Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Peptide containing doai
Utility Patent
1997-03-18
2001-01-02
Kunz, Gary L. (Department: 1647)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Peptide containing doai
C530S324000, C530S326000, C514S013800
Utility Patent
active
06169074
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The invention is directed to peptides which inhibit the release of neurotransmitters from synaptic vesicles. More specifically, the invention provides peptides which mimic the inhibitory effect of
Clostridium botulinum
and
tetani
neurotoxins on the neurosecretory process.
2. History of the Invention
Botulinum neurotoxins (including the A, B, C, D, E, F and G serotypes produced by the anaerobic bacterium
Clostridium botulinum
; collectively, “BoTx”), and Tetanus neurotoxin (produced by the anaerobic bacterium
Clostridium tetani
; “TeTx”) cause temporary paralysis of muscle by blocking the presynaptic release of acetylcholine at the neuromuscular junction. A purified form of BoTxA is used clinically to alleviate chronic muscle spasm (such as occurs in dystonias, cerebal palsy and muscular dystrophy) through injection into the overactive muscle where it produces a dose-related weakness (for reviews of current therapeutical uses of botulinum toxins, see Jankovic,
Curr. Op. Neurol.
7358-366 (1994); Borodic, et al.,
Drug Safety
3:145-152 (1994); and, Hughes,
Drugs
48:888-893 (1994)). BoTxA is also becoming a popular, albeit unapproved, agent for use in minimizing facial wrinkles for those with no interest in aging gracefully (see, e.g., Tanouye, “A Few Wrinkles Still Remain in Quest for Youthful Skin”,
The Wall Street Journal
(Feb. 10, 1997)).
Despite their clinical significance, obstacles to widespread use of Clostridium neurotoxins exist, including instability of the toxins at room temperature, immunogenicity and toxicity-related limitations on their purification and storage.
SUMMARY OF THE INVENTION
The invention provides peptides which mimic certain desirable characteristics of Clostridium neurotoxins while avoiding many of the obstacles to their clinical use. In particular, like Clostridium neurotoxins, the peptides of the invention (excitation-secretion uncoupling peptides, or “ESUPs”) inhibit secretion of neurotransmitters (e.g., acetylcholine) from neuronal vesicles into the neuromuscular junction, thereby lessening muscular spasticity. However, unlike Clostridium neurotoxins, ESUPs do not suffer from the immunogenicity, toxicity and limited availability of their bacterial counterparts.
More specifically, ESUPs display antispastic activity, high specificity and low toxicity in vivo, with therapeutic effects which last for periods ranging from several days to weeks. Thus, therapeutic use of the ESUPs of the invention in lieu of Clostridium neurotoxins for all clinical applications to which such toxins are suited both reduces the incidence of unwanted side effects and allows therapy to be rapidly discontinued if unwanted side effects appear.
Further, due to their lower immunogenicity, administration of ESUPs in lieu of natural toxin reduces the onset of resistance, skin reactions and flu-like symptoms that occasionally accompany BoTx therapy.
ESUPs are also relatively straightforward to produce (by, for example, solid-phase peptide synthesis) as compared to the presently available manufacturing techniques which are used to prepare BoTx compounds for therapeutic use (see, e.g., Hambleton,
J. Neurol.,
239:16-20, 1992). Further, the ESUP manufacturing process does not require the stringent containment conditions involved in toxin production.
The ESUPs of the invention comprise synthetic and purified peptide fragments which correspond in primary structure to peptides which serve as binding domains for the assembly of a ternary protein complex (“docking complex”) which is believed to be critical to neuronal vesicle docking with the cellular plasma membrane prior to neurotransmitter secretion. Preferably, the primary sequence of the ESUPs of the invention also includes amino acids which are identical in sequence to the peptide products of BoTx and TeTx proteolytic cleavage of their respective natural substrates in neuronal cells, or fragments thereof (“proteolytic products”). For optimal activity, ESUPs of the invention have a minimum length of about 20 amino acids and a maximal length of about 28 amino acids.
Thus, in one embodiment of the invention, the ESUPs correspond in primary structure to binding domains in the docking complex, most preferably the region of such binding domains which are involved in the formation of a coiled-coil structure in the native docking complex proteins.
In another embodiment of the invention, the ESUPs further comprise proteolytic products of the cleavage of synaptosomal associated protein (25 kDa; “SNAP-25”) by BoTx serotypes A, E and C.
In an alternative embodiment of the invention, the ESUPs further comprise proteolytic products of the cleavage of vesicle-associated membrane protein (“VAMP-2”, also known in the art as “synpaptobrevin”) by BoTx serotypes B, D, F and G, as well as by TeTx.
In another alternative embodiment of the invention, the ESUPs are mixed with peptides which comprise proteolytic products of the cleavage of syntaxin by BoTx serotype C1.
For use in clinical applications, pharmaceutical compositions of the ESUPs of the invention are disclosed. ESUPs may also be used as pharmaceutical carriers as part of fusion proteins to deliver substances of interest into neural cells in a targeted manner.
REFERENCES:
patent: 95/33850 (1995-12-01), None
patent: 95/32738 (1995-12-01), None
Rudinger, In “Peptide Hormones” (ed. J.A. Parsons) University Park Press, Baltimore, pp. 1-7, 1976.
Schiavo, et al, “Botulinum Neurotoxin Type C Cleaves a Single Lys-Ala Bond within the Carboxyl-terminal Region of Syntaxins”; Journal of Biological Chemistry, May 5, 1995, Vol. 270, No. 18, pp. 10566-10570.
Schiavo, et al., “Identification of the Nerve Terminal Targets of Botulinum Neurotoxin Serotypes A, D and E”; Journal of Biological Chemistry; Nov. 15, 1993, vol. 268, No. 32, pp. 23784-53787.
Yamasaki, et al., “Botulinum Neurotoxin Type G Proteolyses the Ala81-Ala82Bond of Rat Synaptobrevin 2”; aBiochem. Biophys. Res. Comm., Apr. 29, 1994, vol. 200, No. 2, pp. 829-835.
Hayashi, et al., “Synaptic vesicle membrane fusion complex: action of clostridial neurotoxins on assembly”; EMBO Journal 1994, vol. 13, No. 21, pp. 5051-5061.
Canaves Jaume M.
Ferrer-Monteil Antonio V.
Montal Mauricio
Foley & Lardner
Hayes Robert C.
Kunz Gary L.
The Regents of the University of California
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