thereof, wherein X1, X2, X4, X5, X6, X7, L, and R are those defined herein, and at least one of X6 and X7 is not a thiol-containing amino acid. Also provided are methods for using peptides treating pain and for drug abuse intervention.

Description

PEPTIDIC PAIN MODULATORS AND METHODS FOR PRODUCING AND USING THE SAME

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the priority benefit of U.S. Provisional Application No. 63/166,397, filed March 26, 2021, which is incorporated herein by reference in its entirety.

FIELD OF INVENTION

[0002] The present invention relates peptides of the formula: X¹-X²-Gly-X⁴-X⁵-X⁶-X⁷-Trp-L-R (SEQ ID NO: 1) and methods for using the same for the treatment of pain, more specifically compounds, compositions comprising the compounds, and methods for acute and chronic pain relief and acute and chronic interventions for drug abuse.

BACKGROUND OF THE INVENTION

[0003] Pain is a distressing feeling often caused by intense or damaging stimuli. The International Association for the Study of Pain defines pain as "an unpleasant sensory and emotional experience associated with, or resembling that associated with, actual or potential tissue damage." (Raja, S N, et al., PAIN. 2020, Vol. 161(9): 1976-1982). Pain motivates the individual to withdraw from damaging situations, to protect a damaged body part while it heals, and to avoid similar experiences in the future. Most pain resolves once the noxious stimulus is removed and the body has healed, but it may persist despite removal of the stimulus and apparent healing of the body, at which time a pain medication, or an analgesic is needed. Without medical interventions, pains may interfere with a person's quality of life and general functioning.

[0004] In general, pain can be divided into five different types. However, some pain can fit into more than one category, thereby complicating how to categorize pain. The five most common types of pain are acute pain, chronic pain, neuropathic pain, nociceptive pain, and radicular pain. Acute pain typically is relatively short in duration, lasting from minutes to a few three months (sometimes up to six months). Acute pain generally results from injury to a soft- tissue or a temporary illness. Acute pain from an injury can evolve into chronic pain if the injury doesn’t heal correctly. Chronic pain is longer in duration relative to acute pain. Chronic pain can be constant or intermittent. Chronic pain is often due to a health condition, like arthritis, fibromyalgia, or a spine condition.

[0005] Neuropathic pain is result of damage to the nerves or other nervous system. It can affect sensitivity to touch and is a common type of chronic pain. It can be intermittent but also can interfere with normal movement leading to mobility issues. Neuropathic pain can be caused by many different conditions, including: alcoholism, diabetes, HIV or AIDS, multiple sclerosis, joint problems in the spine. In addition, neuropathic pain can also arise as a side effect of chemotherapy.

[0006] Nociceptive pain is generally caused by damage to body tissue, often due to an external injury. People commonly experience nociceptive pain in the musculoskeletal system, which includes the joints, muscles, skin, tendons, and bone. Chronic (long-term) or acute (short term) nociceptive pain can interfere with your daily life and make it difficult to move, causing mobility issues. Examples of injuries that can cause nociceptive pain include, but are not limited to, bruises, burns, cuts, fractures or broken bones, pain caused by repetitive or muscle overuse, and pain caused by joint damage, such as arthritis or sprains. Nociceptive pain can also be caused by an internal problem, such as cancer or a tumor.

[0007] Opioids are often used to treat pain. However, constant opioid treatment is accompanied with serious undesirable effects including drowsiness and mental clouding, nausea and emesis, constipation and in many cases dependence and addiction. Continuous use of opioid therapy also develops analgesic tolerance and hyperalgesia in many patients. These unwanted effects significantly diminish the patients’ quality of life.

[0008] Opioid drugs also are widely used following major surgery and to control pain of terminal diseases such as cancer, but its use is limited by several undesired side effects including nausea, vomiting, constipation, dizziness, system changes (neuroplasticity) due to prolonged pain or treatment by the opioid drugs and the development of tolerance and physical dependence. Because of these limitations there has been extensive search for the novel type of analgesics which have strong pain controlling effect without development of undesired side-effects, such as tolerance and physical dependence. [0009] While a substantial advance has been made in pain treatment, there are still unmet needs for better and more efficacious pain medications that do not have or have a significantly reduced side-effects or with a much-improved safety profile compared to conventional opioid based pain medications.

SUMMARY OF THE INVENTION

[0010] Some aspects of the invention provide peptides and methods for using the same to treat pain as well as methods for producing said peptides. In some embodiments, peptides of the invention comprise an opioid agonist pharmacophore that is covalently linked to an NK1 antagonist pharmacophore. In one particular aspect of the invention, a peptide of the formula:

X¹-X²-Gly-X⁴-X⁵-X⁶-X⁷-Trp-L-R (SEQ ID NO : 1 )

Formula I or a pharmaceutically acceptable salt thereof is provided. In peptide of SEQ ID NO: 1,

X¹ is Tyr, N-alkyl-Tyr, or a substituted Tyr selected from the group consisting of mono- or di-halo-Tyr, mono- or dialkyl-Tyr;

X² is Cys, Ala, hCys, or Pen;

X⁴ is Phe or substituted Phe;

X⁵ is Phe, Leu, Met, Met(O), Gly, Nle, Ser, or substituted Ser;

X⁶ is Pro, Ala, Leu, Cys, hCys, or Pen;

X⁷ is Cys, hCys, Pen, Ser, Leu, Ala, Vai, or Aib;

L is O, NH, or NMe; and

R is benzyl or substituted benzyl, provided at least one of X⁶ and X⁷ is not a thiol-containing amino acid, and wherein optionally a cyclic structure is formed by a disulfide linkage between X² and X⁶ or X² and X⁷. As used herein, the term “thiol-containing amino acid” refers to any natural or synthetic amino acid having a thiol functional group on the side-chain. The terms “halo,” “halogen” and “halide” are used interchangeably herein and refer to fluoro, chloro, bromo, or iodo. The term “alkyl” refers to a saturated linear monovalent hydrocarbon moiety of one to twelve, typically one to six, carbon atoms or a saturated branched monovalent hydrocarbon moiety of three to twelve, typically three to six, carbon atoms. Exemplary alkyl group include, but are not limited to, methyl, ethyl, /?-propyl, 2-propyl, tert-butyl, pentyl, and the like. [0011] Unless explicitly stated, throughout this disclosure each amino acid residue is independently an (L)-isomer or (D)-isomer. As is evident, L is the carboxylate functional group of tryptophan. Thus, if carboxylate functional group of tryptophan is an amide, L is NH or NMe. Whereas if the carboxylate functional group of tryptophan is an ester, then L is O.

[0012] In some embodiments, X² is Cys, homocysteine (hCys), or penicillamine (Pen). Within these embodiments, in some instances one of X⁶ or X⁷ is Cys, hCys, or Pen. In this manner, a cyclic peptide can be formed by a disulfide linkage between X² and one of X⁶ or X⁷. As such, in some cases said peptide of SEQ ID NO: 1 is a cyclic peptide of the formula:

Formula II Formula III

[0013] Still in other embodiments, X⁶ is (L)-Pro, (L)-Ala, (L)-Leu, (L)-Cys, (D)-Cys, (L)-hCys, (D)-hCys, (L)-Pen, or (D)-Pen.

[0014] In further embodiments, X⁷ is (L)-Cys, (D)-Cys, (L)-hCys, (D)-hCys, (L)-Pen, (D)-Pen, (L)-Ser, (L)-Leu, (L)-Ala, (D)-Ala, (L)-Val, (D)-Val, or (L)-Aib;

[0015] Yet in other embodiments, X¹ is selected from the group consisting of (3 ’,5’- dimethyl-Tyr), (2’,6’-dimethyl-Tyr), (3’,5’-difluoro-Tyr), (3’,5’-dichloro-Tyr), (3’,5’-dibromo- Tyr), (2’,6’-difluoro-Tyr), (2’,6’-dichloro-Tyr), and (2’,6’-dibromo-Tyr).

[0016] In other embodiments, X⁴ is Phe.

[0017] Still yet in other embodiments, X⁵ is Met, Met(O) (i.e., methionine sulfoxide), or Nle. In one particular embodiment, X⁵ is Met or Nle.

[0018] In one particular embodiment, X⁶ is a (D)-isomer.

[0019] Still in another particular embodiment, X⁷ is a (D)-isomer.

[0020] Yet in another embodiment, R is selected from the group consisting of mono- or di- substituted haloalkylbenzyl, mono- or di-substituted halobenzyl, and mono- or di-substituted alkylbenzyl. In one specific embodiment, R is selected from the group consisting of benzyl having one or two substituents, wherein each substituent is independently selected from the group consisting of trifluoroalkyl, chloro, fluoro, bromo, and methyl. Still in further embodiment, R is selected from the group consisting of 3,5-ditrifluoromethylbenzyl; 3,5- dimethylbenzyl; 2,4-trifluoromethylbenzyl; 3-trifluoromethyl-benzyl; 4-trifluoromethylbenzyl; 5-trifluoromethylbenzyl; 6-trifluoromethylbenzyl; 3,5-difluorobenzyl; 3, 5 -di chlorobenzyl; 3,5- dibromobenzyl; 2,4-diflurobenzyl; 2,4-dichlorobenzyl; 2,4-dibromobenzyl; 2,4-dimethylbenzyl; and 3, 5 -dimethylbenzyl.

[0021] In other embodiments, X² is a (D)-isomer.

[0022] Still in other embodiments, X² is an (L)-isomer. In one particular embodiment, X² is an (L)-isomer of Cys, homocysteine (hCys), or penicillamine (Pen). Within these embodiments, in some instances one of X⁶ or X⁷ is Cys, hCys, or Pen. In this manner, cyclic peptides of Formula II and III comprise an (L)-isomer of X².

[0023] In further embodiments, said peptide of SEQ ID NO: 1 is a non-cyclic peptide of the formula:

X¹-X²-Gly-X⁴-X⁵-Pro-Leu-Trp-L-R (SEQ ID NO: 4)

Formula IV

[0024] In some embodiments, X² is (D)-Ala.

[0025] Another aspect of the invention provides a method for treating pain or an opioid addiction in a subject, said method comprising administering a therapeutically effective amount of a peptide of the formula:

X¹-X²-Gly-X⁴-X⁵-X⁶-X⁷-Trp-L-R (SEQ ID NO : 1 ) Formula I or a pharmaceutically acceptable salt thereof to a subject in need of such a treatment, where

X² is Cys, Ala, hCys, or Pen;

X⁴ is Phe or substituted Phe;

X⁵ is Phe, Leu, Met, Met(O), Gly, Nle, Ser, or substituted Ser;

X⁶ is Pro, Ala, Leu, Cys, hCys, or Pen; X⁷ is Cys, hCys, Pen, Ser, Leu, Ala, Vai, or Aib;

L is O, NH, or NMe; and

R is benzyl or substituted benzyl, provided at least one of X⁶ and X⁷ is not a thiol-containing amino acid, and wherein optionally a cyclic structure is formed by a disulfide linkage between X² and X⁶ or X² and X⁷.

[0026] In some embodiments, said pain is an acute pain, chronic pain, neuropathic pain, nociceptive pain, radicular pain, or a combination thereof.

[0027] Still in other embodiments, said pain is a chronic pain.

[0028] Yet in other embodiments, said pain is a nociceptive pain.

[0029] In further embodiments, said pain is a neuropathic pain.

[0030] Still yet in other embodiments, said pain is an acute pain.

[0031] In one particular embodiment, X² is cys, hCys, or Pen. Within this embodiment, in some instances, one of X⁶ or X⁷ is Cys, hCys, or Pen. In further instances, said peptide is a cyclic peptide of the formula:

(SEQ ID NO:2) (SEQ ID NO: 3)

Formula II Formula III

It should be readily evident that when said peptide is cyclic peptide of Formula II, then X⁶ is Cys, hCys, or Pen. And when said peptide is cyclic peptide of Formula III, then X⁷ is Cys, hCys, or Pen.

[0032] Yet in other embodiments, said peptide is a non-cyclic peptide of the formula:

X¹-X²-Gly-X⁴-X⁵-Pro-Leu-Trp-L-R (SEQ ID NO: 4)

Formula IV

[0033] In one particular embodiment, X² is (D)-Ala. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] FIG. 1 is a histogram for p (MOP) (h) (agonist effect) of some of the representative peptides of the invention.

[0035] FIG. 2 is a histogram for p (MOP) (h) (agonist effect) of peptide MB301-01 showing ECso = 5.5E-08 M.

[0036] FIG. 3 is a histogram for p (MOP) (h) (agonist effect) of peptide MEG 01 -02 showing EC50 = 5.0E-08 M.

[0037] FIG. 4 is a histogram for p (MOP) (h) (agonist effect) of peptide MEG 01 -04 showing EC50 = 8.7E-06 M.

[0038] FIG. 5 is a histogram for p (MOP) (h) (agonist effect) of peptide MB301-05 showing EC50 > 1.0E-05.

[0039] FIG. 6 is a histogram for p (MOP) (h) (agonist effect) of peptide MB301-08 showing EC50 > 1.0E-05.

[0040] FIG. 7 is a histogram for p (MOP) (h) (agonist effect) of peptide MB301-10 showing EC50 > 1.0E-05.

DETAILED DESCRIPTIONS

[0041] While some new pain medications, such as calcitonin-gene-related peptide (CGRP) antagonists, COX-2 inhibitors, and cannabis, have come into the clinic and become commercially available on the market in recent years, opioid drugs are still widely being used, even though its use is limited by several undesired side effects including nausea, vomiting, constipation, dizziness, system changes (neuroplasticity) due to prolonged pain or treatment by the opioid drugs and the development of tolerance and physical dependence (see, for example, Ananthan, J. Med. Chem., 2004, 47, pp. 1400-1412; Yaksh, Pain, 1982, 11, pp. 293-346; and Ossipov, Biopolymers, 2005, 80, pp. 319-324).

[0042] Opiate drugs work in the brain at a group of opiate receptors. The main opioid receptor is p receptor. Administering receptor agonists can cause full or partial stimulation or effect at the receptor, while administering antagonists blocks the effect of the receptor. It is widely accepted that a p receptor agonist such as morphine has higher antinociceptive activity accompanied with high abuse liability. On the other hand, activation of the 5 opioid receptor has lower analgesic efficacy, but has reduced addictive potential (Kaslo, Eur. J. Pain, 2005, 9, pp. 131-135). It is also generally known that the selective agonists at the 5 opioid receptor have analgesic activity in numerous animal models with fewer adverse effects, though their efficacy is less potent than that of their widely-used p counterparts (Ossipov, Biopolymers, 2005, 80, pp. 319-324). Thus, selective 5 opioid agonists with enhanced analgesic activity are expected as a potent drug candidate for severe pain control.

[0043] Substance P is the preferred ligand for the neurokinin 1 (NK1) receptor and is known to contribute to chronic inflammatory pain and participate in central sensitization and associated hyperalgesia. In the pain states, substance P is known as a major neurotransmitter of pain signals as well as the signals induced by opioid stimulation (Yaksh, Pain, 1981, 11, pp. 293- 346; Ossipov, Biopolymers, 2005, 80, pp. 319-324). Substance P and NK1 receptor expression increases after sustained opioid administration. Also, repeated morphine exposure results in enhanced levels of substance P both in vitro and in vivo, which could induce increased pain; increased pain could require increased pain-relief and thus be manifested as “antinociceptive tolerance” (King, Neurosignals, 2005, 14, pp. 194-205). Interestingly, co-administration of 5/p opioid agonists and a substance P antagonist showed enhanced antinociceptive effect in acute pain states, and in prevention of opioid-induced tolerance in chronic trials. These results suggest that the signals through opioid receptors and neurokinin 1 (NK1) receptors are not independent, but have strong and critical interaction. Moreover, the mice lacking NK1 receptors, the preferred receptor of substance P, didn't show rewarding properties for opiates (Ananlhan, J. Med. Chem., 2004, 47, pp. 1400-1412).

[0044] According to these observations, the use of multimodal combination analgesic therapies or therapies with single molecules possessing multiple analgesic targets has become attractive (Walker, Anesth. Analg., 2002, 95, pp. 674-715). Advantages of hybrid compounds system are developing bioactive compounds designed with a broad spectrum of receptor affinities and single administration of a chimeric compound instead of a specific ration of two different compounds. The two pharmacophores are joined directly or by a linker, which might be working as an address region for both pharmacophores as well as a spacer between them. It should be noted that the designed multivalent/multifunctional ligands have additional rewards over a cocktail of individual drugs for easy administration, a simple ADME property and no drug-drug interactions. Local concentration is also expected to be higher than that in the coadministration of drug cocktails as the expression of the NK1 and opioid receptors as well as the neurotransmitters show a significant degree of overlap in the central nervous system, resulting to synergies in potency and efficacy.

[0045] It has been reported that agonist activities at Mu-type and Delta-type opioid receptors (MOR and DOR, respectively), and antagonist activity at NKI is beneficiary over targeting a single receptor (Hruby et al. U.S. Pat. No. 8,026,218, issued Sept. 27, 2011). This combination addresses several fundamental biological effects such as enhanced potency in acute pain models and inhibition of opioid-induced tolerance in chronic tests using rats. A study revealed that NKI knockout mice did not show the rewarding properties of morphine. Thus, the combination of opioid receptor agonist and NKI receptor antagonist activity may have synergistic effects in the management of prolonged pain states that involve higher substance P activity. Drug combinations have restrictions as therapeutics because of poor patient compliance, difficulties in drug metabolism, distribution, and possible drug-drug interactions.

[0046] The present invention’s approach of drug-design is based on adjacent and overlapping pharmacophores, in which an opioid agonist pharmacophore is placed at the N- terminus and the NKI antagonist pharmacophore sits at the C-terminus of a single peptide derived ligand. The opioid pharmacophore of these multivalent/multifunctional ligands were designed based on the sequences of well-known opioid agonist ligands including enkephalin and Leu-Enkephalin, dermorphin, while the NKI antagonist pharmacophore was adopted from the previously published pharmacophore. See, for example, Hruby et al., U.S. Pat. No. 8,026,218, issued Sept. 27, 2011) for the same kind of activity. Those lead bifunctional compounds are capable to treat neuropathic pain in a rodent model with blood brain barrier permeability, no development of opioid-induce tolerance, and no development of reward liability, supporting the hypothesis that a single ligand containing opioid agonist/NKl antagonist activities is effective against neuropathic pain.

[0047] In particular, the present invention’s approach, termed poly-pharmacological peptide platform (PPP™), focuses on combining these two or three different activities in one ligand which should have appropriate metabolic and pharmacological properties. In this instance, the ligand would have potent analgesic affects not only in acute pain but also in prolonged and neuropathic pain states without or minimized unwanted side effects, ppp™ strategy disclosed herein is well supported by the current understanding of peptide chemistry and biology.

[0048] Ubiquitous of peptides as signaling molecules are widespread in nature: from human beings to animals, from birds to fish, from plants to algae, from bacteria to virus. Where there is a life, you can find existence of peptides. On one hand, as compared to a small molecule, a peptide has less rigidity, but has much more structural molecular spaces that mimics the functions of proteins and can function far beyond the reach of a small molecule, such as mimic protein-protein interactions. A peptide can gain structural rigidity by cyclization, secondary structures, and chemical modifications. On the other hand, compared to proteins, peptides disclosed herein are much smaller in size, but big enough to carry all necessary biological information for signaling and many other functional purposes. Due to their intermediate in size and in structural flexibility, peptides can reach and interact efficiently with multiple pharmacological targets. A peptide, by design and by engineering, can perform many essential functions of a protein.

[0049] Furthermore, as the advances of sciences and technologies, the healthcare of needs of society are demanding higher standards in terms of efficacy of medications and patient’s compliances; on the other hand, the progression of non-communal diseases, including metabolic syndromes (obesity, diabetes and its complications) and cancers (most heterogeneous diseases), demands better approaches and more advanced pharmacological interventions. Many diseases cannot be sufficiently treated with a single drug regimen.

[0050] The invention is based on the belief by the present inventors that a peptide, with innovative design and engineering, can be an effective therapeutic standing high among small molecule drugs and biological therapies. Peptide’s unique structural flexibility and rigidity, and its intermediate, manageable molecular spaces, makes it an idea platform for unimolecular, poly pharmacological therapeutics. With a single peptide molecule, the simultaneous activation of different signaling mechanisms or pharmacological pathways maximizes the therapeutic benefits, minimizes adverse effects, and offers a more balanced pharmacokinetic action profile compared to co-administration of individual drugs, ppp™ is designed based upon a peptide sequence that includes antagonist, agonist, spacer, toxin, special function sequence such as cell membrane penetrating peptides, etc., depending on the targeted disease. The drug candidate is made through chemical synthesis. New advance in technology and pharmaceutical science, both chemical and biological understandings, will help and maintain the status of peptide in pharma as therapeutics, as a way of diagnostics, as a boost of human beings’ health and beauty (cosmetics).

[0051] Unless specifically defined, all technical and scientific terms used herein have the same meaning as commonly understood by a skill artisan in chemistry, biochemistry, cellular biology, molecular biology, and medical sciences.

[0052] All methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, with suitable methods and materials being described herein. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. Further, the materials, methods, and examples are illustrative only and are not intended to be limiting, unless otherwise specified.

[0053] The compounds of the present invention, salts, and derivatives thereof can be incorporated into pharmaceutical compositions suitable for administration. Such compositions typically comprise the compound and a pharmaceutically acceptable carrier. As used herein, “pharmaceutically acceptable carrier” is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions. Modifications can be made to the compound of the present invention to affect solubility or clearance of the compound. These molecules may also be synthesized with D-amino acids to increase resistance to enzymatic degradation. If necessary, the compounds can be coadministered with a solubilizing agent, such as cyclodextran.

[0054] A pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerin, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates, and agents for the adjustment of tonicity such as sodium chloride or dextrose. The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

[0055] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as manitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

[0056] Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, methods of preparation are vacuum drying and freeze-drying that yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile- filtered solution thereof.

[0057] Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.

[0058] For administration by inhalation, the compounds are delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer. Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.

[0059] The compounds can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

[0060] In one embodiment, the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811.

[0061] It is especially advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the invention is dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals.

[0062] The invention also provides a pharmaceutical composition comprising the compound as above described in a pharmaceutical-acceptable carrier.

[0063] The invention also provides a method for treating pain which comprises administering an effective amount of the above-described composition to an individual in need of treatment, as needed, preferably in a dose range of 1 mg/Kg to 200 mg/Kg, and more preferably in a dose range of 0.01 mg/kg to 20 mg/kg.

[0064] The invention also provides a method for forming compound as above described, comprising the steps of solid phase peptide synthesis, cyclization via coupling of appropriate functional groups on solid phase, C-terminal modification and removal of all protecting group in solution phase.

[0065] In some illustrative embodiments, the present disclosure relates to a compound having the formula (I): X¹-X²-Gly-X⁴-X⁵-X⁶-X⁷-Trp-L-R (SEQ ID NO : 1 ) or a pharmaceutically acceptable salt thereof, wherein:

X² is Cys, Ala, hCys, or Pen;

X⁴ is Phe or substituted Phe;

X⁵ is Phe, Leu, Met, Met(O), Gly, Nle, Ser, or substituted Ser;

X⁶ is Pro, Ala, Leu, Cys, hCys, or Pen;

X⁷ is Cys, hCys, Pen, Ser, Leu, Ala, Vai, or Aib;

L is O, NH, or NMe; and

[0066] Unless the context requires otherwise, each amino acid residues of the invention can independently be an (L)-isomer of a (D)-isomer. Moreover, the scope of the invention also includes retro modified peptides, inverse modified peptides, and retro-inverso modified peptides. The term "retro modified" refers to a peptide which is made up of L-amino acids in which the amino acid residues are assembled in opposite direction to the native peptide with respect the which it is retro modified. The term "inverse modified" refers to a peptide which is made up of D-amino acids in which the amino acid residues are assembled in the same direction as the native peptide with respect to which it is inverse modified. The term "retro-inverso modified" refers to a peptide which is made up of D-amino acids in which the amino acid residues are assembled in the opposite direction to the native peptide with respect to which it is retro-inverso modified.

The term "peptide" as used throughout the specification and claims is to be understood to include amino acid chain of any length, typically about 20 amino acid chain length or less, often about 15 amino acid chain length or less, and most often about 10 amino acid chain length or less.

[0067] It should be appreciated that one or more of the amino acids can be replaced with an equivalent amino acid, for example, L (leucine) can be replaced with isoleucine or other hydrophobic side-chain amino acid such as alanine, valine, methionine, etc., and amino acids with polar uncharged side chain can be replaced with other polar uncharged side chain amino acids.

[0068] In some illustrative embodiments, the present disclosure relates to a peptide of Formula (I), or a pharmaceutically acceptable salt thereof. In some embodiments, peptides of Formula (I) are opioid receptor agonists and neurokinin 1 (NK1) receptor antagonists. Still in other embodiments, peptides of Formula (I) are useful as a pain medication.

[0069] Peptides of Formula (I) of the invention include, but are not limited to, the following representative peptides shown in Table 1 :

Table 1. Representative peptides of Formula (I)

[0070] Biological activities of some of the representative peptides of the invention are shown in Table 2 below:

Table 2. Biological Activity of Representative Peptides of Formula (I).

[0071] Another aspect of the invention provides a method for treating a patient suffering from pain (e.g., an acute or chronic pain) by administering to a subject in need of such a treatment a therapeutically effective amount of one or more peptides of Formula I or a pharmaceutically acceptable salt thereof. In some embodiments, the peptide of Formula I is an opioid receptor agonist and a neurokinin 1 (NK1) receptor antagonist.

[0072] Still in other embodiments, methods of the invention include treating a patient suffering from pain (e.g., an acute or chronic pain) by administering to a subject in need of such a treatment a therapeutically effective amount of one or more peptides of Formula II, III, or IV, or a pharmaceutically acceptable salt thereof, or a combination thereof. In some embodiments, the peptide of Formula II, III, or IV is an opioid receptor agonist and a neurokinin 1 (NK1) receptor antagonist.

[0073] Yet in other embodiments, methods of the invention include treating a patient suffering from pain (e.g., an acute or chronic pain) by administering to a subject in need of such a treatment a therapeutically effective amount of one or more peptides of SEQ ID NOS: 5-110, or a pharmaceutically acceptable salt thereof, or a combination thereof. In some embodiments, the peptide of SEQ ID NOS: 5-110 is an opioid receptor agonist and a neurokinin 1 (NK1) receptor antagonist.

[0074] Another aspect of the invention provides a pharmaceutical composition for treating a patient suffering from pain, such as an acute or chronic pain. The pharmaceutical composition comprises one or more peptides of Formula I or a pharmaceutically acceptable salt thereof. In some embodiments, a peptide of Formula (I) or a pharmaceutical salt thereof, is an opioid receptor agonist and a neurokinin 1 (NK1) receptor antagonist. Still in other embodiments, peptide is one or more peptides of SEQ ID NOS: 5-110 or a pharmaceutical salt thereof.

[0075] Synthesis and characterization of Peptides'. All linear peptides were synthesized on solid phase using 2-chlorotrityl chloride resin (loading: 1.02 mmol/g) via Finoc/^lBu approach. All steps during solid phase synthesis were performed in fritted syringes. N-methylation on desired amino acid was performed on solid phase and C-terminal amidation was conducted in solution phase.

[0076] Loading of the first amino acid on the resin'. Chlorotrityl resin (0.102 mmol) was swelled in dry dichloromethane (DCM) for 1 hour at room temperature. After swelling, dry DCM was expelled from the syringe and the resin was washed with DCM (1 mL, 3x1 min). It was then ready for the first amino acid coupling. Pre-generated (by treating with 5.0 equiv. DIPEA) carboxylate of Fmoc-Trp(Boc)-OH (1.2 equiv.) in dry DCM (1.0 mL) was loaded onto the resin by substituting chloride from the resin. After the coupling of first amino acid, methanol (0.1 mL) was added to the mixture and was shaken for 15 minutes in order to cap any unreacted chloride present in the resin. It was then washed with DCM (1 mL, 5x1 min) and DMF (1 mL, 4x1 min).

[0077] Deprotection'. Following the washes, deprotection of Fmoc group was performed. This was done by stirring the resin with 20% piperidine in DMF for 8 minutes, followed by 12 minutes. A DMF wash (1 mL, 1 min) was performed in between the two deprotection steps to remove side products. After the second piperidine treatment, resin washes were performed with DMF (1 mL, 3x1 min), DCM (1 mL, 3x1 min), and DMF (1 mL, 3x1 min) before the next coupling. These steps were repeated after coupling of each Fmoc protected amino acid in the peptide sequence.

[0078] Coupling. For the coupling of the remaining amino acids, HCTU (3.0 equiv. and in case of primary amine) or DCC/HOBt (3.0 equiv.) or HATU/HOAt (3.0 equiv. of each, in case of secondary amine) was used as coupling reagents and DIPEA (6.0 equiv.) as base. All couplings involving primary amines were carried out in DMF while coupling of secondary amine was performed in NMP. Between each coupling, resin washes were performed with DMF (1 mL, 3x1 min), DCM (1 mL, 3x1 min), and DMF (1 mL, 3x1 mill).

[0079] After each coupling or deprotection, the Kaiser/chloranil test was performed to determine whether or not amino acid coupling or Fmoc deprotection was successful. Kaiser tests were run for primary amino acids and chloranil tests for secondary amino acids (e.g. proline and methylated amino acids). A negative test after each coupling suggests that the reaction was complete. After deprotection, the same test should be positive.

[0080] N-Methylation of amino acids'. After Fmoc deprotection of the desired amino acid that will be N-methylated, o-NBS protection, N-methylation, and then o-NBS deprotection were performed. Alternatively, in some cases, a pre-made N-methyl-amino acid building block is used directly in the peptide chain assembly, for example, Fmoc-N-methyl-Tyr(tBu) is used in the peptide chain assembly for SEQ ID NOS:5-18 and 95-102.

[0081] O-NBS protection : After Fmoc deprotection, the resin was washed with DMF,

DCM, then NMP (3x1 min each). NMP was drained out from the syringe. NMP (1 mL) was added to the resin followed by the addition of o-NBS-Cl (4 equiv.) and sym-collidine (10.0 equiv.). It was stirred for 15 minutes. The same step was repeated for one more time after filtering and washing the resin with NMP (1 mL, 1 x1 min) in between. It was then washed with NMP (1 mL, 5x1 min) and then used for N-methylation.

[0082] N-methylation (DBU mediated method)'. DBU (l,8-diazabicyclo(5,4,0)undec-7- ene) (3.0 equiv.) in NMP (1 mL) was treated with the resin for 3 minutes. Afterwards and without filtering, Dimethyl sulfate (DMS) (10.0 equiv.) was added directly to the syringe containing resin and DBU solution and stir for another 3 min. The resin was then filtered and washed with NMP (1 x1 min). This step was repeated once followed by filtration, and washing with NMP (5x1 min). The resultant resin bound peptide with N-methylation on amino acid was used for O-NBS deprotection.

[0083] O-NBS deprotection'. NMP (1 mL), 2-mercaptoethanol (10.0 equiv.), and DBU

(5.0 equiv.) were added to the syringe and the resin was treated for 5 min. The resin was filtered and washed with NMP (1 mL, 1 x1 min). The procedure was repeated one more time and then the resin was filtered and washed with NMP (5x1 min). [0084] Cleaving peptide from the resin'. The resin was stirred on a shaker with 1% TFA

(2 mL/0.102 mmol of starting resin) in DCM (3x5 min) on the shaker. The resin was rinsed in between cleavage with small amounts of DCM. The peptide containing solution was collected in the centrifuge tube. Resin became darker with each TFA treatment. Volatiles were evaporated from the centrifuge tube by flushing the resulting solution with argon.

[0085] C -terminal Amidation'. The crude peptide was dissolved in dry DMF (1 mb) followed by addition of HATU (1.0 equiv.), HOAt (1.0 equiv.), DIPEA (4.0 equiv), and 3,5- bis(trifluoromethyl)benzylamine (1.1 equiv.), respectively and mixture was stirred for overnight. Workup: KHSO4 (0.5 M in H2O, 5 mL) was added to reaction mixture followed by extraction with DCM (3x15 mL). The combined organic extract was taken into a separatory funnel and was washed with brine (1 x15 mL). The organic part was washed with NaHCO, (1 x15 mL) followed by another brine wash. The final organic solution was dried over anhydrous sodium sulfate; gravity filtrated, and then evaporated under pressure to remove DCM in a round bottom flask (RBF).

[0086] Removal o f Roc 'Hu protecting groups'. The crude peptide was treated for 1 h with a cleavage cocktail containing 82.5% TFA, 5% H2O, thioanisol, 5% phenol, and 2.5% 1, 2- ethanedithiol to remove Boc/Bu protecting groups. After 1 h, the solution was flushed with argon to evaporate volatiles.

[0087] Precipitation and HPLC Purification'. Crude peptide is precipitated by three volumes of cold diethyl ether and followed by centrifugation at 3300 rpm (3x5 min). Then washing the precipitate with hexanes and dimethyl ether mixture (30:70, 3x15 mL) gave white precipitate in 80-100% as crude yield. Purification of crudes using RP-HPLC furnished the pure ligands in 20-40% overall yield for lilear peptides and 10-20% overall yield for cyclic peptides.

[0088] Methods for in vitro Study

[0089] hNKl/CHO Cell Membrane Preparation and Radioligand Binding Assay: Recombinant hNKl/CHO cells were grown to confluency in 37° C., 95% air and 5% CO2, humidified atmosphere, in a Forma Scientific (Thermo Forma, OH) incubator in Ham's F12 medium supplemented with 10% fetal bovine serum, 100 U/mL penicillin, 100 pg/mL streptomycin, and 500 pg/mL geneticin. The confluent cell monolayers were then washed with Ca²⁺, Mg²⁺- deficient phosphate-buffered saline (PD buffer) and harvested in the same buffer containing 0.02% EDTA. After centrifugation at 2700 rpm for 12 min, the cells were homogenized in ice-cold 10 mM Tris-HCl and 1 mM EDTA, pH 7.4, buffer. A crude membrane fraction was collected by centrifugation at 18000 rpm for 12 min at 4 ° C., the pellet was suspended in 50 mM Tris-Mg buffer, and the protein concentration of the membrane preparation was determined by using Bradford assay.

[0090] Bradford assay: Six different concentrations of the test compound were each incubated, in duplicates, with 20 pg of membrane homogenate, and 0.5 nM [³H] SP (135 Ci/mmol, Perkin-Elmer, United States) in 1 mL final volume of assay buffer (50 mM Tris, pH 7.4, containing 5 mM MgCh, 50 ug/mL bacitracin, 30 pM bestatin, 10 pMcaptopril, and 100 pM phenylmethylsulfonylfluoride) SP at 10 uM was used to define the nonspecific binding. The samples were incubated in a shaking water bath at 25° C. for 20 min. The reaction was terminated by rapid filtration through Whatman grade GF/B filter paper (Gaithersburg, Md.) presoaked in 1% polyethyleneirnine, washed four times each with 2 mL of cold saline, and the filter bound radioactivity was determined by liquid scintillation counting (Beckman LS5000 TD).

[0091] Data Analysis: Analysis of data collected from three independent experiments performed in duplicates is done using GraphPad Prizm 4 software (GraphPad, San Diego, Calif). Log IC50 values for each test compound were determined from nonlinear regression. The inhibition constant (Ki) was calculated from the antilogarithmic IC50 value by the Cheng and Prusoff equation.

[0092] Agonist and Antagonist Testing: Compounds were tested as agonists by adding cumulatively to the bath until a full dose-response curve was constructed or to a concentration of 1 M. Compounds were tested as antagonists by adding to the bath 2 minutes before beginning the cumulative agonist dose- response curves of the delta (DPDPE) or mu (PL-017) opioid agonists.

[0093] Analysis: Percentage inhibition was calculated using the average tissue contraction height for 1 min preceding the addition of the agonist divided by the contraction height 3 min after exposure to the dose of agonist. IC50 values represent the mean of not less than 4 tissues. IC50 and E_max estimates were determined by computerized nonlinear least-squares analysis (FlashCale). [0094] In vitro metabolic stability. A stock solution (50 mg/mL in DMSO) of each compound in study was made. It was diluted 1000-fold into rat plasma (Pel-Freez Biologicals, Rogers, AK) resulting in an incubation concentration of 50 pg/mL. Incubation temperature was 37° C. 200 uL of aliquots were pipetted out at different time points (i.e. 1 min, 10 min, 30 min, 1 h, 2 h, 4h, 6 h, 81 i, and 24 h). 300 uL of acetonitrile was added to it and vortexed followed by centrifugation at 15000 rpm for 15 min. The supernatant was taken and analyzed for the remaining amount of parent compound using RP-HPLC (Vydac 218TP Cl 8 I Op, Length: 250 mm, ID: 4.6 mm). Each sample was run twice and each time in duplet.

[0095] Additional objects, advantages, and novel features of this invention will become apparent to those skilled in the art upon examination of the following examples thereof, which are not intended to be limiting. In the Examples, procedures that are constructively reduced to practice are described in the present tense, and procedures that have been carried out in the laboratory are set forth in the past tense.

EXAMPLES

[0096] The following abbreviations are used: Ac: acetyl; Boc: tert-butyloxycarbonyl; BOP: (benzotriazol- l-yloxy)-tris(dimethylamino) phosphonium hexafluorophosphate; Bz: benzoyl; Bzl/Bn: benzyl; Dab: 1,4-diaminobutyric acid; Dap: 1,3 -diaminopropionic acid; DBU: l,8-diazabicyclo(5,4,0)undec-7-ene; DCC: dicyclohexyl-carbodiimide; DIC: diisopropylcarbodiimide; DCM: dichloromethane; DIC: diisopropyl carbodiimide; DIE A: diisopropylethylamine; DMAP: 4-(A, A-dimethylamino)pyridine; DMF: V, V-dimethyl formamide; DMS: Dimethyl sulfate; DMSO: dimethyl-sulfoxide; EDT: 1,2-ethane-dithiol; Et: ethyl; Fmoc: 9- fluor-enylmethoxy carbonyl; HBTU: C-benzo-triazolyl-A,A,A’,A’-tetramethyluronium hexafluorophosphate; HO At: l-Hydroxy-7-azabenzotriazole (); HOBt: hydroxybenzotriazole; hCys: homocysteine; iPr: isopropyl; IPA: isopropyl alcohol; Me: methyl; Mmt: 4-mthoxytrityl; 2Nal: 2-naphthylalanine; INal: 1 -naphthylalanine; Nle: norleucine; NMM: N- methylmorpholine; NMP: A-methyl-pyrrolidone; Orn: ornithine; Pbf: 2,2,4,6,7-pentamethyl- dihydrobenzofurane-5-sulfonyl; PBS: phosphate buffered saline; Pen: penicillamine; PyBOP: (benzotriazol- 1 -yloxy)-tris(pyrrolidino)-phosphonium hexafluoro-phosphate; PyBrOP: bromotris(pyrrolidino)phosphonium hexafluorophosphate; tBu: tert-butyl; TFA: trifluoroacetic acid; TFE: trifluroethanol; THF: tetrahydrofuran; TIS: triisopropyl silane; Trt: trityl; all common amino acids are expressed as three letter symbols or otherwise specified.

[0097] Mass Spectroscopy (MS) Analysis: Preparation of compounds of the present invention as described in the following examples is meant to be illustrative rather than limiting. In each of these examples, the observed molecular weight is reported as a de-convoluted value. The de-convoluted value is derived from the formula MW (observed) = n(m/z)-n, where m/z represents the charged ion (positive mode) and n is the number of charges of the specific species. When multiple charged species are present in the mass spectrum, the observed molecular weight is reported as an average.

[0098] General Method of Peptide Synthesis, Cyclic Structure Formation, and Salt Exchange: Peptides were synthesized using solid phase peptide synthesis chemistry known in the art. The cyclic structure of those peptides was established, for a disulfide, by using air oxidation, or iodine oxidation in the presence of acidic acid, or for a bisthioether ring, by nucleophilic substitution using a bis(halomethyl) aryl compound, typically using 1.3 equivalents of a bis(bromomethyl) aryl compound, in the presence of a base, such as 15 mM ammonium bicarbonate solution.

[0099] While the present invention illustrates preparation of one particular peptide linkage, other peptide linkages that are within the scope of the present invention can be readily prepared using procedures disclosed in, for example, commonly assigned U.S. Patent Application No. 15/121,270, filed August 24, 2016, now U.S. Patent No. 11,141,452, issued October 12, 2021. Furthermore, other peptides linkages that are within the scope of the present invention can readily be prepared by one skilled in the art having read the present disclosure along with the commonly assigned U.S. patent applications that are incorporated by reference herein.

[0100] Purification, Salt Form Conversion, and Final Product Characterization: Final products were purified by reverse phased HPLC and further characterized by analytical HPLC and mass spectroscopy. Peptides purified from reverse phased HPLC were usually in trifluoroacetic acid (TFA) form. This salt was typically converted to a more pharmaceutically friendly salt form, such as acetic acid or hydrochloric acid salt form. In some instance, the final product is preferred as an acetate salt, or a hydrochloride salt, or tartaric acid salt, or even a citric acid salt. Converting a peptide in TFA salt to a hydrochloric acid salt could be achieved by repeated lyophilization of the peptide in TFA salt in a dilute hydrochloric acid solution. For conversion of a peptide in TFA salt to an acetate salt, typically the following process was used. Strong anion exchange resin (chloride form, substitution 3 mmole/g, water content 50%, using 2 grams of resin per gram of peptide) was first washed three times with milli Q water, then three times with 1 N NaOH solution three times, 5 min/time, and then five times with milli Q water, 5 min/time. The resin was further washed with 75% ethanol water until the pH reaches about 7.4. This resin was treated with 10% acetic acid solution three times, five minutes each time. The resin was then washed with 1% acetic acid solution three times, five minutes each time. The resin was ready for the salt conversion of the purified peptide.

[0101] The purified, lyophilized peptide was dissolved in 1% acetic acid solution and added to the prepared resin described above. The mixture was agitated or magnetically stirred at room temperature for 1 h. The supernatant was separated. The resin was washed three times with 1% acetic acid solution. The supernatant and the washing solution were combined, filtered through a 0.22 pm membrane and lyophilized, to afford a peptide in acetate salt.

[0102] Example 1, Synthesis of MB301-01, Tyr-D-Ala-Gly-Phe-Met-Pro-Leu-Trp-NH- Bn(3',5'-(CF₃)2) (SEQ ID NO: 111)

[0103] The peptide chain was assembled by standard Fmoc chemistry using a 2-chloro- trityl resin (substitution capacity 0.4 mmol/gram). Briefly, 1 g of 2-chlorotrityl resin is swollen in 10 mL of DCM for ten minutes and then washed four times with dry DMF. Then add to the resin a solution of two equivalents of Fmoc-Trp(Boc)-OH dissolved in dry DMF and five equivalents of DIEA. The resin mixture is agitated at room temperature for an hour. Reaction solution is drained from the resin bed and the resin is washed with DMF twice. Then 10 mL of a mixture of DCM/MeOH/DIEA (80: 15:5, v/v) is added to the resin and the mixture is shaken at room temperature for ten minutes. The solution is drained from the resin and wash the resin three time with DMF. 10 mL of 25% piperidine in DMF is added to the resin and shake for 20 minutes at room temperature. Repeat the process once. The resin is washed well before coupling the next amino acid: 3 eq of Fmoc-Leu-OH is activated with HOBt/DIC in DMF, add 5 eq of DIEA. The coupling reaction is allowed to proceed for one to one and half hour and negative Ninhydrin test suggests reaction completion. This was followed by Fmoc removal using 10 mL of 25% piperidine in DMF for 20 min. The following residues were coupled sequentially using three equivalents of Fmoc protected amino acid residue: Fmoc-Pro-OH, Fmoc-Met-OH, Fmoc- Phe-OH, Fmoc-Gly-OH, Fmoc-D-Ala-OH, and Fmoc-Tyr(tBu)-OH.

[0104] After the coupling of last residue Fmoc-Tyr(tBu), the fully protected peptide acid is cleaved from the resin using 1% TFA in DCM for 1 h at room temperature. The cleavage process is repeated once and the solvents are removed by rotary evaporation and the fully protected crude peptide acid is lyophilized. 500 mg of the lyophilized crude peptide acid is activated with HOBt/DIC/DIEA in about 15 mL of dry DMF and coupled with 100 mg of 3,5- bis(trifluoromethyl) benzyl amine (MW 243.1 Da, 3 eq). The reaction is allowed to proceed for two hours at room temperature under magnetic stirring. Then 5 mL of piperidine is added to the reaction mixture and the reaction mixture is stirred for another 20 min to remove the Fmoc protection group. The crude reaction product is precipitated out with cold ether and lyophilized.

[0105] The crude lyophilized coupling product is fully deprotected using 10 mL of the cleavage cocktail: TFA:EDT:TIS:H2O:thioanisole:phenol (81.5:2.5:1 :5:5:5, v/v) at room temperature for Ih. Once the reaction is complete, the crude final product is precipitated out with t-butyl methyl ether. The final product is purified to homogeneity using a preparative HPLC and lyophilized to afford 181. 8 mg of Tyr-D-Ala-Gly-Phe-Met-Pro-Leu-Trp-NH-Bn(3',5'-(CF3)2) (SEQ ID NO: 111) at a HPLC purity of 99.10%, and an overall yield of 29.6%.

[0106] Example 2, Synthesis of MB301-02, N-methyl-Tyr-D-Ala-Gly-Phe-Met-Pro- Leu-Trp-NH-Bn(3',5'-(CF₃)2) (SEQ ID NO: 6)

[0107] The peptide chain was assembled by standard Fmoc chemistry using a 2-chloro- trityl resin (substitution capacity 0.4 mmol/gram). Briefly, 1 g of 2-chlorotrityl resin is swollen in 10 mL of DCM for ten minutes and then washed four times with dry DMF. Then add to the resin a solution of two equivalents of Fmoc-Trp(Boc)-OH dissolved in dry DMF and five equivalents of DIEA. The resin mixture is agitated at room temperature for an hour. Reaction solution is drained from the resin bed and the resin is washed with DMF twice. Then 10 mL of a mixture of DCM/MeOH/DIEA (80: 15:5, v/v) is added to the resin and the mixture is shaken at room temperature for ten minutes. The solution is drained from the resin and wash the resin three time with DMF. 10 mL of 25% piperidine in DMF is added to the resin and shake for 20 minutes at room temperature. Repeat the process once. The resin is washed well before coupling the next amino acid: 3 eq of Fmoc-Leu-OH is activated with HOBt/DIC in DMF, add 5 eq of DIEA. The coupling reaction is allowed to proceed for one to one and half hour and negative Ninhydrin test suggests reaction completion. This was followed by Fmoc removal using 10 mL of 25% piperidine in DMF for 20 min. The following residues were coupled sequentially using three equivalents of Fmoc protected amino acid residue: Fmoc-Pro-OH, Fmoc-Met-OH, Fmoc- Phe-OH, Fmoc-Gly-OH, Fmoc-D-Ala-OH, and Fmoc-(N-methyl)-Tyr(tBu)-OH.

[0108] After the coupling of last residue Fmoc-(N-methyl)-Tyr(tBu), the fully protected peptide acid is cleaved from the resin using 1% TFA in DCM for 1 h at room temperature. The cleavage process is repeated once and the solvents are removed by rotary evaporation and the fully protected crude peptide acid is lyophilized. 500 mg of the lyophilized crude peptide acid is activated with HOBt/DIC/DIEA in about 15 mL of dry DMF and coupled with 100 mg of 3,5- bis(trifluoromethyl) benzyl amine (MW 243.1 Da, 3 eq). The reaction is allowed to proceed for two hours at room temperature under magnetic stirring. Then 5 mL of piperidine is added to the reaction mixture and the reaction mixture is stirred for another 20 min to remove the Fmoc protection group. The crude reaction product is precipitated out with cold ether and lyophilized.

[0109] The crude lyophilized coupling product is fully deprotected using 10 mL of the cleavage cocktail: TFA:EDT:TIS:H2O:thioanisole:phenol (81.5:2.5:1 :5:5:5, v/v) at room temperature for Ih. Then the crude final product is precipitated out with t-butyl methyl ether for a few times. The final product is purified to homogeneity using a preparative HPLC and lyophilized to afford 22.4 mg of MB301-02, N-methyl-Tyr-D-Ala-Gly-Phe-Met-Pro-Leu-Trp- NH-Bn(3',5'-(CF3)2) (SEQ ID NO:6) at a HPLC purity of 97.38%, and an overall yield of 3.6%.

[0110] Example 3, Synthesis of MB301-03, Tyr-[Cys-Gly-Phe-Met-Pro-Cys]-Trp-NH- Bn(3',5'-(CF₃)2) (SEQ ID NO:19)

[0111] The peptide chain was assembled by standard Fmoc chemistry using a 2-chloro- trityl resin (substitution capacity 0.4 mmol/gram). Briefly, 1 g of 2-chlorotrityl resin is swollen in 10 mL of DCM for ten minutes and then washed four times with dry DMF. Then add to the resin a solution of two equivalents of Fmoc-Trp(Boc)-OH dissolved in dry DMF and five equivalents of DIEA. The resin mixture is agitated at room temperature for an hour. Reaction solution is drained from the resin bed and the resin is washed with DMF twice. Then 10 mL of a mixture of DCM/MeOH/DIEA (80: 15:5, v/v) is added to the resin and the mixture is shaken at room temperature for ten minutes. The solution is drained from the resin and wash the resin three time with DMF. 10 mL of 25% piperidine in DMF is added to the resin and shake for 20 minutes at room temperature. Repeat the process once. The resin is washed well before coupling the next amino acid: 3 eq of Fmoc-Cys(Trt)-OH is activated with HOBt/DIC in DMF, add 5 eq of DIEA. The coupling reaction is allowed to proceed for one to one and half hour and negative Ninhydrin test suggests reaction completion. This was followed by Fmoc removal using 10 mL of 25% piperidine in DMF for 20 min. The following residues were coupled sequentially using three equivalents of Fmoc protected amino acid residue: Fmoc-Pro-OH, Fmoc-Met-OH, Fmoc- Phe-OH, Fmoc-Gly-OH, Fmoc-Cys(Trt)-OH, and Fmoc-Tyr(tBu)-OH.

[0112] After the coupling of last residue Fmoc-Tyr(tBu), the fully protected peptide acid is cleaved from the resin using 1% TFA in DCM for 1 h at room temperature. The cleavage process is repeated once and the solvents are removed by rotary evaporation and the fully protected crude peptide acid is lyophilized. 500 mg of the lyophilized crude peptide acid is activated with HOBt/DIC/DIEA in about 15 mL of dry DMF and coupled with 100 mg of 3,5- bis(trifluoromethyl) benzyl amine (MW 243.1 Da, 3 eq). The reaction is allowed to proceed for two hours at room temperature under magnetic stirring. Then 5 mL of piperidine is added to the reaction mixture and the reaction mixture is stirred for another 20 min to remove the Fmoc protection group. The crude reaction product is precipitated out with cold ether and lyophilized.

[0113] The crude lyophilized coupling linear product is fully deprotected using 10 mL of the cleavage cocktail: TFA:EDT:TIS:H2O:thioanisole:phenol (81.5:2.5: 1:5:5:5, v/v) at room temperature for 1 h. Then the crude linear peptide is precipitated out with t-butyl methyl ether a few times and lyophilized.

[0114] The lyophilized crude linear peptide is then redissolved in 5% acetic acid solution of acetonitrile/water (10:90, v/v) at about 2 mg/mL. An iodine methanol solution (3.6 g of Iodine in 500 mL of methanol) is added to the peptide solution dropwise under constant stirring until the reaction solution becomes light yellow. A small amount of ascorbic acid solution (lOOmg/mL) is added to the reaction mixture to reduce/neutralize the extra iodine. The final cyclized peptide is purified to homogeneity using a preparative HPLC and lyophilized to afford 100.4 mg of BPT1089002-3, MB301-03, Tyr-[Cys-Gly-Phe-Met-Pro-Cys]-Trp-NH-Bn(3',5'- (CF₃)₂) (SEQ ID NO: 19) at a HPLC purity of 98.03%, and an overall yield of 16.4%. [0115] Example 4, Synthesis of MB301-04, Tyr-[Cys-Gly-Phe-Met-Cys]-Leu-Trp-NH- Bn(3',5'-(CF₃)2) (SEQ ID NO:20)

[0116] The peptide chain was assembled by standard Fmoc chemistry using a 2-chloro- trityl resin (substitution capacity 0.4 mmol/gram). Briefly, 1 g of 2-chlorotrityl resin is swollen in 10 mL of DCM for ten minutes and then washed four times with dry DMF. Then add to the resin a solution of two equivalents of Fmoc-Trp(Boc)-OH dissolved in dry DMF and five equivalents of DIEA. The resin mixture is agitated at room temperature for an hour. Reaction solution is drained from the resin bed and the resin is washed with DMF twice. Then 10 mL of a mixture of DCM/MeOH/DIEA (80: 15:5, v/v) is added to the resin and the mixture is shaken at room temperature for ten minutes. The solution is drained from the resin and wash the resin three time with DMF. 10 mL of 25% piperidine in DMF is added to the resin and shake for 20 minutes at room temperature. Repeat the process once. The resin is washed well before coupling the next amino acid: 3 eq of Fmoc-Leu-OH is activated with HOBt/DIC in DMF, add 5 eq of DIEA. The coupling reaction is allowed to proceed for one to one and half hour and negative Ninhydrin test suggests reaction completion. This was followed by Fmoc removal using 10 mL of 25% piperidine in DMF for 20 min. The following residues were coupled sequentially using three equivalents of Fmoc protected amino acid residue: Fmoc- Cys(Trt)-OH, Fmoc-Met-OH, Fmoc-Phe-OH, Fmoc-Gly-OH, Fmoc-Cys(Trt)-OH, and Fmoc-Tyr(tBu)-OH.

[0117] After the coupling of last residue Fmoc-Tyr(tBu), the fully protected peptide acid is cleaved from the resin using 1% TFA in DCM for 1 h at room temperature. The cleavage process is repeated once and the solvents are removed by rotary evaporation and the fully protected crude peptide acid is lyophilized. 500 mg of the lyophilized crude peptide acid is activated with HOBt/DIC/DIEA in about 15 mL of dry DMF and coupled with 100 mg of 3,5- bis(trifluoromethyl) benzyl amine (MW 243.1 Da, 3 eq). The reaction is allowed to proceed for two hours at room temperature under magnetic stirring. Then 5 mL of piperidine is added to the reaction mixture and the reaction mixture is stirred for another 20 min to remove the Fmoc protection group. The crude reaction product is precipitated out with cold ether and lyophilized.

[0118] The crude lyophilized coupling linear product is fully deprotected using 10 mL of the cleavage cocktail: TFA:EDT:TIS:H2O:thioanisole:phenol (81.5:2.5: 1:5:5:5, v/v) at room temperature for 1 h. Then the crude linear peptide is precipitated out with t-butyl methyl ether a few times and lyophilized.

[0119] The lyophilized crude linear peptide is then redissolved in 5% acetic acid solution of acetonitrile/water (10:90, v/v) at about 2 mg/mL. An iodine methanol solution (3.6 g of Iodine in 500 mL of methanol) is added to the peptide solution dropwise under constant stirring until the reaction solution becomes light yellow. A small amount of ascorbic acid solution (lOOmg/mL) is added to the reaction mixture to reduce/neutralize the extra iodine. The final cyclized peptide is purified to homogeneity using a preparative HPLC and lyophilized to afford 28.3 mg of BPT1089002-4, MB301-04, Tyr-[Cys-Gly-Phe-Met-Cys]-Leu-Trp-NH-Bn(3',5'- (CF₃)2) (SEQ ID NO:20) at a HPLC purity of 98.07%, and an overall yield of 4.6%.

[0120] Example 5, Synthesis of MB301-05, Tyr-[Cys-Gly-Phe-Met-Pro-hCys]-Trp-NH- Bn(3',5'-(CF₃)2) (SEQ ID NO:21)

[0121] The peptide chain was assembled by standard Fmoc chemistry using a 2-chloro- trityl resin (substitution capacity 0.4 mmol/gram). Briefly, 1 g of 2-chlorotrityl resin is swollen in 10 mL of DCM for ten minutes and then washed four times with dry DMF. Then add to the resin a solution of two equivalents of Fmoc-Trp(Boc)-OH dissolved in dry DMF and five equivalents of DIEA. The resin mixture is agitated at room temperature for an hour. Reaction solution is drained from the resin bed and the resin is washed with DMF twice. Then 10 mL of a mixture of DCM/MeOH/DIEA (80: 15:5, v/v) is added to the resin and the mixture is shaken at room temperature for ten minutes. The solution is drained from the resin and wash the resin three time with DMF. 10 mL of 25% piperidine in DMF is added to the resin and shake for 20 minutes at room temperature. Repeat the process once. The resin is washed well before coupling the next amino acid: 3 eq of Fmoc-hCys(Trt)-OH is activated with HOBt/DIC in DMF, add 5 eq of DIEA. The coupling reaction is allowed to proceed for one to one and half hour and negative Ninhydrin test suggests reaction completion. This was followed by Fmoc removal using 10 mL of 25% piperidine in DMF for 20 min. The following residues were coupled sequentially using three equivalents of Fmoc protected amino acid residue: Fmoc-Pro-OH, Fmoc-Met-OH, Fmoc- Phe-OH, Fmoc-Gly-OH, Fmoc-Cys(Trt)-OH, and Fmoc-Tyr(tBu)-OH.

[0122] After the coupling of last residue Fmoc-Tyr(tBu), the fully protected peptide acid is cleaved from the resin using 1% TFA in DCM for 1 h at room temperature. The cleavage process is repeated once and the solvents are removed by rotary evaporation and the fully protected crude peptide acid is lyophilized. 500 mg of the lyophilized crude peptide acid is activated with HOBt/DIC/DIEA in about 15 mL of dry DMF and coupled with 100 mg of 3,5- bis(trifluoromethyl) benzyl amine (MW 243.1 Da, 3 eq). The reaction is allowed to proceed for two hours at room temperature under magnetic stirring. Then 5 mL of piperidine is added to the reaction mixture and the reaction mixture is stirred for another 20 min to remove the Fmoc protection group. The crude reaction product is precipitated out with cold ether and lyophilized.

[0123] The crude lyophilized coupling linear product is fully deprotected using 10 mL of the cleavage cocktail: TFA:EDT:TIS:H2O:thioanisole:phenol (81.5:2.5: 1:5:5:5, v/v) at room temperature for 1 h. Then the crude linear peptide is precipitated out with t-butyl methyl ether a few times and lyophilized.

[0124] The lyophilized crude linear peptide is then redissolved in 5% acetic acid solution of acetonitrile/water (10:90, v/v) at about 2 mg/mL. An iodine methanol solution (3.6 g of Iodine in 500 mL of methanol) is added to the peptide solution dropwise under constant stirring until the reaction solution becomes light yellow. A small amount of ascorbic acid solution (lOOmg/mL) is added to the reaction mixture to reduce/neutralize the extra iodine. The final cyclized peptide is purified to homogeneity using a preparative HPLC and lyophilized to afford 24.70 mg of BPT1089002-5, MB301-05, Tyr-[Cys-Gly-Phe-Met-Pro-hCys]-Trp-NH-Bn(3',5'- (CF₃)₂) (SEQ ID NO:21) at a HPLC purity of 98.04%, and an overall yield of 4.0%.

[0125] Example 6, Synthesis of MB301-06, Tyr-[Cys-Gly-Phe-Met-hCys]-Leu-Trp- NH-Bn(3',5'-(CF₃)₂) (SEQ ID NO:22)

[0126] The peptide chain was assembled by standard Fmoc chemistry using a 2-chloro- trityl resin (substitution capacity 0.4 mmol/gram). Briefly, 1 g of 2-chlorotrityl resin is swollen in 10 mL of DCM for ten minutes and then washed four times with dry DMF. Then add to the resin a solution of two equivalents of Fmoc-Trp(Boc)-OH dissolved in dry DMF and five equivalents of DIEA. The resin mixture is agitated at room temperature for an hour. Reaction solution is drained from the resin bed and the resin is washed with DMF twice. Then 10 mL of a mixture of DCM/MeOH/DIEA (80: 15:5, v/v) is added to the resin and the mixture is shaken at room temperature for ten minutes. The solution is drained from the resin and wash the resin three time with DMF. 10 mL of 25% piperidine in DMF is added to the resin and shake for 20 minutes at room temperature. Repeat the process once. The resin is washed well before coupling the next amino acid: 3 eq of Fmoc-Leu-OH is activated with HOBt/DIC in DMF, add 5 eq of DIEA. The coupling reaction is allowed to proceed for one to one and half hour and negative Ninhydrin test suggests reaction completion. This was followed by Fmoc removal using 10 mL of 25% piperidine in DMF for 20 min. The following residues were coupled sequentially using three equivalents of Fmoc protected amino acid residue: Fmoc- hCys(Trt)-OH, Fmoc-Met-OH, Fmoc-Phe-OH, Fmoc-Gly-OH, Fmoc-Cys(Trt)-OH, and Fmoc-Tyr(tBu)-OH.

[0127] After the coupling of last residue Fmoc-Tyr(tBu), the fully protected peptide acid is cleaved from the resin using 1% TFA in DCM for 1 h at room temperature. The cleavage process is repeated once and the solvents are removed by rotary evaporation and the fully protected crude peptide acid is lyophilized. 500 mg of the lyophilized crude peptide acid is activated with HOBt/DIC/DIEA in about 15 mL of dry DMF and coupled with 100 mg of 3,5- bis(trifluoromethyl) benzyl amine (MW 243.1 Da, 3 eq). The reaction is allowed to proceed for two hours at room temperature under magnetic stirring. Then 5 mL of piperidine is added to the reaction mixture and the reaction mixture is stirred for another 20 min to remove the Fmoc protection group. The crude reaction product is precipitated out with cold ether and lyophilized.

[0128] The crude lyophilized coupling linear product is fully deprotected using 10 mL of the cleavage cocktail: TFA:EDT:TIS:H2O:thioanisole:phenol (81.5:2.5: 1:5:5:5, v/v) at room temperature for 1 h. Then the crude linear peptide is precipitated out with t-butyl methyl ether a few times and lyophilized.

[0129] The lyophilized crude linear peptide is then redissolved in 5% acetic acid solution of acetonitrile/water (10:90, v/v) at about 2 mg/mL. An iodine methanol solution (3.6 g of Iodine in 500 mL of methanol) is added to the peptide solution dropwise under constant stirring until the reaction solution becomes light yellow. A small amount of ascorbic acid solution (lOOmg/mL) is added to the reaction mixture to reduce/neutralize the extra iodine. The final cyclized peptide is purified to homogeneity using a preparative HPLC and lyophilized to afford 44.0 mg of BPT1089002-6, MB301-06, Tyr-[Cys-Gly-Phe-Met-hCys]-Leu-Trp-NH-Bn(3',5'- (CF₃)₂) (SEQ ID NO:22) at a HPLC purity of 98.69%, and an overall yield of 7.2%.

[0130] Example 7, Synthesis of MB301-07, Tyr-[Cys-Gly-Phe-Nle-Pro-Cys]-Trp-NH- Bn(3',5'-(CF₃)2) (SEQ ID NO:35) [0131] The peptide chain was assembled by standard Fmoc chemistry using a 2-chloro- trityl resin (substitution capacity 0.4 mmol/gram). Briefly, 1 g of 2-chlorotrityl resin is swollen in 10 mL of DCM for ten minutes and then washed four times with dry DMF. Then add to the resin a solution of two equivalents of Fmoc-Trp(Boc)-OH dissolved in dry DMF and five equivalents of DIEA. The resin mixture is agitated at room temperature for an hour. Reaction solution is drained from the resin bed and the resin is washed with DMF twice. Then 10 mL of a mixture of DCM/MeOH/DIEA (80: 15:5, v/v) is added to the resin and the mixture is shaken at room temperature for ten minutes. The solution is drained from the resin and wash the resin three time with DMF. 10 mL of 25% piperidine in DMF is added to the resin and shake for 20 minutes at room temperature. Repeat the process once. The resin is washed well before coupling the next amino acid: 3 eq of Fmoc-Cys(Trt)-OH is activated with HOBt/DIC in DMF, add 5 eq of DIEA. The coupling reaction is allowed to proceed for one to one and half hour and negative Ninhydrin test suggests reaction completion. This was followed by Fmoc removal using 10 mL of 25% piperidine in DMF for 20 min. The following residues were coupled sequentially using three equivalents of Fmoc protected amino acid residue: Fmoc-Pro-OH, Fmoc-Nle-OH, Fmoc- Phe-OH, Fmoc-Gly-OH, Fmoc-Cys(Trt)-OH, and Fmoc-Tyr(tBu)-OH.

[0132] After the coupling of last residue Fmoc-Tyr(tBu), the fully protected peptide acid is cleaved from the resin using 1% TFA in DCM for 1 h at room temperature. The cleavage process is repeated once and the solvents are removed by rotary evaporation and the fully protected crude peptide acid is lyophilized. 500 mg of the lyophilized crude peptide acid is activated with HOBt/DIC/DIEA in about 15 mL of dry DMF and coupled with 100 mg of 3,5- bis(trifluoromethyl) benzyl amine (MW 243.1 Da, 3 eq). The reaction is allowed to proceed for two hours at room temperature under magnetic stirring. Then 5 mL of piperidine is added to the reaction mixture and the reaction mixture is stirred for another 20 min to remove the Fmoc protection group. The crude reaction product is precipitated out with cold ether and lyophilized.

[0133] The crude lyophilized coupling linear product is fully deprotected using 10 mL of the cleavage cocktail: TFA:EDT:TIS:H2O:thioanisole:phenol (81.5:2.5: 1:5:5:5, v/v) at room temperature for 1 h. Then the crude linear peptide is precipitated out with t-butyl methyl ether a few times and lyophilized. [0134] The lyophilized crude linear peptide is then redissolved in 5% acetic acid solution of acetonitrile/water (10:90, v/v) at about 2 mg/mL. An iodine methanol solution (3.6 g of Iodine in 500 mL of methanol) is added to the peptide solution dropwise under constant stirring until the reaction solution becomes light yellow. A small amount of ascorbic acid solution (lOOmg/mL) is added to the reaction mixture to reduce/neutralize the extra iodine. The final cyclized peptide is purified to homogeneity using a preparative HPLC and lyophilized to afford 23.7 mg of BPT1089002-7, MB301-07, Tyr-[Cys-Gly-Phe-Nle-Pro-Cys]-Trp-NH-Bn(3',5'- (CF₃)2) (SEQ ID NO:35) at a HPLC purity of 95.15%, and an overall yield of 3.8%.

[0135] Example 8, Synthesis of MB301-04, Tyr-[Cys-Gly-Phe-Nle-Cys]-Leu-Trp-NH- Bn(3',5'-(CF₃)₂) (SEQ ID NO: 36)

[0136] The peptide chain was assembled by standard Fmoc chemistry using a 2-chloro- trityl resin (substitution capacity 0.4 mmol/gram). Briefly, 1 g of 2-chlorotrityl resin is swollen in 10 mL of DCM for ten minutes and then washed four times with dry DMF. Then add to the resin a solution of two equivalents of Fmoc-Trp(Boc)-OH dissolved in dry DMF and five equivalents of DIEA. The resin mixture is agitated at room temperature for an hour. Reaction solution is drained from the resin bed and the resin is washed with DMF twice. Then 10 mL of a mixture of DCM/MeOH/DIEA (80: 15:5, v/v) is added to the resin and the mixture is shaken at room temperature for ten minutes. The solution is drained from the resin and wash the resin three time with DMF. 10 mL of 25% piperidine in DMF is added to the resin and shake for 20 minutes at room temperature. Repeat the process once. The resin is washed well before coupling the next amino acid: 3 eq of Fmoc-Leu-OH is activated with HOBt/DIC in DMF, add 5 eq of DIEA. The coupling reaction is allowed to proceed for one to one and half hour and negative Ninhydrin test suggests reaction completion. This was followed by Fmoc removal using 10 mL of 25% piperidine in DMF for 20 min. The following residues were coupled sequentially using three equivalents of Fmoc protected amino acid residue: Fmoc- Cys(Trt)-OH, Fmoc-Nle-OH, Fmoc-Phe-OH, Fmoc-Gly-OH, Fmoc-Cys(Trt)-OH, and Fmoc-Tyr(tBu)-OH.

[0137] After the coupling of last residue Fmoc-Tyr(tBu), the fully protected peptide acid is cleaved from the resin using 1% TFA in DCM for 1 h at room temperature. The cleavage process is repeated once and the solvents are removed by rotary evaporation and the fully protected crude peptide acid is lyophilized. 500 mg of the lyophilized crude peptide acid is activated with HOBt/DIC/DIEA in about 15 mL of dry DMF and coupled with 100 mg of 3,5- bis(trifluoromethyl) benzyl amine (MW 243.1 Da, 3 eq). The reaction is allowed to proceed for two hours at room temperature under magnetic stirring. Then 5 mL of piperidine is added to the reaction mixture and the reaction mixture is stirred for another 20 min to remove the Fmoc protection group. The crude reaction product is precipitated out with cold ether and lyophilized.

[0138] The crude lyophilized coupling linear product is fully deprotected using 10 mL of the cleavage cocktail: TFA:EDT:TIS:H2O:thioanisole:phenol (81.5:2.5: 1:5:5:5, v/v) at room temperature for 1 h. Then the crude linear peptide is precipitated out with t-butyl methyl ether a few times and lyophilized.

[0139] The lyophilized crude linear peptide is then redissolved in 5% acetic acid solution of acetonitrile/water (10:90, v/v) at about 2 mg/mL. An iodine methanol solution (3.6 g of Iodine in 500 mL of methanol) is added to the peptide solution dropwise under constant stirring until the reaction solution becomes light yellow. A small amount of ascorbic acid solution (lOOmg/mL) is added to the reaction mixture to reduce/neutralize the extra iodine. The final cyclized peptide is purified to homogeneity using a preparative HPLC and lyophilized to afford 18.3 mg of BPT1089002-4, MB301-04, Tyr-[Cys-Gly-Phe-Nle-Cys]-Leu-Trp-NH-Bn(3',5'- (CF₃)₂) (SEQ ID NO:36) at a HPLC purity of 95.02%, and an overall yield of 3.0%.

[0140] Example 9, Synthesis of MB301-09, Tyr-[hCys-Gly-Phe-Nle-Pro-Cys]-Trp-NH- Bn(3',5'-(CF₃)₂) (SEQ ID NO:39)

[0141] The peptide chain was assembled by standard Fmoc chemistry using a 2-chloro- trityl resin (substitution capacity 0.4 mmol/gram). Briefly, 1 g of 2-chlorotrityl resin is swollen in 10 mL of DCM for ten minutes and then washed four times with dry DMF. Then add to the resin a solution of two equivalents of Fmoc-Trp(Boc)-OH dissolved in dry DMF and five equivalents of DIEA. The resin mixture is agitated at room temperature for an hour. Reaction solution is drained from the resin bed and the resin is washed with DMF twice. Then 10 mL of a mixture of DCM/MeOH/DIEA (80: 15:5, v/v) is added to the resin and the mixture is shaken at room temperature for ten minutes. The solution is drained from the resin and wash the resin three time with DMF. 10 mL of 25% piperidine in DMF is added to the resin and shake for 20 minutes at room temperature. Repeat the process once. The resin is washed well before coupling the next amino acid: 3 eq of Fmoc-Cys(Trt)-OH is activated with HOBt/DIC in DMF, add 5 eq of DIEA. The coupling reaction is allowed to proceed for one to one and half hour and negative Ninhydrin test suggests reaction completion. This was followed by Fmoc removal using 10 mL of 25% piperidine in DMF for 20 min. The following residues were coupled sequentially using three equivalents of Fmoc protected amino acid residue: Fmoc-Pro-OH, Fmoc-Nle-OH, Fmoc- Phe-OH, Fmoc-Gly-OH, Fmoc-hCys(Trt)-OH, and Fmoc-Tyr(tBu)-OH.

[0142] After the coupling of last residue Fmoc-Tyr(tBu), the fully protected peptide acid is cleaved from the resin using 1% TFA in DCM for 1 h at room temperature. The cleavage process is repeated once and the solvents are removed by rotary evaporation and the fully protected crude peptide acid is lyophilized. 500 mg of the lyophilized crude peptide acid is activated with HOBt/DIC/DIEA in about 15 mL of dry DMF and coupled with 100 mg of 3,5- bis(trifluoromethyl) benzyl amine (MW 243.1 Da, 3 eq). The reaction is allowed to proceed for two hours at room temperature under magnetic stirring. Then 5 mL of piperidine is added to the reaction mixture and the reaction mixture is stirred for another 20 min to remove the Fmoc protection group. The crude reaction product is precipitated out with cold ether and lyophilized.

[0143] The crude lyophilized coupling linear product is fully deprotected using 10 mL of the cleavage cocktail: TFA:EDT:TIS:H2O:thioanisole:phenol (81.5:2.5: 1:5:5:5, v/v) at room temperature for 1 h. Then the crude linear peptide is precipitated out with t-butyl methyl ether a few times and lyophilized.

[0144] The lyophilized crude linear peptide is then redissolved in 5% acetic acid solution of acetonitrile/water (10:90, v/v) at about 2 mg/mL. An iodine methanol solution (3.6 g of Iodine in 500 mL of methanol) is added to the peptide solution dropwise under constant stirring until the reaction solution becomes light yellow. A small amount of ascorbic acid solution (lOOmg/mL) is added to the reaction mixture to reduce/neutralize the extra iodine. The final cyclized peptide is purified to homogeneity using a preparative HPLC and lyophilized to afford 34.9 mg of BPT1089002-9, MB301-09, Tyr-[hCys-Gly-Phe-Nle-Pro-Cys]-Trp-NH-Bn(3',5'- (CF₃)₂) (SEQ ID NO:39) at a HPLC purity of 97.77%, and an overall yield of 5.7%.

[0145] Example 10, Synthesis of MB301-04, Tyr-[hCys-Gly-Phe-Nle-Cys]-Leu-Trp- NH-Bn(3',5'-(CF₃)₂) (SEQ ID NO:40) [0146] The peptide chain was assembled by standard Fmoc chemistry using a 2-chloro- trityl resin (substitution capacity 0.4 mmol/gram). Briefly, 1 g of 2-chlorotrityl resin is swollen in 10 mL of DCM for ten minutes and then washed four times with dry DMF. Then add to the resin a solution of two equivalents of Fmoc-Trp(Boc)-OH dissolved in dry DMF and five equivalents of DIEA. The resin mixture is agitated at room temperature for an hour. Reaction solution is drained from the resin bed and the resin is washed with DMF twice. Then 10 mL of a mixture of DCM/MeOH/DIEA (80: 15:5, v/v) is added to the resin and the mixture is shaken at room temperature for ten minutes. The solution is drained from the resin and wash the resin three time with DMF. 10 mL of 25% piperidine in DMF is added to the resin and shake for 20 minutes at room temperature. Repeat the process once. The resin is washed well before coupling the next amino acid: 3 eq of Fmoc-Leu-OH is activated with HOBt/DIC in DMF, add 5 eq of DIEA. The coupling reaction is allowed to proceed for one to one and half hour and negative Ninhydrin test suggests reaction completion. This was followed by Fmoc removal using 10 mL of 25% piperidine in DMF for 20 min. The following residues were coupled sequentially using three equivalents of Fmoc protected amino acid residue: Fmoc-hCys(Trt)-OH, Fmoc-Nle-OH, Fmoc-Phe-OH, Fmoc-Gly-OH, Fmoc-Cys(Trt)-OH, and Fmoc-Tyr(tBu)-OH.

[0147] After the coupling of last residue Fmoc-Tyr(tBu), the fully protected peptide acid is cleaved from the resin using 1% TFA in DCM for 1 h at room temperature. The cleavage process is repeated once and the solvents are removed by rotary evaporation and the fully protected crude peptide acid is lyophilized. 500 mg of the lyophilized crude peptide acid is activated with HOBt/DIC/DIEA in about 15 mL of dry DMF and coupled with 100 mg of 3,5- bis(trifluoromethyl) benzyl amine (MW 243.1 Da, 3 eq). The reaction is allowed to proceed for two hours at room temperature under magnetic stirring. Then 5 mL of piperidine is added to the reaction mixture and the reaction mixture is stirred for another 20 min to remove the Fmoc protection group. The crude reaction product is precipitated out with cold ether and lyophilized.

[0148] The crude lyophilized coupling linear product is fully deprotected using 10 mL of the cleavage cocktail: TFA:EDT:TIS:H2O:thioanisole:phenol (81.5:2.5: 1:5:5:5, v/v) at room temperature for 1 h. Then the crude linear peptide is precipitated out with t-butyl methyl ether a few times and lyophilized. [0149] The lyophilized crude linear peptide is then redissolved in 5% acetic acid solution of acetonitrile/water (10:90, v/v) at about 2 mg/mL. An iodine methanol solution (3.6 g of Iodine in 500 mL of methanol) is added to the peptide solution dropwise under constant stirring until the reaction solution becomes light yellow. A small amount of ascorbic acid solution (lOOmg/mL) is added to the reaction mixture to reduce/neutralize the extra iodine. The final cyclized peptide is purified to homogeneity using a preparative HPLC and lyophilized to afford 31.5 mg of BPT1089002-10, MB301-10, Tyr-[hCys-Gly-Phe-Nle-Cys]-Leu-Trp-NH-Bn(3',5'- (CF₃)2) (SEQ ID NO:40) at a HPLC purity of 99.47%, and an overall yield of 5.1%.

[0150] Example 11, Synthesis of MB301-11, (2’,6’-dimethyl)-Tyr-D-Ala-Gly-Phe- Met-Pro-Leu-Trp-NH-Bn(3',5'-(CF₃)₂) (SEQ ID NO: 104)

[0151] The peptide chain was assembled by standard Fmoc chemistry using a 2-chloro- trityl resin (substitution capacity 0.4 mmol/gram). Briefly, 1 g of 2-chlorotrityl resin is swollen in 10 mL of DCM for ten minutes and then washed four times with dry DMF. Then add to the resin a solution of two equivalents of Fmoc-Trp(Boc)-OH dissolved in dry DMF and five equivalents of DIEA. The resin mixture is agitated at room temperature for an hour. Reaction solution is drained from the resin bed and the resin is washed with DMF twice. Then 10 mL of a mixture of DCM/MeOH/DIEA (80: 15:5, v/v) is added to the resin and the mixture is shaken at room temperature for ten minutes. The solution is drained from the resin and wash the resin three time with DMF. 10 mL of 25% piperidine in DMF is added to the resin and shake for 20 minutes at room temperature. Repeat the process once.

[0152] The resin is washed well before coupling the next amino acid: 3 eq of Fmoc-Leu- OH is activated with HOBt/DIC in DMF, add 5 eq of DIEA. The coupling reaction is allowed to proceed for one to one and half hour and negative Ninhydrin test suggests reaction completion. This was followed by Fmoc removal using 10 mL of 25% piperidine in DMF for 20 min. The following residues were coupled sequentially using three equivalents of Fmoc protected amino acid residue: Fmoc-Pro-OH, Fmoc-Met-OH, Fmoc-Phe-OH, Fmoc-Gly-OH, Fmoc-D-Ala-OH, and Fmoc-(2’,6’-dimethyl)-Tyr(tBu)-OH.

[0153] After the coupling of last residue Fmoc-(2’,6’-dimethyl)-Tyr(tBu), the fully protected peptide acid is cleaved from the resin using 1% TFA in DCM for 1 h at room temperature. The cleavage process is repeated once and the solvents are removed by rotary evaporation and the fully protected crude peptide acid is lyophilized. 500 mg of the lyophilized crude peptide acid is activated with HOBt/DIC/DIEA in about 15 mL of dry DMF and coupled with 100 mg of 3,5-bis(trifluoromethyl) benzyl amine (MW 243.1 Da, 3 eq). The reaction is allowed to proceed for two hours at room temperature under magnetic stirring. Then 5 mL of piperidine is added to the reaction mixture and the reaction mixture is stirred for another 20 min to remove the Fmoc protection group. The crude reaction product is precipitated out with cold ether and lyophilized.

[0154] The crude lyophilized coupling product is fully deprotected using 10 mL of the cleavage cocktail: TFA:EDT:HS:H2O:thioanisole:phenol (81.5:2.5:1 :5:5:5, v/v) at room temperature for Ih. Then the crude final product is precipitated out with t-butyl methyl ether for a few times. The final product is purified to homogeneity using a preparative HPLC and lyophilized to afford 110.4 mg of MB301-11, 2’,6’-dimethyl-Tyr-D-Ala-Gly-Phe-Met-Pro-Leu- Trp-NH-Bn(3',5'-(CF3)2) at a HPLC purity of 98.94%, and an overall yield of 18.1%.

[0155] Example 12, Synthesis of MB301-12, (3’,5’-dimethyl)-Tyr-D-Ala-Gly-Phe- Met-Pro-Leu-Trp-NH-Bn(3',5'-(CF₃)2) (SEQ ID NO: 103)

[0156] The peptide chain was assembled by standard Fmoc chemistry using a 2-chloro- trityl resin (substitution capacity 0.4 mmol/gram). Briefly, 1 g of 2-chlorotrityl resin is swollen in 10 mL of DCM for ten minutes and then washed four times with dry DMF. Then add to the resin a solution of two equivalents of Fmoc-Trp(Boc)-OH dissolved in dry DMF and five equivalents of DIEA. The resin mixture is agitated at room temperature for an hour. Reaction solution is drained from the resin bed and the resin is washed with DMF twice. Then 10 mL of a mixture of DCM/MeOH/DIEA (80: 15:5, v/v) is added to the resin and the mixture is shaken at room temperature for ten minutes. The solution is drained from the resin and wash the resin three time with DMF. 10 mL of 25% piperidine in DMF is added to the resin and shake for 20 minutes at room temperature. Repeat the process once.

[0157] The resin is washed well before coupling the next amino acid: 3 eq of Fmoc-Leu- OH is activated with HOBt/DIC in DMF, add 5 eq of DIEA. The coupling reaction is allowed to proceed for one to one and half hour and negative Ninhydrin test suggests reaction completion. This was followed by Fmoc removal using 10 mL of 25% piperidine in DMF for 20 min. The following residues were coupled sequentially using three equivalents of Fmoc protected amino acid residue: Fmoc-Pro-OH, Fmoc-Met-OH, Fmoc-Phe-OH, Fmoc-Gly-OH, Fmoc-D-Ala-OH, and Fmoc-(3’,5’-dimethyl)-Tyr(tBu)-OH.

[0158] After the coupling of last residue Fmoc-(2’,6’-dimethyl)-Tyr(tBu), the fully protected peptide acid is cleaved from the resin using 1% TFA in DCM for 1 h at room temperature. The cleavage process is repeated once and the solvents are removed by rotary evaporation and the fully protected crude peptide acid is lyophilized. 500 mg of the lyophilized crude peptide acid is activated with HOBt/DIC/DIEA in about 15 mL of dry DMF and coupled with 100 mg of 3,5-bis(trifluoromethyl) benzyl amine (MW 243.1 Da, 3 eq). The reaction is allowed to proceed for two hours at room temperature under magnetic stirring. Then 5 mL of piperidine is added to the reaction mixture and the reaction mixture is stirred for another 20 min to remove the Fmoc protection group. The crude reaction product is precipitated out with cold ether and lyophilized.

[0159] The crude lyophilized coupling product is fully deprotected using 10 mL of the cleavage cocktail: TFA:EDT:TIS:H2O:thioanisole:phenol (81.5:2.5:1 :5:5:5, v/v) at room temperature for Ih. Then the crude final product is precipitated out with t-butyl methyl ether for a few times. The final product is purified to homogeneity using a preparative HPLC and lyophilized to afford 75.2 mg ofMB301-12, (3’,5’-dimethyl)-Tyr-D-Ala-Gly-Phe-Met-Pro-Leu- Trp-NH-Bn(3',5'-(CF3)2) (SEQ ID NO: 103) at a HPLC purity of 98.94%, and an overall yield of 12.3%.

[0160] Example 13, Synthesis of MB301-13, (3’,5’-difluoro-Tyr)-D-Ala-Gly-Phe-Met- Pro-Leu-Trp-NH-Bn(3',5'-(CF₃)2) (SEQ ID NO: 105)

[0161] The peptide chain was assembled by standard Fmoc chemistry using a 2-chloro- trityl resin (substitution capacity 0.4 mmol/gram). Briefly, 1 g of 2-chlorotrityl resin is swollen in 10 mL of DCM for ten minutes and then washed four times with dry DMF. Then add to the resin a solution of two equivalents of Fmoc-Trp(Boc)-OH dissolved in dry DMF and five equivalents of DIEA. The resin mixture is agitated at room temperature for an hour. Reaction solution is drained from the resin bed and the resin is washed with DMF twice. Then 10 mL of a mixture of DCM/MeOH/DIEA (80: 15:5, v/v) is added to the resin and the mixture is shaken at room temperature for ten minutes. The solution is drained from the resin and wash the resin three time with DMF. 10 mL of 25% piperidine in DMF is added to the resin and shake for 20 minutes at room temperature. Repeat the process once.

[0162] The resin is washed well before coupling the next amino acid: 3 eq of Fmoc-Leu- OH is activated with HOBt/DIC in DMF, add 5 eq of DIEA. The coupling reaction is allowed to proceed for one to one and half hour and negative Ninhydrin test suggests reaction completion. This was followed by Fmoc removal using 10 mL of 25% piperidine in DMF for 20 min. The following residues were coupled sequentially using three equivalents of Fmoc protected amino acid residue: Fmoc-Pro-OH, Fmoc-Met-OH, Fmoc-Phe-OH, Fmoc-Gly-OH, Fmoc-D-Ala-OH, and Fmoc-(3 ’ , 5 ’ -difluoro)-Tyr(tBu)-OH.

[0163] After the coupling of last residue Fmoc-(3’,5’-difluoro)-Tyr(tBu), the fully protected peptide acid is cleaved from the resin using 1% TFA in DCM for 1 h at room temperature. The cleavage process is repeated once and the solvents are removed by rotary evaporation and the fully protected crude peptide acid is lyophilized. 500 mg of the lyophilized crude peptide acid is activated with HOBt/DIC/DIEA in about 15 mL of dry DMF and coupled with 100 mg of 3,5-bis(trifluoromethyl) benzyl amine (MW 243.1 Da, 3 eq). The reaction is allowed to proceed for two hours at room temperature under magnetic stirring. Then 5 mL of piperidine is added to the reaction mixture and the reaction mixture is stirred for another 20 min to remove the Fmoc protection group. The crude reaction product is precipitated out with cold ether and lyophilized.

[0164] The crude lyophilized coupling product is fully deprotected using 10 mL of the cleavage cocktail: TFA:EDT:TIS:H2O:thioanisole:phenol (81.5:2.5:1 :5:5:5, v/v) at room temperature for Ih. Then the crude final product is precipitated out with t-butyl methyl ether for a few times. The final product is purified to homogeneity using a preparative HPLC and lyophilized to afford 54.8 mg ofMB301-13, (3’,5’-difluoro-Tyr)-D-Ala-Gly-Phe-Met-Pro-Leu- Trp-NH-Bn(3',5'-(CF3)2) (SEQ ID NO: 105) at a HPLC purity of 98.95%, and an overall yield of 9.0%.

[0165] BioAnalysis and expression of results — Cellular and Nuclear Receptor Functional Assays [0166] In addition to those cited references, the following procedure is used for assay. The results are expressed as a percent of control agonist response or inverse agonist response: measured response/control response *100 and as a percent inhibition of control agonist response:

100 - (measured response/control response *100) obtained in the presence of the test compounds.

[0167] The EC50 values (concentration producing a half-maximal response) and IC50 values (concentration causing a half-maximal inhibition of the control agonist response) were determined by non-linear regression analysis of the concentration-response curves generated with mean replicate values using Hill equation curve fitting:

Y = D + [A-D] / [l+(C7C₅o)^l,H] where Y = response, A = left asymptote of the curve, D = right asymptote of the curve, C = compound concentration, and Cso= ECso or IC50, and nH = slope factor.

[0168] This analysis was performed using software developed at Cerep (Hill software) and validated by comparison with data generated by the commercial software SigmaPlot ® 4.0 for Windows ® (© 1997 by SPSS Inc.).

[0169] For the antagonists, the apparent dissociation constants (KB) were calculated using the modified Cheng Prusoff equation:

KB = IC5O/(1+(A/ECSOA)) where A = concentration of reference agonist in the assay, and ECSOA = EC50 value of the reference agonist (Wang, J.B. et al., FEBS Lett., 1994, 338, pp. 217-222; Eistetter, H.R. et al. Glia, 1992, 6, pp 89-95).

[0170] Some of the representative biological assay data are shown in FIGS. 1-7 and also on Table 3. In particular, FIGS. 1-7 are histograms for p (MOP) (h) (agonist effect) of some of the representative peptides of the invention, and Table 3 shows NK1 antagonist activity and opioid m MOP agonistic activity of some of the representative peptides of the invention.

Table 3. Biological activities of exemplified compounds: NK1 antagonist activity and Opioid m MOP agonistic activity

[0171] The foregoing discussion of the invention has been presented for purposes of illustration and description. The foregoing is not intended to limit the invention to the form or forms disclosed herein. Although the description of the invention has included description of one or more embodiments and certain variations and modifications, other variations and modifications are within the scope of the invention, e.g., as may be within the skill and knowledge of those in the art, after understanding the present disclosure. It is intended to obtain rights which include alternative embodiments to the extent permitted, including alternate, interchangeable and/or equivalent structures, functions, ranges or steps to those claimed, whether or not such alternate, interchangeable and/or equivalent structures, functions, ranges or steps are disclosed herein, and without intending to publicly dedicate any patentable subject matter. All references cited herein are incorporated by reference in their entirety.

Claims

What is Claimed is:

1. A peptide of the formula:

X¹-X²-Gly-X⁴-X⁵-X⁶-X⁷-Trp-L-R (SEQ ID NO : 1 )

Formula I or a pharmaceutically acceptable salt thereof, wherein

X² is Cys, Ala, hCys, or Pen;

X⁴ is Phe or substituted Phe;

X⁵ is Phe, Leu, Met, Met(O), Gly, Nle, Ser, or substituted Ser;

X⁶ is Pro, Ala, Leu, Cys, hCys, or Pen;

X⁷ is Cys, hCys, Pen, Ser, Leu, Ala, Vai, or Aib;

L is O, NH, or NMe; and

2. The peptide according to claim 1, wherein X² is cys, hCys, or Pen.

3. The peptide according to claim 2, wherein one of X⁶ or X⁷ is Cys, hCys, or Pen.

4. The peptide according to claim 3, wherein said peptide of SEQ ID NO: 1 is a cyclic peptide of the formula:

Formula II Formula III

5. The peptide according to claim 1, wherein X⁶ is L-Pro, L-Ala, L-Leu, L-Cys, D- Cys, hCys, D-hCys, L-Pen, or D-Pen.

6. The peptide according to claim 1, wherein X⁷ is L-Cys, D-Cys, L-hCys, D-hCys, L-Pen, D-Pen, L-Ser, L-Leu, L-Ala, D-Ala, L-Val, D-Val, or L-Aib;

7. The peptide according to claim 1, wherein X¹ is selected from the group consisting of (3’,5’-dimethyl-Tyr), (2’,6’-dimethyl-Tyr), (3’,5’-difluoro-Tyr), (3’,5’-dichloro- Tyr), (3’,5’-dibromo-Tyr), (2’,6’-difluoro-Tyr), (2’,6’-dichloro-Tyr), and (2’,6’-dibromo-Tyr).

8. The peptide according to claim 1, wherein X⁴ is Phe.

9. The peptide according to claim 1, wherein X⁵ is Met, Met(O), or Nle.

10. The peptide according to claim 1, wherein X⁶ is a (D)-isomer.

11. The peptide according to claim 1, wherein X⁷ is a (D)-isomer.

12. The peptide according to claim 1, wherein R is selected from the group consisting of mono- or di-substituted haloalkylbenzyl, mono- or di-substituted halobenzyl, and mono- or disubstituted alkylbenzyl.

13. The peptide according to claim 12, wherein R is selected from the group consisting of benzyl having one or two substituents, wherein each substituent is independently selected from the group consisting of trifluoroalkyl, chloro, fluoro, bromo, and methyl.

14. The peptide according to claim 13, wherein R is selected from the group consisting of 3,5-ditrifluoromethylbenzyl; 3,5-dimethylbenzyl; 2,4-trifluoromethylbenzyl; 3- trifluoromethyl-benzyl; 4-trifluoromethylbenzyl; 5-trifluoromethylbenzyl; 6- trifluoromethylbenzyl; 3, 5 -difluorobenzyl; 3,5-dichlorobenzyl; 3, 5 -dibromobenzyl; 2,4- diflurobenzyl; 2,4-dichlorobenzyl; 2,4-dibromobenzyl; 2,4-dimethylbenzyl; and 3,5- dimethylbenzyl.

15. The peptide according to claim 1, wherein said peptide of SEQ ID NO: 1 is a non- cyclic peptide of the formula:

X¹-X²-Gly-X⁴-X⁵-Pro-Leu-Trp-L-R (SEQ ID NO: 4)

Formula IV

16. The peptide according to claim 15, wherein X² is (D)-Ala.

17. The peptide according to claim 1, wherein said peptide is selected from the group consisting of SEQ ID NOS: 5-110.

SUBSTITUTE SHEET (RULE 26)

18. The peptide according to claim 17, wherein said peptide is selected from the group consisting of: (3’,5’-dimethyl-Tyr)-[Cys-Gly-Phe-Met-Cys]-Leu-Trp-NH-Bn(3',5'-(CF₃)₂) (SEQ ID NO: 112); (3’,5’-dimethyl-Tyr)-[Cys-Gly-Phe-Met-Pro-hCys]-Trp-NH-Bn(3',5'-(CF₃)₂) (SEQ ID NO: 113); (2’,6’-dimethyl-Tyr)-[Cys-Gly-Phe-Met-Cys]-Leu-Trp-NH-Bn(3',5'-(CF₃)₂) (SEQ ID NO: 114); (2’,6’-dimethyl-Tyr)-[Cys-Gly-Phe-Met-Pro-hCys]-Trp-NH-Bn(3',5'-(CF₃)₂) (SEQ ID NO: 115); (N-methyl-Tyr)-D-Ala-Gly-Phe-Met-Pro-Leu-Trp-NH-Bn(3',5'-(CF₃)₂) (SEQ ID NO:6); (2’,6’-dimethyl-Tyr)-D-Ala-Gly-Phe-Met-Pro-Leu-Trp-NH-Bn(3',5'-(CF₃)₂) (SEQ ID NO: 104); (3’,5’-dimethyl-Tyr)-D-Ala-Gly-Phe-Met-Pro-Leu-Trp-NH-Bn(3',5'-(CF₃)₂) (SEQ ID NO: 103); (3’,5’-difluoro-Tyr)-D-Ala-Gly-Phe-Met-Pro-Leu-Trp-NH-Bn(3',5'-(CF₃)₂) (SEQ ID NO:105); Tyr-[Cys-Gly-Phe-Met-Pro-Cys]-Trp-NH-Bn(3',5'-(CF₃)₂) (SEQ ID NO: 19); Tyr-[Cys-Gly-Phe-Met-Cys]-Leu-Trp-NH-Bn(3',5'-(CF₃)₂) (SEQ ID NO:20); Tyr-[Cys-Gly-Phe-Met-Pro-hCys]-Trp-NH-Bn(3',5'-(CF₃)₂) (SEQ ID NO:21); Tyr-[Cys-Gly-Phe-Met-hCys]-Leu-Trp-NH-Bn(3',5'-(CF₃)₂) (SEQ ID NO:22); Tyr-[Cys-Gly-Phe-Nle-Pro-Cys]-Trp-NH-Bn(3',5'-(CF₃)₂) (SEQ ID NO:35); Tyr-[Cys-Gly-Phe-Nle-Cys]-Leu-Trp-NH-Bn(3',5'-(CF₃)₂) (SEQ ID NO:36); Tyr-[hCys-Gly-Phe-Nle-Pro-Cys]-Trp-NH-Bn(3',5'-(CF₃)₂) (SEQ ID NO:39); and Tyr-[hCys-Gly-Phe-Nle-Cys]-Leu-Trp-NH-Bn(3',5'-(CF₃)₂) (SEQ ID NO:40).

19. A method for treating pain or an opioid addiction in a subject, said method comprising administering a therapeutically effective amount of a peptide of the formula:

X¹-X²-Gly-X⁴-X⁵-X⁶-X⁷-Trp-L-R (SEQ ID NO : 1 ) or a pharmaceutically acceptable salt thereof to a subject in need of such a treatment, wherein

X² is Cys, Ala, hCys, or Pen;

X⁴ is Phe or substituted Phe;

X⁵ is Phe, Leu, Met, Met(O), Gly, Nle, Ser, or substituted Ser;

X⁶ is Pro, Ala, Leu, Cys, hCys, or Pen;

X⁷ is Cys, hCys, Pen, Ser, Leu, Ala, Vai, or Aib;

SUBSTITUTE SHEET (RULE 26) L is O, NH, or NMe; and

20. The method of claim 19 wherein said pain is an acute pain, chronic pain, neuropathic pain, nociceptive pain, radicular pain, or a combination thereof.

21. The method of claim 20, wherein said pain is a chronic pain.

22. The method of claim 20, wherein said pain is a nociceptive pain.

23. The method of claim 20, wherein said pain is a neuropathic pain.

24. The method of claim 20, wherein said pain is an acute pain.

25. The method of claim 19, wherein X² is cys, hCys, or Pen.

26. The method of claim 25, wherein one of X⁶ or X⁷ is Cys, hCys, or Pen.

27. The method of claim 26, wherein said peptide is a cyclic peptide of the formula:

(SEQ ID NO:2) (SEQ ID NO:3)

II III

28. The method of claim 19, wherein said peptide is a non-cyclic peptide of the formula:

X¹-X²-Gly-X⁴-X⁵-Pro-Leu-Trp-L-R (SEQ ID NO: 4)

29. The method of claim 28, wherein X² is (D)-Ala.

SUBSTITUTE SHEET (RULE 26)