Selenocosmia huwena toxin and analgesic uses thereof

Details Selenocosmia huwena toxin and analgesic uses thereof Selenocosmia huwena toxin and analgesic uses thereof

2001-02-13

2003-12-30

Carlson, Karen Cochran (Department: 1653)

Drug, bio-affecting and body treating compositions

Designated organic active ingredient containing

Peptide containing doai

C514S002600, C530S350000, C435S069100, C435S320100, C435S440000, C435S094000, C435S007100, C435S007200, C536S023100, C424S009100, C424S278100

Reexamination Certificate

active

06670329

ABSTRACT:

CROSS-REFERENCE TO RELATED APPLICATIONS
Pursuant to 35 U.S.C. 35 §119, this application claims priority from China application No. 00104254.8, filed Apr. 11, 2000.
BACKGROUND
Analgesics and other medicines that relieve pain are critical therapeutics that vastly improve the quality of life of sufferers and their caretakers. Analgesics not only relieve pain, but also the dread, tension, anxiety and unpleasant perceptions inflicted by acute pain. Existing analgesics can be classified into at least two categories, non-steroidal anti-inflammatory drugs (NSAIDs), and narcotic analgesics. NSAIDs (e.g., aspirin) inhibit the synthesis of prostaglandin, thereby decreasing the sensitivity of nerve endings. NSAIDs are primarily used to relieve inflammatory pain and other kinds of dull pain. Narcotic analgesics (e.g., morphine) act on &mgr;-type opium receptors of the central nerve system, mainly in the thalamencephalon and cortex, to relieve sharp pain, as well as dull pain. Although widely used to treat pain, e.g., in late stage cancer patients, and post-operative patients, narcotic analgesics are highly addictive and are associated with severe withdrawal symptoms. Their dosage has to be increased continuously to sustain their analgesic effects. Further narcotic analgesics have little if any therapeutic value for pain caused by neurotrosis.
SUMMARY
The invention is based, in part, on the discovery that purified HWAP-I polypeptide of the Chinese bird spider,
Selenocosmia huwena,
is a very effective analgesic. Accordingly, the invention features a method of reducing perceived pain in a subject. The method includes administering to the subject an effective amount of a purified polypeptide having an amino acid sequence at least 70%, 80%, 90%, 92%, or 95% identical to SEQ ID NO:1. The purified polypeptide can be HWAP-I which has the amino acid sequence of SEQ ID NO:1. The polypeptide can be administered as an epidural or as a parenteral solution. In addition, the purified polypeptide can be applied locally (e.g., by injection or topical application). The purified polypeptide can be formulated as a pharmaceutical composition, e.g., with a pharmaceutically acceptable carrier, in a sterile solution, e.g., a sterile saline solution.
In another aspect, the invention features a method of inhibiting calcium channel activity in a subject. The method includes administering to the subject an effective amount of a purified polypeptide having an amino acid sequence at least 70%, 80%, 90%, 92%, or 95% identical to SEQ ID NO:1. The purified polypeptide can be HWAP-I which has the amino acid sequence of SEQ ID NO:1. The polypeptide can be administered as an epidural or as a parenteral solution. In addition, the purified polypeptide can be applied locally (e.g., by injection or topical application). The purified polypeptide can be formulated as a pharmaceutical composition, e.g., with a pharmaceutically acceptable carrier, in a sterile solution, e.g., a sterile saline solution. The method can reduce the amount of calcium channel activity in a local area of a subject, e.g., at least 10%, 20%, 30%, 40%, 50%, 70% or more using an assay described herein.
In another aspect, the invention features an article of manufacture. The article includes: i) a container; ii) a purified polypeptide, disposed in the container, and having an amino acid sequence at least 70%, 80%, 90%, 92%, or 95% identical to SEQ ID NO:1; and iii) a label, disposed on the container and having instructions for administration of the purified polypeptide. The purified polypeptide can be HWAP-I which has the amino acid sequence of SEQ ID NO:1. The purified polypeptide can be formulated as a pharmaceutical composition, e.g., with a pharmaceutically acceptable carrier, in a sterile solution, e.g., a sterile saline solution. The instructions can provide directions for administration of the purified polypeptide to a subject, e.g., for epidural, intrathecal, parenteral, or local administration. The instructions can indicate that the treatment is relief of pain in a subject or to reduce calcium channel activity in a subject.
In still another aspect, the invention features isolated nucleic acids that include a sequence at least 70%, 80%, 85%, 90%, 92%, 95% identical to SEQ ID NO:2 or 3. The isolated nucleic acid can be identical to SEQ ID NO:2 or 3. Further, the isolated nucleic acid can have a strand that hybridizes under high stringency conditions to a single-stranded probe, the sequence of which consists of SEQ ID NO:2 or 3 or the complement of SEQ ID NO:2 or 3. Also included are nucleic acids further containing a heterologous promoter operably linked to the HWAP-I-related sequence. The heterologous promoter can be a prokaryotic or eukaryotic promoter, and/or, an inducible promoter. The invention further features a method of providing a calcium channel inhibitor. The method includes: providing cells having a heterologous nucleic acid sequence encoding a polypeptide having an amino acid sequence at least 70%, 80%, 90%, or 95% identical to SEQ ID NO:1 and optionally a heterologous promoter operably linked to the encoding sequence; culturing the cells in a medium; extracting the polypeptide from the cells or the medium to thereby provide a calcium channel inhibitor.
A “purified polypeptide”, as used herein, refers to a polypeptide that has been separated from other proteins, lipids, and nucleic acids with which it is naturally associated. The polypeptide can constitutes at least 10%, 20% 50%, 70%, 80% or 95% of the purified preparation by dry weight.
An “isolated nucleic acid” is a nucleic acid the structure of which is not identical to that of any naturally occurring nucleic acid, or to that of any fragment of a naturally occurring genomic nucleic acid spanning more than three separate genes. The term therefore covers, for example, (a) a DNA which has the sequence of part of a naturally occurring genomic DNA molecule but is not flanked by both of the coding sequences that flank that part of the molecule in the genome of the organism in which it naturally occurs; (b) a nucleic acid incorporated into a vector or into the genomic DNA of a prokaryote or eukaryote in a manner such that the resulting molecule is not identical to any naturally occurring vector or genomic DNA; (c) a separate molecule such as a cDNA, a genomic fragment, a fragment produced by polymerase chain reaction (PCR), or a restriction fragment; and (d) a recombinant nucleotide sequence that is part of a hybrid gene, i.e., a gene encoding a fusion protein. Specifically excluded from this definition are nucleic acids present in mixtures of different (i) DNA molecules, (ii) transfected cells, or (iii) cell clones: e.g., as these occur in a DNA library such as a cDNA or genomic DNA library.
The “percent identity” of two amino acid sequences or of two nucleic acids is determined using the algorithm of Karlin and Altschul
Proc. Natl. Acad. Sci.
USA 87:2264-68, 1990, modified as in Karlin and Altschul
Proc. Natl. Acad. Sci.
USA 90:5873-77, 1993. Such an algorithm is incorporated into the NBLAST and XBLAST programs (version 2.0) of Altschul, et al.
J. Mol. Biol.
215:403-10, 1990. BLAST nucleotide searches can be performed with the NBLAST program, score=100, wordlength-12 to obtain nucleotide sequences homologous to the nucleic acid molecules of the invention. BLAST protein searches can be performed with the XBLAST program, score=50, wordlength=3 to obtain amino acid sequences homologous to the protein molecules of the invention. Where gaps exist between two sequences, Gapped BLAST can be utilized as described in Altschul et al.,
Nucleic Acids Res.
25(17):3389-3402, 1997. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. See resources of the National Center for Biotechnology Information (NCBI), Bethesda Md.
As used herein, the term “hybridizes under stringent conditions” refers to conditions for hybridization in 6× sodium chloride/sodium citrate (SSC) at about 45° C., follow

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