Novel chimeric analgesic peptides   

FIELD OF THE INVENTION

The present invention relates generally to methods and compositions for the treatment of pain. More specifically, the present invention relates to novel chimeric peptides for the treatment of pain.

BACKGROUND OF THE INVENTION

Two million people in the United States suffer from chronic pain. Pain is caused by a highly complex perception of an aversive or unpleasant sensation. The sensation of pain begins with noxious stimulation of free nerve endings, which leads to activation of different types of nociceptive afferent fibers. These fibers include Aδ fibers and C fibers. Aδ fibers are small diameters, thinly myelinated fibers that transmit sharp, prickling pain. C fibers are unmyelinated and conduct more slowly and transmit dull aching pain. Repeated stimulation of pain fibers can lead to hyperalgesia, or a lowering of the threshold for activation of nociceptors.

Primary afferent fibers Aδ or C from the damaged periphery synapse release a variety of chemical mediators. These mediators include glutamate and substance P (“SP”), a nociceptive peptide. SP has long been recognized and identified as a neurotransmitter intimately associated with the transfer of painful or nociceptive stimuli from peripheral receptive fields into the CNS. This neuropeptide is involved in pain signaling and the maintenance of the chronic pain state. SP is the prototypic member of a family of related peptides named tachykinins, all of which were initially characterized by contractile activity on isolated smooth muscle preparations. SP is also found in the brain, spinal cord, spinal ganglia, and intestine of all vertebrates, including man.

SP is present in small-diameter sensory fibers that mediate nociceptive inputs in the spinal cord, and it specifically excites nociceptive neurons in this region. SP is released in the spinal cord in vivo, upon activation of nociceptive primary sensory fibers. Direct application of microgram doses of SP into the lumbar spinal subarachnoid produces hyperalgesia, i.e., an increased sensitivity to pain. The release of SP can be blocked by administration of morphine and opioid peptides in vivo and in vitro. For example, intrathecal administration of morphine blocks the hyperalgesic effects of exogenously administered SP. See, Hyden and Wilcox, Eur. J. Pharmacol., 86-95-98 (1983); and J. Pharmacol. Exp. Ther. 226: 398-404 (1983).

While opioids can be effective for the treatment of chronic pain, they frequently have side effects, including respiratory depression, urinary retention, nausea and vomiting, pruritis, and sedation. Moreover, repeated daily administration of opioids eventually produces tolerance, whereby the dose of the drug must be increased in order to maintain adequate analgesia, and may also initiate physical dependence. If tolerance develops and the level of opioids is insufficient, withdrawal symptoms such as diarrhea, sweating, tremors, anxiety, and fever may result. These concerns have prompted a search for new analgesics with limited side effects and that show decreased susceptibility to tolerance.

SUMMARY

The present invention provides a novel chimeric peptide having an opioid moiety that binds to an opioid receptor and a nociceptive moiety that binds to a nociceptive receptor, such as NK1. The opioid moiety may be directed to any of the opioid receptor types, including the μ, δ, or κ receptor

For example, the chimeric peptide can include an μ-receptor binding opioid moiety and an NK1-binding SP moiety. In one embodiment this chimeric peptide has the sequence:

(SEQ ID NO: 42) Tyr-Pro-Phe-Phe-Gly-Leu-Met-NH2.

The chimeric peptides may be designed to have a plurality of SP moieties and a plurality of opioid moieties. The plurality of opioid moieties may be directed to the same receptor type, or, alternatively, the plurality of opioid moieties may be directed to different opioid receptor types.

The invention provides pharmaceutical compositions including chimeric peptides and a pharmaceutically acceptable carrier useful for the treatment of pain.

The invention also provides a method of treating pain by administering the chimeric peptide capable of binding to both an opioid receptor and the NK1 receptor admixed with a pharmaceutically acceptable carrier, such as pharmaceutical sterile saline. The peptide may be administered intrathecally (IT), intracerebrovertricularlly (ICV) or systemically, for example, intraperitoneally (IP). Solubility of the chimeric peptides may be enhanced by admixture with a solubilizing agent, for example, cyclodextran. In a alternative embodiment, a chimeric peptide is administered in conjunction with one or more non-chimeric opioid drugs.

Among the advantages of the invention is that the chimeric peptides produce effective analgesia yet inhibit the development of tolerance.

The details of one or more embodiments of the invention are set forth in the accompanying description below. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. Other features, objects, and advantages of the invention will be apparent from the description and from the claims. In the specification and the appended claims, the singular forms include plural referents unless the context clearly dictates otherwise. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Unless expressly stated otherwise, the techniques employed or contemplated herein are standard methodologies well known to one of ordinary skill in the art. The examples of embodiments are for illustration purposes only. All patents and publications cited in this specification are incorporated herein by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic representation of the chimeric peptide ESP7.

FIG. 2 is a schematic representation of the chimeric peptide ESP6.

FIG. 3 is a graph illustrating the binding affinity of ESP7 to the μ receptor.

FIG. 4 is a graph illustrating the binding affinity of ESP7 to the NK1 receptor.

FIG. 5 is a graph illustrating the analgesic effect in rats overtime of 1.0 μg of ESP7 administered intrathecally.

FIG. 6 is a graph illustrating the analgesic effect in rats over time of 0.2 μg of ESP7 administered intrathecally.

FIG. 7 is a graph illustrating the analgesic effect in rats of 0.05 μg of ESP7 administered intrathecally.

FIG. 8 is a graph illustrating the analgesic effect in rats over time of 0.2 μg of ESP7 antagonized with on days 2 and 4 with 0.2 μg of naltrexone.

FIG. 9 is a graph illustrating the analgesic effect in rats over time of 1.0 μg of ESP7 antagonized with RP67580 on days 1-4.

FIG. 10 is a graph illustrating the analgesic effect in rats over time of 0.1 μg of ESP7 administered intracerebroventricularly.

FIG. 11 is a graph illustrating the analgesic effect in rats over time of 1 mg of ESP7 administered intraperitoneally.

DETAILED DESCRIPTION

The present invention provides a chimeric peptide having an opioid receptor binding moiety and a nociceptive receptor binding moiety (e.g., Substance P). The chimeric peptide molecules may be designed to bind to any of the opioid receptors known to be involved in pain mediation. See review in Lipkowski and Cary, Peptides: Synthesis, Structures, and Applications, Gutte, ed., Academic Press pp. 297-320 (1995), incorporated herein by reference. While the opioid peptides frequently exhibit some cross reactivity with the different receptor types, they can be generally characterized by the degree of affinity for a particular receptor type. These receptors include the p receptor, the μ receptor and the κ receptor.

The separate moieties may be chemically synthesized and purified or isolated from natural sources and then chemically cross-linked to form the chimeric peptide. Alternatively, the chimera can be chemically synthesized as one molecule. In another embodiment, chimeric peptides are produced by recombinant DNA techniques and isolated from cells or tissue sources by an appropriate purification scheme using standard protein purification techniques. The invention also relates to derivatives, fragments, homologs, analogs and variants of these peptides.

Chimeric peptides, and individual moieties or analogs and derivatives thereof, can be chemically synthesized. A variety of protein synthesis methods are common in the art, including synthesis using a peptide synthesizer. See, e.g., Peptide Chemistry, A Practical Textbook, Bodasnsky, Ed. Springer-Verlag, 1988; Merrifield, Science 232: 241-247 (1986); Barany, et al, Intl. Peptide Protein Res. 30: 705-739 (1987); Kent, Ann. Rev. Biochem. 57:957-989 (1988), and Kaiser, et al, Science 243: 187-198 (1989). The peptides are purified so that they are substantially free of chemical precursors or other chemicals using standard peptide purification techniques. The language “substantially free of chemical precursors or other chemicals” includes preparations of peptide in which the peptide is separated from chemical precursors or other chemicals that are involved in the synthesis of the peptide. In one embodiment the language “substantially free of chemical precursors or other chemicals” includes preparations of peptide having less than about 30% (by dry weight) of chemical precursors or non-peptide chemicals, more preferably less than about 20% chemical precursors or non-peptide chemicals, still more preferably less than about 10% chemical precursors or non-peptide chemicals, and most preferably less than about 5% chemical precursors or non-peptide chemicals.

Chemical synthesis of peptides facilitates the incorporation of modified or unnatural amino acids, including D-amino acids and other small organic molecules. Replacement of one or more L-amino acids in a peptide with the corresponding D-amino acid isoforms can be used to increase the resistance of peptides to enzymatic hydrolysis, and to enhance one or more properties of biologically active peptides, i.e., receptor binding, functional potency or duration of action. See, e.g., Doherty, et al., 1993. J Med. Chem. 36: 2585-2594; Kirby, et al., 1993. J. Med. Chem. 36:3802-3808; Morita, et al., 1994. FEBS Lett. 353: 84-88; Wang, et al., 1993. Int. J. Pept. Protein Res. 42:392-399; Fauchere and Thiunieau, 1992. Adv. Drug Res. 23:127-159.

Introduction of covalent cross-links into a peptide sequence can conformationally and topographically constrain the peptide backbone. This strategy can be used to develop peptide analogs of the chimeric peptides with increased potency, selectivity and stability. Because the conformational entropy of a cyclic peptide is lower than its linear counterpart, adoption of a specific conformation may occur with a smaller decrease in entropy for a cyclic analog than for an acyclic analog, thereby making the free energy for binding more favorable. Macrocyclization is often accomplished by forming an amide bond between the peptide N- and C-termini, between a side chain and the N- or C-terminus [e.g., with K3Fe(CN)6 at pH 8.5] (Samson et al., Endocrinology, 137: 5182-5185 (1996)), or between two amino acid side chains. See, e.g., DeGrado, Adv Protein Chem, 39: 51-124 (1988). Disulfide bridges are also introduced into linear sequences to reduce their flexibility. See, e.g., Rose, et al., Adv Protein Chem, 37:1-109 (1985); Mosberg et al., Biochem Biophys Res Commun, 106: 505-512 (1982). Furthermore, the replacement of cysteine residues with penicillamine (Pen, 3-mercapto-(D) valine) has been used to increase the selectivity of some opioid-receptor interactions. Lipkowski and Carr, Peptides: Synthesis, Structures, and Applications, Gutte, ed., Academic Press pp. 287-320 (1995).

A number of other methods have been used successfully to introduce conformational constraints into peptide sequences in order to improve their potency, receptor selectivity and biological half-life. These include the use of (i)Cα-methylamino acids (see, e.g., Rose, et al., Adv Protein Chem, 37: 1-109 (1985); Prasad and Balaram, CRC Crit Rev Biochem, 16: 307-348 (1984)); (ii) Nα-methylamino acids (see, e.g., Aubry, et al., Int J Pept Protein Res, 18: 195-202 (1981); Manavalan and Momany, Biopolymers, 19: 1943-1973 (1980)); and (iii) α,β-unsaturated amino acids (see, e.g., Bach and Gierasch, Biopolymers, 25: 5175-S192 (1986); Singh, et al., Biopolymers, 26: 819-829 (1987)). These and many other amino acid analogs are commercially available, or can be easily prepared. Additionally, replacement of the C-terminal acid with an amide can be used to enhance the solubility and clearance of a peptide.

Alternatively, the peptides may be obtained by methods well-known in the art for recombinant peptide expression and purification. A DNA molecule encoding a chimeric peptide can be generated. The DNA sequence is deduced from the protein sequence based on known codon usage. See, e.g., Old and Primrose, Principles of Gene Manipulation 3rd ed., Blackwell Scientific Publications, 1985; Wada et al., Nucleic Acids Res. 20: 2111-2118 (1992). Preferably, the DNA molecule includes additional sequence, e.g., recognition sites for restriction enzymes which facilitate its cloning into a suitable cloning vector, such as a plasmid. The invention provides the nucleic acids comprising the coding regions, non-coding regions, or both, either alone or cloned in a recombinant vector, as well as oligonucleotides and related primer and primer pairs corresponding thereto. Nucleic acids may be DNA, RNA, or a combination thereof. Vectors of the invention may be expression vectors. Nucleic acids encoding chimeric peptides may be obtained by any method known within the art (e.g., by PCR amplification using synthetic primers hybridizable to the 3′- and 5′-termini of the sequence and/or by cloning from a cDNA or genomic library using an oligonucleotide sequence specific for the given gene sequence, or the like). Nucleic acids can also be generated by chemical synthesis.

The invention relates to nucleic acids hybridizable—or complementary—to the nucleic acids encoding the chimeric peptides. In particular the invention provides the inverse complement to nucleic acids hybridizable to the encoding nucleic acids (i.e., the inverse complement of a nucleic acid strand has the complementary sequence running in reverse orientation to the strand so that the inverse complement would hybridize with few or no mismatches to the nucleic acid strand). Nucleic acid molecules encoding derivatives and analogs of a chimeric peptide, or antisense nucleic acids to the same are additionally provided.

Any of the methodologies known within the relevant art regarding the insertion of nucleic acid fragments into a vector may be used to construct expression vectors that contain a chimeric gene comprised of the appropriate transcriptional/translational control signals and peptide-coding sequences. Promoter/enhancer sequences within expression vectors may use plant, animal, insect, or fungus regulatory sequences, as provided in the invention.

A host cell can be any prokaryotic or eukaryotic cell. For example, the peptide can be expressed in bacterial cells such as E. coli, insect cells, fungi or mammalian cells (such as Chinese hamster ovary cells (CHO) or COS cells). Other suitable host cells are known to those skilled in the art. In one embodiment, a nucleic acid encoding the peptide is expressed in mammalian cells using a mammalian expression vector. Examples of mammalian expression vectors include pCD1M8 (Seed (1987) Nature 329:840) and pMT2PC (Kaufman et al. (1987) EMBO J 6: 187-195). Furthermore, transgenic animals containing nucleic acids that encode a chimeric peptide may also be used to express peptides of the invention.

Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques. As used herein, the terms “transformation” and “transfection” are intended to refer to a variety of art-recognized techniques for introducing foreign nucleic acid (e.g., DNA) into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, or electroporation. Suitable methods for transforming or transfecting host cells can be found in Sambrook, et al. (Molecular Cloning: A Laboratory Manual. 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989), and other laboratory manuals.

More commonly, the host cells, can be used to produce (i.e., overexpress) peptide in culture. Accordingly, the invention flier provides methods for producing the peptide using the host cells of the invention. In one embodiment, the method comprises culturing the host cell of invention (into which a recombinant expression vector encoding the peptide has been introduced) in a suitable medium such that peptide is produced. The method fiercer involves isolating peptide from the medium or the host cell. Ausubel et al., (Eds). In: Current Protocols in Molecular Biology. J. Wiley and Sons, New York, N.Y. 1998.

An “isolated” or “purified” recombinant peptide or biologically active portion thereof is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which the peptide of interest is derived. The language “substantially free of cellular material” includes preparations in which the peptide is separated from cellular components of the cells from which it is isolated or recombinantly produced. In one embodiment, the language “substantially free of cellular material” includes preparations of peptide having less than about 30% (by dry weight) of peptide other than the desired peptide (also referred to herein as a “contaminating protein”), more preferably less than about 20% of contaminating protein, still more preferably less than about 10% of contaminating protein, and most preferably less than about 5% contaminating protein. When the peptide or biologically active portion thereof is recombinantly produced, it is also preferably substantially free of culture medium, i.e., culture medium represents less than about 20%, more preferably less than about 10%, and most preferably less than about 5% of the volume of the peptide preparation.

Cells engineered to over-express a chimeric peptide can also be introduced in vivo for therapeutic purposes by any method known in the art, including, but not limited to, implantation or transplantation of cells into a host subject, wherein the cells may be “naked” or encapsulated prior to implantation. Cells may be screened prior to implantation for various characteristics including, but not limited to, the level of peptide secreted, stability of expression, and the like.

The present invention also pertains to variants of the peptides that function as either agonists (mimetics) or as antagonists. Variants of a parent peptides can be generated by mutagenesis, e.g., discrete point mutation. An agonist of a parent peptide can retain substantially the same, or a subset of, the biological activities of the naturally occurring form of the parent peptide. An antagonist of the peptide can inhibit one or more of the activities of the naturally occurring form of the parent peptide by, for example, competitively binding to the receptor. Thus, specific biological effects can be elicited by treatment with a variant with a limited function. In one embodiment, treatment of a subject with a variant having a subset of the biological activities of the naturally occurring form of the peptide has fewer side effects in a subject relative to treatment with the naturally occurring form of the parent peptide.

Preferably, the analog, variant, or derivative peptides are functionally active. As utilized herein, the term “functionally active” refers to species displaying one or more known functional attributes of a full-length peptide. “Variant” refers to a polynucleotide or polypeptide differing from the polynucleotide or polypeptide of the present invention, but retaining essential properties thereof. Generally, variants are overall closely similar, and in many regions, identical to the polynucleotide or polypeptide of the present invention. The variants may contain alterations in the coding regions, non-coding regions, or both.

Variants of the peptides that function as either agonists (mimetics) or as antagonists can be identified by screening combinatorial libraries of mutants of the parent peptide for peptide agonist or antagonist activity. In one embodiment a variegated library of variants is generated by combinatorial mutagenesis at the nucleic acid level and is encoded by a variegated gene library. A variegated library of variants can be produced by, for example, enzymatically ligating a mixture of synthetic oligonucleotides into gene sequences such that a degenerate set of potential sequences is expressible as individual peptides, or alternatively, as a set of larger fusion proteins (e.g., for phage display) containing the set of sequences therein. There are a variety of methods which can be used to produce libraries of potential variants from a degenerate oligonucleotide sequence. Chemical synthesis of a degenerate gene sequence can be performed in an automatic DNA synthesizer, and the synthetic gene then ligated into an appropriate expression vector. Use of a degenerate set of genes allows for the provision, in one mixture, of all of the sequences encoding the desired set of potential sequences. Methods for synthesizing degenerate oligonucleotides are known in the art (see, e.g., Narang (1983) Tetrahedron 39:3; Itakura et al. (1984)Annu Rev Biochem 53:323; Itakura et al. (1984) Science 198:1056; Ike et al. (1983) Nucl Acid Res 11:477.

Derivatives and analogs of the chimeric peptides or individual moieties can be produced by various methods known within the art. For example, the polypeptide sequences may be modified by any of numerous methods known within the art. See e.g., Sambrook, et al. 1990. Molecular Cloning: A Laboratory Manual, 2nd ed., (Cold Spring Harbor Laboratory Press; Cold Spring Harbor, N.Y.). Manipulations can include by glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, linkage to an antibody molecule or other cellular ligand, and the like. Any of the numerous chemical modification methodologies known within the art may be utilized including, but not limited to, specific chemical cleavage by cyanogen bromide, trypsin, chymotrypsin, papain, V8 protease, NaBH4, acetylation, formylation, oxidation, reduction, metabolic synthesis in the presence of tunicamycin, etc. In one embodiment, the peptide is modified by the incorporation of a heterofunctional reagent, wherein such heterofunctional reagents may be used to connect the opioid moiety to the nociceptive moiety.

Derivatives, fragments, and analogs provided herein are defined as sequences of at least 6 (contiguous) nucleic acids or at least 4 (contiguous) amino acids, a length sufficient to allow for specific hybridization in the case of nucleic acids or for specific recognition of an epitope in the case of amino acids, respectively. Fragments are, at most, one nucleic acid-less or one amino acid-less than the wild type full length sequence. Derivatives and analogs may be full length or other than full length, if said derivative or analog contains a modified nucleic acid or amino acid, as described infra. Derivatives or analogs of the chimeric peptides include, but are not limited to, molecules comprising regions that are substantially homologous in various embodiments, of at least 30%, 40%, 50%, 60%, 70%, 80%, 90% or preferably 95% amino acid identity when: (i) compared to an amino acid sequence of identical size; (ii) compared to an aligned sequence in that the alignment is done by a computer homology program known within the art (e.g., Wisconsin GCG software) or (iii) the encoding nucleic acid is capable of hybridizing to a sequence encoding the aforementioned peptides under stringent (preferred), moderately stringent, or non-stringent conditions. See, e.g., Ausubel, et al., Current Protocols in Molecular Biology, John Wiley and Sons, New York, N.Y., 1993.

Derivatives of the chimeric peptides may be produced by alteration of their sequences by substitutions, additions or deletions that result in functionally-equivalent molecules. Thus, the invention includes DNA sequences that encode substantially the same amino acid sequence. In another embodiment, one or more amino acid residues within the sequence of interest may be substituted by another amino acid of a similar polarity and net charge, thus resulting in a silent alteration. Substitutes for an amino acid within the sequence may be selected from other members of the class to which the amino acid belongs. For example, nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine. Polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine. Positively charged (basic) amino acids include arginine, lysine and histidine. Negatively charged (acidic) amino acids include aspartic acid and glutamic acid.

In particular embodiments, the chimeric peptides, and fragments, derivatives, homologs or analogs thereof, are related to animals (e.g., mouse, rat pig, cow, dog, monkey, frog), or human opioids. Homologs (i.e., nucleic acids encoding peptides derived from species other than human) or other related sequences (e.g., paralogs) can also be obtained by low, moderate or high stringency hybridization with all or a portion of the particular human sequence as a probe using methods well known in the art for nucleic acid hybridization and cloning. See, e.g., Ausubel et al., (eds.), 1993, Current Protocols in Molecular Biology, John Wiley and Sons, NY; and Kriegler, 1990, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY.

In one embodiment, a nucleic acid sequence that is hybridizable to a nucleic acid sequence (or a complement of the foregoing) encoding the chimeric peptides, or a derivative of the same, under conditions of high stringency is provided: Step 1: Filters containing DNA are pretreated for a hours to overnight at 65° C. in buffer composed of 6×SSC, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.02% BSA, and 500 μg/ml denatured salmon sperm DNA. Step 2: Filters are hybridized for 48 hours at 65° C. in the above prehybridization mixture to which is added 100 mg/ml denatured salmon sperm DNA and 5-20×106 cpm of 32P-labeled probe. Step 3: Filters are washed for 1 hour at 37° C. in a solution containing 2×SSC, 0.01% PVP, 0.01% Ficoll, and 0.01% BSA. This is followed by a wash in 0.1×SSC at 50° C. for 45 minutes. Step 4: Filters are autoradiographed. Other conditions of high stringency that may be used are well known in the art.

In a second embodiment, a nucleic acid sequence that is hybridizable to a nucleic acid sequence (or a complement thereof) encoding the chimeric peptides, or derivatives, under conditions of moderate stringency is provided: Step 1: Filters containing DNA are pretreated for 6 hours at 55° C. in a solution containing 6×SSC, 5×Denhardt\'s solution, 0.5% SDS and 100 mg/ml denatured salmon sperm DNA. Step 2: Filters are hybridized for 18-20 hours at 55° C. in the same solution with 5-20×106 cpm 32P-labeled probe added. Step 3: Filters are washed at 370° C. for 1 hour in a solution containing 2×SSC 0.1% SDS, then washed twice for 30 minutes at 60° C. in a solution containing 1×SSC and 0.1% SDS. Step 4: Filters are blotted dry and exposed for autoradiography. Other conditions of moderate stringency that may be used are well-known in the art.

In a third embodiment, a nucleic acid that is hybridizable to a nucleic acid sequence disclosed in this invention or to a nucleic acid sequence encoding a the aforementioned peptides, or fragments, analogs or derivatives under conditions of low stringency: Step 1: Filters containing DNA are pretreated for 6 hours at 40° C. in a solution containing 35% formamide, 5×SSC, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.1% PVP, 0.1% Ficoll, 11% BSA, and 500 μg/ml denatured salmon sperm DNA. Step 2: Filters are hybridized for 18-20 hours at 40° C. in the same solution with the addition of 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 μg/ml salmon sperm DNA, 10% (wt/vol) dextran sulfate, and 5-20×106 cpm 32P-labeled probe. Step 3: Filters are washed for 1.5 hours at 55° C. in a solution containing 2×SSC, 25 mM Tris-HCl (pH 7.4), 5 mM EDTA, and 0.1% SDS. The wash solution is replaced with fresh solution and incubated an additional 1.5 hours at 60° C. Step 4: Filters are blotted dry and exposed for autoradiography. If necessary, filters are washed for a third time at 65-68° C. and re-exposed to film. Other conditions of low stringency that may be used are well known in the art (e.g. as employed for cross-species hybridizations). See also Shilo and Weinberg, Proc Natl Acad Sci USA 78: 6789-6792 (1981).

Peptides with Affinity for the μ Receptor

The exogenous opioid peptide agonists for the μ receptor type include those listed in Table 1: α-endorphin, endomorphin-1, endomorphin-2, dermorphin, β-casomorphin (bovine or human), Morphiceptin, Leu-enkephalin, Met-enkephalin, DALDA, and PL107. Modifications of the peptides have resulted in very selective μ receptor ligands. These modifications can include amidation of the carboxyl terminus (—NH2), the use of (D) amino acids in the peptide (e.g. DALDA), incorporation of small non-peptidyl moieties, as well as the modification of the amino acids themselves (e.g. alkylation or esterification of side chain R-groups). As in, for example, the compound DAMGO: Tyr-(D)Ala-Gly-Phe-NHCH2CH2OH.

TABLE 1 μ receptor SEQ ID NO: agonist Sequence  1 α-endorphin Tyr-Gly-Gly-Phe-Met-Thr-Ser-Glu- Ser-Gln-Thr-Pro-Leu-Val-Thr-NH2  2 endomorphin-1 Tyr-Pro-Trp-Phe-NH2  3 endomorphin-2 Tyr-Pro-Phe-Phe-NH2  4 dermorphin Tyr-(D)Ala-Phe-Gly-Tyr-Pro-Ser-NH2  5 β-casomorphin Tyr-Pro-Phe-Pro-Gly-Pro-Ile (bovine)  6 β-casomorphin Tyr-Pro-Phe-Val-Glu-Pro-Ile (human)  7 Morphiceptin Tyr-Pro-Phe-Pro-NH2  8 Leu-enkephalin Tyr-Gly-Gly-Phe-Leu  9 Met-enkephalin Tyr-Gly-Gly-Phe-Met 10 DALDA Tyr-(D)Arg-Phe-Lys-NH2 11 PL017 Tyr-Pro-(N-Me)Phe-(D)Pro-NH2 Peptides with Affinity for the δ Receptor

Other suitable opioid peptide moieties include the δ receptor agonists listed in Table 2. Those with the highest receptor selectivity generally are enkephalin-derived peptides. For example, DADLE has a three to ten fold higher selectivity for the δ receptor than the μ receptor. Modifications of the parent enkephalin sequence results in two groups of peptide analogs. The first group is a series of linear analogs, for example, DSLET. The second group, all rigid cyclic analogs, includes DPDPE (where Pen is penicillamine, or 3-mercapto-(D)Valine). In binding assays, these analogs show an 100-fold affinity for the δ receptor over the μ-receptor and a 1000-fold increase over the κ-receptor. Additional pseudopeptide analogs, either linear or cyclic, also display high selectivity to the δ receptor, for example Tyr-Tic-Phe-Phe, where Tic is L-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid Schiller, et al., J. Med. Chem. 36: 3182-3187 (1993).

TABLE 2 SEQ δ receptor

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