U.S. patent application number 10/828623 was filed with the patent office on 2006-05-18 for novel chimeric analgesic peptides.
This patent application is currently assigned to New England Medical Center Hospitals, Inc.. Invention is credited to Daniel B. Carr, Richard Kream, Andrzej W. Lipkowski, Aleksandra Misicka-Kesik.
Application Number | 20060105947 10/828623 |
Document ID | / |
Family ID | 23699975 |
Filed Date | 2006-05-18 |
United States Patent
Application |
20060105947 |
Kind Code |
A1 |
Carr; Daniel B. ; et
al. |
May 18, 2006 |
Novel chimeric analgesic peptides
Abstract
The present invention provides a novel chimeric peptide
containing an opioid peptide moiety and a nociceptive peptide
moiety for producing analgesia
Inventors: |
Carr; Daniel B.; (Chestnut
Hill, MA) ; Lipkowski; Andrzej W.; (Warsaw, PL)
; Kream; Richard; (Roslindale, MA) ;
Misicka-Kesik; Aleksandra; (Piastow, PL) |
Correspondence
Address: |
CHOATE, HALL & STEWART LLP
TWO INTERNATIONAL PLACE
BOSTON
MA
02110
US
|
Assignee: |
New England Medical Center
Hospitals, Inc.
Medical Research Center of the Polish Academy of
Sciences
|
Family ID: |
23699975 |
Appl. No.: |
10/828623 |
Filed: |
April 21, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
09428692 |
Oct 28, 1999 |
6759520 |
|
|
10828623 |
Apr 21, 2004 |
|
|
|
Current U.S.
Class: |
530/402 ;
514/18.3; 530/399 |
Current CPC
Class: |
C07K 7/22 20130101; A61P
25/00 20180101; C07K 19/00 20130101; A61K 38/00 20130101 |
Class at
Publication: |
514/012 ;
530/399 |
International
Class: |
C07K 14/675 20060101
C07K014/675; A61K 38/22 20060101 A61K038/22 |
Claims
1-23. (canceled)
24. A method for treating pain in a mammal, said method comprising
administering to said mammal a chimeric peptide comprising an
agonist opioid receptor binding moiety at its N-terminus and an
agonist Substance P receptor binding moiety at its C-terminus, in
an amount sufficient to induce analgesia in said mammal.
25. The method of claim 24 wherein, in the peptide, the agonist
opioid receptor binding moiety is a .mu., .delta. or .kappa.
agonist opioid receptor binding moiety.
26. The method of claim 25 wherein, in the peptide, the agonist
opioid receptor binding moiety is a p agonist opioid receptor
binding moiety.
27. The method of claim 26 wherein, in the peptide, the N-terminal
amino acid residue of said opioid receptor binding moiety is a free
amine.
28. The method of claim 27 wherein, in the peptide, the N-terminal
amino acid residue of said opioid receptor binding moiety is
Tyr.
29. The method of claim 28 wherein, in the peptide, said opioid
receptor binding moiety is a peptide having any one of SEQ ID Nos:
1-11, or N-terminal fragment thereof.
30. The method of claim 28 wherein, in the peptide, said opioid
receptor binding moiety is endomorphin 1, endomorphin 2, or
N-terminal fragment thereof.
31. The method of claim 30 wherein, in the peptide, said opioid
receptor binding moiety is a peptide having SEQ ID No: 2 or 3, or
N-terminal fragment thereof.
32. The method of claim 26 wherein, in the peptide, said agonist
Substance P receptor binding moiety comprises Substance P, or
C-terminal Substance P fragment thereof.
33. The method of claim 26 wherein, in the peptide, the --COOH
moiety of the C-terminal amino acid residue of said Substance P
receptor binding moiety is protected.
34. The method of claim 33 wherein, in the peptide, the --COOH
moiety of the C-terminal amino acid residue of said Substance P
receptor binding moiety is amidated.
35. The method of claim 34 wherein, in the peptide, the C-terminal
amino acid residue of said Substance P receptor binding moiety is
Met-NH.sub.2.
36. The method of claim 35 wherein, in the peptide, said Substance
P receptor binding moiety is a peptide having any one of SEQ ID
Nos: 21, 36 and 38-41, or C-terminal fragment thereof.
37. The method of claim 26 wherein, in the peptide, the opioid
receptor binding moiety is endomorphin 1, endomorphin 2, or
N-terminal fragment thereof; and the Substance P receptor binding
moiety is Substance P, or C-terminal fragment thereof.
38. The method of claim 26 wherein the peptide has SEQ ID No:
42.
39. The method of claim 26 wherein the peptide has SEQ ID No:
43.
40. The method of claim 24 wherein the method of administration is
selected from the group consisting of intrathecal,
intracerebroventricular and systemic administration.
41. The method of claim 24 wherein the peptide is administered with
a solubilizing agent.
42. The method of claim 41 wherein the solubilizing agent is
cyclodextran.
Description
FIELD OF THE INVENTION
[0001] 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
[0002] 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 AS fibers and C fibers. AS fibers are small diameter,
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.
[0003] Primary afferent fibers AS 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.
[0004] 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 primer 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).
[0005] 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 OF THE INVENTION
[0006] 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
NK.sub.1. The opioid moiety may be directed to any of the opioid
receptor types, including the .mu., .delta., or .kappa.
receptor
[0007] For example, the chimeric peptide can include an Preceptor
binding opioid moiety and an NK.sub.1-binding SP moiety. In one
embodiment this chimeric peptide has the sequence:
Tyr-Pro-Phe-Phe-Gly-Leu-Met-NH.sub.2 (SEQ ID NO:42).
[0008] 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.
[0009] The invention provides pharmaceutical compositions including
chimeric peptides and a pharmaceutically acceptable carrier useful
for the treatment of pain.
[0010] The invention also provides a method of treating pain by
administering the chimeric peptide capable of binding to both an
opioid receptor and the NK.sub.1 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 admixtre 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.
[0011] Among the advantages of the invention is that the chimeric
peptides produce effective analgesia yet inhibit the development of
tolerance.
[0012] 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
[0013] FIG. 1 is a schematic representation of the chimeric peptide
ESP7.
[0014] FIG. 2 is a schematic representation of the chimeric peptide
ESP6.
[0015] FIG. 3 is a graph illustrating the binding affinity of ESP7
to the .mu. receptor.
[0016] FIG. 4 is a graph illustrating the binding affinity of ESP7
to the NK.sub.1 receptor.
[0017] FIG. 5 is a graph illustrating the analgesic effect in rats
over time of 1.0 .mu.g of ESP7 administered intrathecally.
[0018] FIG. 6 is a graph illustrating the analgesic effect in rats
over time of 0.2 .mu.g of ESP7 administered intrathecally.
[0019] FIG. 7 is a graph illustrating the analgesic effect in rats
of 0.05 .mu.g of ESP7 administered intrathecally.
[0020] FIG. 8 is a graph illustrating the analgesic effect in rats
over time of 0.2 .mu.g of ESP7 antagonized with on days 2 and 4
with 0.2 .mu.g of naltrexone.
[0021] FIG. 9 is a graph illustrating the analgesic effect in rats
over time of 1.0 .mu.g of ESP7 antagonized with RP67580 on days
1-4.
[0022] FIG. 10 is a graph illustrating the analgesic effect in rats
over time of 0.1 .mu.g of ESP7 administered
intracerebroventricularly.
[0023] FIG. 11 is a graph illustrating the analgesic effect in rats
over time of 1 mg of ESP7 administered intraperitoneally.
DETAILED DESCRIPTION OF THE INVENTION
[0024] 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 Carr,
Peptides: Synthesis, Structures, and Applications, Gutte, ed.,
Academic Press pp. 287-320 (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 .mu. receptor, the .delta.
receptor and the .kappa. receptor.
[0025] 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.
Chemical Synthesis
[0026] 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.
J. 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.
[0027] 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: 8488; Wang, et al., 1993.
Int. J. Pepi. Protein Res. 42: 392-399; Fauchere and Thiunieau,
1992. Adv. Drug Res. 23: 127-159.
[0028] 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 K.sub.3Fe(CN).sub.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).
[0029] 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) C.sub..alpha.-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)
N.sub..alpha.-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)
.alpha.,.beta.-unsaturated amino acids (see, e.g., Bach and
Gierasch, Biopolymers, 25: 5175S192 (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.
Recombinant Peptides
[0030] 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 3.sup.rd 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.
[0031] 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.
[0032] 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.
[0033] 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 pCDM8 (Seed (1987) Nature 329:840) and pMT2PC
(Kaulinan 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.
[0034] 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.
[0035] More commonly, the host cells, can be used to produce (ie.,
over-express) peptide in culture. Accordingly, the invention
further 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 further
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.
[0036] 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.
[0037] 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.
Production of Derivatives and Analogs
[0038] 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.
[0039] 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.
[0040] 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.
[0041] 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,
NaBH.sub.4, 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.
[0042] 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 fill 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.
[0043] 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.
[0044] 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.
[0045] 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 8 hours to overnight at
65.degree. C. in buffer composed of 6.times.SSC, 50 mM Tris-HCl (pH
7.5), 1 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.02% BSA, and 500
.mu.g/ml denatured salmon sperm DNA. Step 2: Filters are hybridized
for 48 hours at 65.degree. C. in the above prehybridization mixture
to which is added 100 mg/ml denatured salmon sperm DNA and
5-20.times.10.sup.6 cpm of .sup.32P-labeled probe. Step 3: Filters
are washed for 1 hour at 37.degree. C. in a solution containing
2.times.SSC, 0.01% PVP, 0.01% Ficoll, and 0.01% BSA. This is
followed by a wash in 0.1.times.SSC at 50.degree. C. for 45
minutes. Step 4: Filters are autoradiographed. Other conditions of
high stringency that may be used are well known in the art.
[0046] 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.degree. C. in a solution containing
6.times.SSC, 5.times. 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.degree. C. in the same solution with
5-20.times.106 cpm .sup.32P-labeled probe added. Step 3: Filters
are washed at 37.degree. C. for 1 hour in a solution containing
2.times.SSC, 0.1% SDS, then washed twice for 30 minutes at
60.degree. C. in a solution containing 1.times.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.
[0047] 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.degree. C. in a solution containing 35% formamide,
5.times.SSC, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.1% PVP, 0.1%
Ficoll, 1% BSA, and 500 .mu.g/ml denatured salmon sperm DNA Step 2:
Filters are hybridized for 18-20 hours at 40.degree. C. in the same
solution with the addition of 0.02% PVP, 0.02% Ficoll, 0.2% BSA,
100 .mu.g/ml salmon sperm DNA, 10% (wt/vol) dextian sulfate, and
5-20.times.106 cpm .sup.32P-labeled probe. Step 3: Filters are
washed for 1.5 hours at 55.degree. C. in a solution containing
2.times.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.degree. C. Step 4: Filters are blotted
dry and exposed for autoradiography. If necessary, filters are
washed for a third time at 65-68.degree. 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).
Design of Chimeric Peptides
Peptides with Affinity for the .mu. Receptor
[0048] The exogenous opioid peptide agonists for the 11 receptor
type include those listed in Table 1: .alpha.-endorphin,
endomorphin-1, endomorphin-2, dermorphin, .beta.-casomorphin
(bovine or human), Morphiceptin, Leu-enkephalin, Met-enkephalin,
DALDA, and PL107. Modifications of the peptides have resulted in
very selective .mu. receptor ligands. These modifications can
include amidation of the carboxyl terminus (--NH.sub.2), 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-NHCH.sub.2CH.sub.2OH. TABLE-US-00001 TABLE 1 SEQ
ID .mu. receptor NO: agonist Sequence 1 .alpha.-endorphin
Tyr-Gly-Gly-Phe-Met-Thr-Ser-Glu-
Ser-Gln-Thr-Pro-Leu-Val-Thr-NH.sub.2 2 endomorphin-1
Tyr-Pro-Trp-Phe-NH.sub.2 3 endomorphin-2 Tyr-Pro-Phe-Phe-NH.sub.2 4
dermorphin Tyr-(D)Ala-Phe-Gly-Tyr-Pro-Ser- NH.sub.2 5
.beta.-casomorphin Tyr-Pro-Phe-Pro-Gly-Pro-Ile (bovine) 6
.beta.-casomorphin Tyr-Pro-Phe-Val-Glu-Pro-Ile (human) 7
Morphiceptin Tyr-Pro-Phe-Pro-NH.sub.2 8 Leu-enkephalin
Tyr-Gly-Gly-Phe-Leu 9 Met-enkephalin Tyr-Gly-Gly-Phe-Met 10 DALDA
Tyr-(D)Arg-Phe-Lys-NH.sub.2 11 PL017
Tyr-Pro-(N-Me)Phe-(D)Pro-NH.sub.2
Peptides With affinity for the .delta. Receptor
[0049] Other suitable opioid peptide moieties include the .delta.
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
.delta. receptor than the .mu. 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 .delta. receptor over the .mu.-receptor
and a 1000-fold increase over the .kappa.-receptor. Additional
pseudopeptide analogs, either linear or cyclic, also display high
selectivity to the .delta. 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-US-00002 TABLE 2 SEQ ID .delta. receptor NO: agonists
Sequence 12 DADLE Tyr-(D)Ala-Gly-Phe-(D)Leu 13 DSLET
Tyr-(D)Ser-Gly-Phe-Leu-Thr 14 DPDPE
.sub.-------------------------------------- | |
Tyr-(D)Pen-Gly-Phe-(D)Pen 15 deltorphin I
Tyr-(D)Ala-Phe-Asp-Val-Val-Gly- NH.sub.2 16 deltorphin II
Tyr-(D)Ala-Phe-Glu-Val-Val-Gly- NH.sub.2 17 dermenkephalin
Tyr-(D)Met-Phe-His-Leu-Met-Asp- NH.sub.2
Peptides With Affinity for the .kappa. Receptor
[0050] Opioid moieties also include Dynorphin ("DYN") related
peptides, which are endogenous peptide agonists for the .kappa.
receptor. Some representative peptides are shown in Table 3. The
propeptide, pro-dynorphin, is processed into peptides of different
lengths and with different receptor selectivities. Several of these
peptides, including Dynorphin A, DYN(1-8), and DYN(1-13) are found
in the CNS of vertebrates in physiologically significant
concentrations. Several dynorphin analogs have been generated by
substitution of D-amino acids at position 8 (Ile) or 10 (Pro).
Additionally cyclic dynorphin analogs with high K receptor
selectivity have been generated: e.g.,
Tyr-Gly-Gly-Phe-Leu-Arg-Arg-Cys-Arg-Pro-Lys-Leu-Cys-NH, (SEQ ID NO:
44), where the two Cysteines are engaged in a disulfide bond, to
create a six amino acid ring. TABLE-US-00003 TABLE 3 SEQ ID .kappa.
receptor NO: agonists Sequence 18 Dynorphin A
Tyr-Gly-Gly-Phe-Leu-Arg-Arg-Ile-Arg-
Pro-Lys-Leu-Lys-Trp-Asp-Asn-Gln 19 DYN (1-8)
Tyr-Gly-Gly-Phe-Leu-Arg-Arg-Ile 20 DYN (1-13)
Tyr-Gly-Gly-Phe-Leu-Arg-Arg-Ile-Arg- Pro-Lys-Leu-Lys
Peptides With Affinity for NK.sub.1 Receptor: Substance P
Peptides
[0051] The SP moiety of the chimeric peptide is designed to bind to
the NK.sub.1 receptor. SP is an 11 amino acid peptide, which has a
number of different natural and synthetic analogs. A representative
group is shown in Table 4, below. A number of SP amino-terminal
fragments and modified peptides have a high degree of specificity
for the NK.sub.1 receptor relative to NK.sub.2 and NK.sub.3
receptors. This specificity can be increased by esterification of
the carboxy terminal amide. Other modifications include the
generation of cyclic molecules (e.g. via Cys-Cys disulfide
bridges), the incorporation of non-peptidyl moieties (e.g.
spirolactones as discussed by Ward in J. Med. Chem. 33: 1848-1851
(1990)). Additionally, SP and SP analogs can be made more stable by
using D-amino acids. A representative listing of SP and its related
family of compounds is provided in Table 4 below. TABLE-US-00004
TABLE 4 SEQ ID NO: Compound Sequence 21 SP
Arg-Pro-Lys-Pro-Gln-Gln-Phe- Phe-Gly-Leu-Met-NH.sub.2 22 SP-Glycine
Arg-Pro-Lys-Pro-Gln-Gln-Phe- Phe-Gly-Leu-Met-Gly-NH.sub.2 23
SP-Glycine-Lysine Arg-Pro-Lys-Pro-Gln-Gln-Phe-
Phe-Gly-Leu-Met-Gly-Lys-NH.sub.2 24 SP-Glycine-Lysine-
Arg-Pro-Lys-Pro-Gln-Gln-Phe- Arginine Phe-Gly-Leu-Met-Gly-Lys-Arg-
NH.sub.2 25 SP-Glycine Methyl Arg-Pro-Lys-Pro-Gln-Gln-Phe- Ester
Phe-Gly-Leu-Met-Gly-O.sup.me 26 SP-Glycine-Lycine-
Arg-Pro-Lys-Pro-Gln-Gln-Phe- Methyl Ester
Phe-Gly-Leu-Met-Gly-Lys-O.sup.me 27 SP-Glycine-Lysine-
Arg-Pro-Lys-Pro-Gln-Gln-Phe- Arginine Methyl
Phe-Gly-Leu-Met-Gly-Lys-Arg- Ester O.sup.me 28 SP-Glycine-Elthyl
Arg-Pro-Lys-Pro-Gln-Gln-Phe- Ester Phe-Gly-Leu-Met-Gly-O.sup.eth 29
SP-Glycine-Lysine Arg-Pro-Lys-Pro-Gln-Gln-Phe- Ethyl Ester
Phe-Gly-Leu-Met-Gly-Lys-O.sup.eth 30 SP-Glycine-Lysine-
Arg-Pro-Lys-Pro-Gln-Gln-Phe- Arginine Ethyl
Phe-Gly-Leu-Met-Gly-Lys-Arg- Ester O.sup.eth 31 SP/1-4#
Arg-Pro-Lys-Pro-NH.sub.2 32 SP/1-7# Arg-Pro-Lys-Pro-Gln-Gln-Phe-
NH.sub.2 33 SP/1-9# Arg-Pro-Lys-Pro-Gln-Gln-Phe- Phe-Gly-NH.sub.2
34 [D-Pro2, D-Phe7, Arg-(D)Pro-Lys-Pro-Gln-Gln- D-Trp9]-SP
(D)Phe-Phe-(D)Trp-Leu-Met- NH.sub.2 35 [D-Pro2, D-Phe7,
Arg-(D)Pro-Lys-Pro-Gln-Gln- D-Trp9]-SP- (D)Phe-Phe-(D)Trp-Leu-Met-
(Glycine) Gly-NH.sub.2 36 [D-Pro2, D-Trp7,
Arg-(D)Pro-Lys-Pro-Gln-Gln- D-Trp9]-SP (D)Trp-Phe-(D)Trp-Leu-Met-
NH.sub.2 37 [D-Pro2, D-Trp7, Arg-(D)Pro-Lys-Pro-Gln-Gln-
D-Trp9]-SP- (D)Trp-Phe-(D)Trp-Leu-Met- Glycine Gly-NH.sub.2 38
[Cys3, Cys6, Tyr8, Arg-Pro-Cys-Pro-Gln-Cys-Phe- Pro10]-SP
Tyr-Gly-Pro-Met-NH.sub.2 39 [Glu6]-SP/6-11
Glu-Phe-Phe-Gly-Leu-Met-NH.sub.2 40 Septide
Glu-Phe-Phe-Pro-Leu-Met-NH.sub.2 41 Sanktide
HOOC-CH.sub.2-CH.sub.2-CO-Asp-Phe-(N-
Me)Phe-Gly-Leu-Met-NH.sub.2
[0052] If the target of the chimeric peptide is the .mu. receptor,
the opioid agonist moiety is chosen from those shown to be
selective for that receptor, e.g. those in Table 1. If the opioid
target receptor is the .delta. receptor, the opioid agonist moiety
is selected from the group consisting of DADLE, DSLET, DPDPE,
deltorphin I, deltorphin II and dermenkephalin. If the target
opioid receptor of the chimeric peptide is the K-receptor, the
opioid agonist moiety is selected from the group consisting of the
dynorphin peptides. The chimeric peptide may be synthesized to have
a plurality of opioid moieties. These opioid moieties may be
directed to any combination of the opioid receptors or may be
directed to the same receptor type. Furthermore, a chimera may be
synthesized to contain a plurality of SP moieties per each opioid
moiety. In one embodiment, the novel chimeric peptide is ESP7, SEQ
ID NO:42 (FIG. 1). Because it includes endomorphin-2 at the
N-terminus and SP (7-11) at the C-terminus, ESP7 is designed to
bind to the .mu. receptor and the NK.sub.1 receptor. One ESP7
derivative is ESP6, or Pro 5 ESP7:
Tyr-Pro-Phe-Phe-Pro-Leu-Met-NH.sub.2 (FIG. 2, SEQ ID NO:43).
Pharmaceutical Compositions
[0053] The chimeric peptides of the invention, and derivatives can
be incorporated into pharmaceutical compositions suitable for
administration. Such compositions typically comprise the peptide
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 peptide of the present invention to affect solubility
or clearance of the peptide. These molecules may also be
synthesized with D-amino acids to increase resistance to enzymatic
degradation. If necessary, the chimeric peptides can be
co-administered 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, rintradernal, or
subcutaneous application can include the following components: a
sterile diluent such as water for injection, saline solution, fixed
oils, polyethylene glycols, glycerine, 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, Cremophor EL.TM. (BASF, Parsippany, N.J.) 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 (e.g., chimeric peptide) 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.
[0059] 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.
[0060] 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.
[0061] 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 from Alza Corporation and Nova
Pharmaceuticals, Inc. 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.
[0062] 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 are 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.
[0063] Nucleic acid molecules encoding the chimeric peptides of the
invention can be inserted into vectors and used as gene therapy
vectors. Gene therapy vectors can be delivered to a subject by, for
example, intravenous injection, local administration (see U.S. Pat.
No. 5,328,470) or by stereotactic injection (see e.g., Chen et al.
(1994) PNAS 91:3054-3057). The pharmaceutical preparation of the
gene therapy vector can include the gene therapy vector in an
acceptable diluent, or can comprise a slow release matrix in which
the gene delivery vehicle is imbedded. Alternatively, where the
complete gene delivery vector can be produced intact from
recombinant cells, e.g., retroviral vectors, the pharmaceutical
preparation can include one or more cells that produce the gene
delivery system.
[0064] The pharmaceutical compositions can be included in a
container, pack, or dispenser together with instructions for
administration.
Treatment of Pain
[0065] The invention further provides methods of treating a mammal
for pain by administering a pharmaceutical composition (as
described above) in order to produce analgesia in the patient One
method to assess the analgesic properties of the chimeric peptides
is the tail flick test, which is administered to rats following
intrathecal, intracerebroventricular, and intraperitoneal
administration. The effects of opioid antagonists (e.g.,
naltrexone) and NK.sub.1 antagonists (e.g., RP67580) on the
activity of the peptides can be assessed according to methods
common in the art.
[0066] In order that this invention may be better understood, the
following examples are set forth These examples are for the
purposes of illustration only and are not to be construed as
limiting the scope of this invention in any manner.
EXAMPLE 1
In Vitro Binding of ESP7 to Opioid and SP Receptors in Rat Brain
Preparations
[0067] In order to assess the binding affinity of ESP7 to opioid
and SP receptor, binding assays to opioid and SP receptors were
performed with crude rat brain plasma membranes prepared using a
modified procedure of Zadina Zadina et al., Life Sci, 55: 461-466
(1994). These assays showed that ESP7 has a strong affinity for
both the .mu. receptor and the NK.sub.1 receptor in rat brain.
[0068] For binding assays to opioid receptors, frozen rat brains
(-80.degree. C.) were homogenized in 40 volumes of standard Tris
buffer (50 mM Tris HCl (pH 7.4), 0.2 mg/ml BSA, 2.5 mM EDTA, 40
.mu.g/ml bacitracin, 30 .mu.g/ml bestatin and 5 mM MgCl.sub.1) and
centrifuged at 15,000.times.g for 20 minutes. 100 mM NaCl was added
to the buffer, in order to remove endogenous ligands, and the
centrifugation was repeated After a wash with standard buffer, the
membrane preparation was finally resuspended in 10 volumes of
incubation buffer (standard buffer with 4 .mu.g/ml leupeptin and 2
.mu.g/ml chymostatin). The same procedure was followed for the SP
receptor except the wash with NaCl was eliminated and 5 mM
MgCl.sub.2 was replaced with 3 mM MnCl.sub.2. The brain homogenates
were used on the day of preparation.
[0069] Binding assays for the .mu. receptor were performed at
4.degree. C. for 90 minutes, as described in Zadina et al., Life
Sci, 55: 461-466 (1994). A final volume of 0.35 mL was used which
contained incubation buffer (described above), brain homogenate,
and 1.85 nM [.sup.3H]DAMGO with or without competing peptide (DAMGO
or ESP7). Nonspecific binding was determined with 10 .mu.M DAMGO.
After incubation the samples were filtered on a Brandell-Harvester
using an appropriate GF/B filter soaked in 50 mM Tris HCl (pH 7.4)
and 0.5% PEI. Scintillation fluid was added to the filters in order
to solubilize the membranes, and a beta-counter was used to
quantify radioactivity.
[0070] A procedure similar to the opioid binding assay was followed
for the SP receptor except the SP assays were performed at room
temperature for 75 minutes using 23 fmol of [.sup.125I]BH-SP. 10
.mu.M SP defined the non-specific binding. After filtration, the
radioactivity was determined on a gamma-counter.
[0071] As seen in the FIG. 3, DAMGO had a K.sub.d of approximately
3 nM (FIG. 8). ESP7 had a K.sub.d of approximately 300 nM,
illustrating that ESP7 possesses significant affinity for the .mu.
receptor. As seen in FIG. 4, SP had a K.sub.d of approximately 0.03
nM, while ESP7 had a K.sub.d of approximately 200 nM. Thus, ESP7
has significant affinity for the NK.sub.1 receptor, and, as
expected, ESP7 bound specifically to and had significant binding
affinity for both the .mu. receptor and the NK.sub.1 receptor.
EXAMPLE 2
Characterization of the Analgesic Properties of ESP7
[0072] ESP7 was tested clinically in rats to determine analgesic
effect and tolerance. The classical tail flick test was used to
measure pain response and thermal pain was mimicked using a heat
source. This system was controlled using standard opioids. The drug
was administered with cyclodextran to increase solubility of the
peptide in an aqueous solution.
[0073] 2.1 Intrathecal Administration of ESP7 in Rats and the
Effects of Naltrexone and RP67580 Blockades
[0074] Intrathecal administration of ESP7 produced long-lasting
analgesia without any significant development of tolerance. The
opioid antagonist naltrexone blocked this analgesia, indicating
that the analgesia was opioid in nature. Additionally, when the SP
portion was antagonized with RP67580, an NK.sub.1 antagonist,
tolerance to the drug developed within three days. These results
indicate that the SP moiety of ESP7 does not contribute to the
analgesia, but rather plays an integral role in preventing the
development of tolerance.
[0075] Adult male Sprague Dawley rats (200-250 g) were implanted
with chronic indwelling intrathecal catheters using a modified
protocol of Yaksh and Rudy, Physiol. Behav., 17: 1031-1036 (1976).
Catheters were made of silastic tubing, had an inside diameter of
0.012'' and an outside diameter of 0.025'', and measured a total of
11.5 cm with 7.5 cm inserted into the intrathecal space to level
T13-LI. The rats were anesthetized throughout the surgery with 5.0%
isoflurane. The catheter was inserted through the alanto-occipital
membrane and into the intrathecal space using a guide wire. Sutures
were used to secure the placement of the catheter. The rats were
allowed to recover from surgery for 3-4 days and any rats with
neurological impairment were not used for analgesic measurements.
Rats were housed separately in a 12 hr light-dark cycle with free
access to food and water. During their recovery from surgery, rats
were habituated to the laboratory environment and analgesic testing
apparatus.
[0076] For measurement of the thermal anti-nociceptive properties
of the peptides of interest, the tail flick test was employed. Rats
were first habituated to the tail flick chamber. During testing,
the rats were placed in the chamber and a light source, which
generated heat, was directed at their tail. The latency to remove
the tail was recorded. The baseline latency was approximately 3.5
sec and the cutoff latency was 10 sec to avoid tissue damage. Three
measurements were made at each pre- and post-treatment time point
and the results were averaged. Responses were expressed as %
maximum possible effect: % .times. .times. MPE = post-treatment
latency - baseline latency cutoff time - baseline .times. 100
##EQU1## After testing, the rats were sacrificed and the correct
placement of the catheter was verified by dissection of the spinal
cord.
[0077] Rats were given doses of 1.0 .mu.g (FIG. 5), 0.2 .mu.g (FIG.
6), and 0.05 .mu.g (FIG. 7) of ESP7. The desired concentration of
the compound (in 10 .mu.l) was injected into the catheter followed
by 10 .mu.l saline flush to fill the dead volume. ESP7 was combined
with two molecules of cyclodextrin (ESP7+2CD) to increase
solubility. Ultimately, cyclodextrin can form reversible complexes
with lipophilic compounds such as ESP7 to increase their
solubility, decrease their clearance from the spinal cord and
enhance their duration of action.
[0078] As shown in FIG. 5, the 1.0 .mu.g dose produced a low level
of prolonged analgesia for five days. More interestingly, no
tolerance developed to the effects of ESP7+2CD (p>0.05). As
shown in FIG. 6, the analgesia produced by 0.2 .mu.g remained at
the same level for five days (p>0.05). As shown in FIG. 7,
however, some tolerance did appear to develop at the 0.05 .mu.g
dose on day 5 (p=0.014). As a control 1.0 .mu.g of 2-cyclodextrin
was administered intrathecally with no significant effect
(p>0.05) (data not shown).
[0079] To examine whether the analgesia produced was opioid in
nature, the opioid receptor was antagonized with naltrexone. On the
days indicated below, naltrexone, was administered 10 min prior to
ESP7+2CD. As shown in FIG. 8, 0.2 .mu.g ESP7+2CD produced analgesia
on Days 1, 3, and 5, but not on Days 2 and 4 when naltrexone was
administered (p=0.0042). Similar results were seen with the 1.0
.mu.g of ESP7+2CD, where naltrexone again significantly blocked the
analgesia (p=0.0009) (data not shown). Naltrexone actually produced
some hyperalgesia when given with both doses of ESP7+2CD, unmasking
the nociceptive activity of SP. In addition, the naltrexone
blockade was reversible once the drug had been removed. A control
experiment illustrated that naltrexone alone produced no change in
analgesia (p>0.05)(data not shown).
[0080] To examine whether the reduced tolerance exhibited in rats
treated with ESP7 was SP mediated, the NK.sub.1 receptor was
antagonized with RP67580, a specific NK.sub.1 antagonist with high
affinity for the rat NK.sub.1 receptor. RP67580 (250 pmol) was
administered IT prior to 1.0 .mu.g ESP7+2CD. As seen in FIG. 9,
ESP7+2CD produced significant analgesia on Day 1, but tolerance
developed to this analgesia within three days (p<0.0001). Slight
hyperalgesia was present on Day 4. On Day 5, the NK.sub.1
antagonist was removed and a partial rescue of the analgesia
occurred. The level of analgesia on Day 5 reached a level similar
to Day 2 (p>0.05). RP67580 alone had no effect on the level of
analgesia (p>0.05). Therefore, ESP7 administered intrathecally
was able to induce analgesia while minimizing the development of
tolerance.
[0081] 2.2 Intracerebroventricular Administration of ESP7 in
Rats
[0082] Adult male Sprague Dawley rats weighing 200-250 g were use
Before surgery, the rats were anesthesized with 0.2-0.3 mL of
xylazine (10%) and ketamine (90%). Rats were positioned in a
stereotaxic apparatus and the bregma was located. To reach the
lateral ventricle, a hole was drilled 0.8 mm caudal and 1.4-1.5 mm
left or right of the bregma. The catheter was inserted 4.5 mm deep
into the brain and 4.0 cm of polyethylene tubing was connected to
the end to the catheter. Screws and dental cement were used to
secure the catheter in place. After suturing the skin, the rats
were allowed to recover from surgery for 4-5 days. Each rat was
housed separately in a 12 hr light-ark cycle with free access to
food and water. Rats with any neurological problems were not used
in the analgesic testing.
[0083] The tail flick assay was used to measure analgesia as
described above in Example 2.1. Briefly, the rats were first
habituated to the tail flick chamber. During testing, the rats were
placed in the chamber and a light source, which generated heat, was
directed at their tail. The latency to remove the tail was recorded
The baseline latency was approximately 3.5 sec and the cutoff
latency was 10 sec to avoid tissue damage. Three measurements were
made at each pre- and post-treatment time point and the results
were averaged. Responses were expressed as % maximum possible
effect (MPE): % .times. .times. MPE = post-treatment latency -
baseline latency cutoff time - baseline .times. 100 ##EQU2## After
resting the rats were sacrificed and the correct placement of the
catheter was verified.
[0084] As shown in FIG. 10, 0.1 .mu.g ESP7+2CD produced a low level
of analgesia that dissipated after one hour. ESP7 (1.0 .mu.g) also
produced analgesia (data not shown).
[0085] 2.3 Intraperitoneal Administration of ESP7 in Rats
[0086] ESP7 was administered intraperitoneally in order to assess
the effectiveness of ESP7 systemically. The tail flick assay was
used to measure analgesia as described above in Example 2.1.
Briefly, the rats were first habituated to the tail flick chamber.
During testing, the rats were placed in the chamber and a light
source, which generated heat, was directed at their tail. The
latency to remove the tail was recorded. The baseline latency was
approximately 3.5 sec and the cutoff latency was 10 sec to avoid
tissue damage. Three measurements were made at each pre- and
post-treatment time point and the results were averaged. Responses
were expressed as % maximum possible effect (MPE): % .times.
.times. MPE = post-treatment latency - baseline latency cutoff time
- baseline .times. 100 ##EQU3##
[0087] As seen in FIG. 11, 1 mg of ESP7 produced analgesia as seen
with intrathecal administration. In addition, 3 mg of ESP7+2CD also
produced analgesia similar to that seen with intrathecal
administration (data not shown). A dose of 1 mg of ESP7+2CD was
ineffective, thus a dose of 3 mg was chosen to account for the
presence of two molecules of cyclodextrin (data not shown).
Equivalents
[0088] From the foregoing detailed description of the specific
embodiments of the invention, it should be apparent that unique
chimeric analgesic peptides have been described. Although
particular embodiments have been disclosed herein in detail, this
has been done by way of example for purposes of illustration only,
and is not intended to be limiting with respect to the scope of the
appended claims which follow. In particular, it is contemplated by
the inventor that various substitutions, alterations, and
modifications may be made to the invention without departing from
the spirit and scope of the invention as defined by the claims. For
instance, the choice of the particular opioid moiety, or the
particular SP moiety is believed to be a matter of routine for a
person of ordinary skill in the art with knowledge of the
embodiments described herein.
Sequence CWU 1
1
43 1 15 PRT Artificial Human 1 Thr Gly Gly Phe Met Thr Ser Glu Ser
Gln Thr Pro Leu Val Thr 1 5 10 15 2 4 PRT Artificial Human 2 Tyr
Pro Trp Phe 1 3 4 PRT Artificial Human 3 Tyr Pro Phe Phe 1 4 7 PRT
Artificial Human 4 Tyr Ala Phe Gly Tyr Pro Ser 1 5 5 7 PRT
Artificial Bovine 5 Tyr Pro Phe Pro Gly Pro Ile 1 5 6 7 PRT
Artificial Human 6 Tyr Pro Phe Val Glu Pro Ile 1 5 7 4 PRT
Artificial Human 7 Tyr Pro Phe Pro 1 8 5 PRT Artificial Human 8 Tyr
Gly Gly Phe Leu 1 5 9 5 PRT Artificial Human 9 Tyr Gly Gly Phe Met
1 5 10 4 PRT Artificial Human 10 Tyr Arg Phe Lys 1 11 4 PRT
Artificial Human 11 Tyr Pro Phe Pro 1 12 5 PRT Artificial Human 12
Tyr Ala Gly Phe Leu 1 5 13 6 PRT Artificial Human 13 Tyr Ser Gly
Phe Leu Thr 1 5 14 5 PRT Artificial Human 14 Tyr Xaa Gly Phe Xaa 1
5 15 7 PRT Artificial Human 15 Tyr Ala Phe Asp Val Val Gly 1 5 16 7
PRT Artificial Human 16 Tyr Ala Phe Glu Val Val Gly 1 5 17 7 PRT
Artificial Human 17 Tyr Met Phe His Leu Met Asp 1 5 18 16 PRT
Artificial Human 18 Tyr Gly Gly Phe Leu Arg Arg Ile Arg Pro Lys Leu
Lys Trp Asp Asn 1 5 10 15 19 8 PRT Artificial Human 19 Tyr Gly Gly
Phe Leu Arg Arg Ile 1 5 20 13 PRT Artificial Human 20 Tyr Gly Gly
Phe Leu Arg Arg Ile Arg Pro Lys Leu Lys 1 5 10 21 11 PRT Artificial
Human 21 Arg Pro Lys Pro Gln Gln Phe Phe Gly Leu Met 1 5 10 22 12
PRT Artificial Human 22 Arg Pro Lys Pro Gln Gln Phe Phe Gly Leu Met
Gly 1 5 10 23 13 PRT Artificial Human 23 Arg Pro Lys Pro Gln Gln
Phe Phe Gly Leu Met Gly Lys 1 5 10 24 14 PRT Artificial Human 24
Ala Pro Lys Pro Gln Gln Phe Phe Gly Leu Met Gly Lys Arg 1 5 10 25
12 PRT Artificial Human 25 Arg Pro Lys Pro Gln Gln Phe Phe Gly Leu
Met Gly 1 5 10 26 13 PRT Artificial Human 26 Arg Pro Lys Pro Gln
Gln Phe Phe Gly Leu Met Gly Lys 1 5 10 27 14 PRT Artificial Human
27 Arg Pro Lys Pro Gln Gln Phe Phe Gly Leu Met Gly Lys Arg 1 5 10
28 12 PRT Artificial Human 28 Arg Pro Lys Pro Gln Gln Phe Phe Gly
Leu Met Gly 1 5 10 29 13 PRT Artificial Human 29 Arg Pro Lys Pro
Gln Gln Phe Phe Gly Leu Met Gly Lys 1 5 10 30 14 PRT Artificial
Human 30 Arg Pro Lys Pro Gln Gln Phe Phe Gly Leu Met Gly Lys Arg 1
5 10 31 4 PRT Artificial Human 31 Arg Pro Lys Pro 1 32 7 PRT
Artificial Human 32 Arg Pro Lys Pro Gln Gln Phe 1 5 33 9 PRT
Artificial Human 33 Arg Pro Lys Pro Gln Gln Phe Phe Gly 1 5 34 11
PRT Artificial Human 34 Arg Pro Lys Pro Gln Gln Phe Phe Trp Leu Met
1 5 10 35 12 PRT Artificial Human 35 Arg Pro Lys Pro Gln Gln Phe
Phe Trp Leu Met Gly 1 5 10 36 11 PRT Artificial Human 36 Arg Pro
Lys Pro Gln Gln Trp Phe Trp Leu Met 1 5 10 37 12 PRT Artificial
Human 37 Arg Pro Lys Pro Gln Gln Trp Phe Trp Leu Met Gly 1 5 10 38
11 PRT Artificial Human 38 Arg Pro Cys Pro Gln Cys Phe Tyr Gly Pro
Met 1 5 10 39 6 PRT Artificial Human 39 Glu Phe Phe Gly Leu Met 1 5
40 6 PRT Artificial Human 40 Glu Phe Phe Pro Leu Met 1 5 41 6 PRT
Artificial Human 41 Asp Phe Phe Gly Leu Met 1 5 42 7 PRT Artificial
Human 42 Tyr Pro Phe Phe Gly Leu Met 1 5 43 4 PRT Artificial Human
43 Tyr Ala Gly Phe 1
* * * * *