&agr;-bungarotoxin molecules and uses thereof

Details &agr;-bungarotoxin molecules and uses thereof &agr;-bungarotoxin molecules and uses thereof

2001-02-23

2004-06-22

Carlson, Karen Cochrane (Department: 1653)

Drug, bio-affecting and body treating compositions

Designated organic active ingredient containing

Peptide containing doai

Reexamination Certificate

active

06753315

ABSTRACT:

FIELD OF THE INVENTION
This invention relates to modified &agr;-bungarotoxin molecules and to methods for selectively inhibiting neurotransmitter receptors using modified and unmodified &agr;-bungarotoxin molecules. More specifically, the invention relates to the use of &agr;-bungarotoxin compositions for the localized inhibition of neuronal and/or muscle cell function.
BACKGROUND OF THE INVENTION
At the neuromuscular junction, the nicotinic acetyicholine receptor (nAChR) mediates muscle contraction through the binding at the muscle membrane surface of acetyicholine, which is released from the skeletal motorneuron. There is growing evidence that nAChRs in the nervous system play critical roles in a wide variety of physiological responses and pathological states (Sargent,
Annu. Rev. Neurosci.
16:403-443, 1993; McGehee & Role,
Annu. Rev. Physiol.
57:521-546, 1995; Boyd,
Crit. Rev. Toxicol.
27:299-318, 1997),
and mediate the behavioral effects of nicotine (Dani & Ileinemann,
Neuron
16:905-908, 1996).
The class of snake venom proteins known as a-neurotoxins, which are competitive antagonists of nAChRs, have been extremely useful tools in the study of the al-subunit containing muscle-type nAChR and the &agr;7-subunit containing neuronal nAChR (Endo & Tamiya, in
Snake Toxins
A.L. Harvey, Ed., pp 165-222, Pergamon Press, Inc., New York, N.Y., 1991; Chiappinelli, in
Natural and Synthetic Neurotoxins,
A. Harvey, ed., pp.65-128, Academic Press, New York, N.Y., 1995). &agr;-Neurotoxins have a highly conserved fold, due primarily to four invariant disulfide bonds and are classified as either “short” with 60-62 residues and four disulfides, or “long” with 66-74 residues and a fifth disulfide. The long &agr;-neurotoxin, &agr;-bungarotoxin (&agr;Bgtx), from the venom of
Bungarus multicinctus,
has played a critical role in the biochemical purification and characterization of nAChRs from muscle (e.g., Changeux et al.,
Proc. Natl.Acad Sci. USA
67:1241-1247, 1970) and brain (Whiting & Lindstrom,
Biochem.
25:2082-2093, 1986; Whiting & Lindstrom,
Proc. Nati. Acad Sci. USA
84:595-599, 1987
) due to the essentially irreversible binding of aBgtx to these receptors.
Venom-derived &agr;Bgtx has been a critically important tool in biochemical studies of nAChRs largely because of its high affinity and nearly irreversible binding characteristics (Chiappinelli, 1995). Similar biochemical studies of most neuronal nAChRs, apart from &agr;7 subunit-containing receptors, have been hampered by the lack of similar high-affinity probes. &kgr;-Bungarotoxin blocks cholinergic transmission in peripheral nervous tissue (e.g., Chiappinelli et al.,
Toxicon
34:1243-1256, 1996) and has a high specificity for &agr;3&bgr;2 receptors (Grant et al.,
Biochem.
37:12166-12171, 1998), but this toxin is in relatively short supply. Recently, two naturally-occurring toxins isolated from the venom of Conus snails have been shown to be selective for &agr;3&bgr;2 and &agr;3&bgr;4 nAChRs (Cartier et al.,
J. Biol. Chem.
271:7522-7528, 1996; Luo et al.,
J. Neurosci.
18:8571-8579, 1998) although their utility may be limited by their very rapid dissociation kinetics. Thus there is a need for additional well characterized molecular probes of high specificity for investigation of the properties of nAChRs, particularly &agr;7 subunit-containing nAChRs.
Bacterial, snake and snail toxins typically affect neurotransmitter uptake and/or release at neuromuscular synapses. Despite their lethality, a variety of these toxins are used as therapeutic agents in human disease. For example, treatment of certain neuromuscular disorders involving local muscle spasticity or dystonia (see, e.g., U.S. Pat. Nos. 5,721,215; 5,677,308; 5,562,907, 5,053,005, etc.) involves injection of a chemodenervating agent, currently a botulinum toxin preparation (BOTOX®, Allergan, Irvine, Calif.), directly into the muscle, using, for example, a fine gauge teflon-coated needle under electromyographic control to aid the physician in locating the intended intramuscular locus of the injection. A sufficient dose of the toxin acts on striated muscle to block release of the acetylcholine neurotransmitter from the presynaptic membrane resulting in varying degrees of effective denervation of the muscle in regions contacted by the toxin (i.e., causing local paralysis). This results in an increase in post-synaptic acetylcholinesterase activity and an increase in the population of acetylcholine receptors, effects which occur as a characteristic physiological response to denervation. After a period of days, the axon terminals develop sprouting, and over a period of several months, collateral motor axons establish new neuromuscular connections with the muscle fiber. As neuromuscular junctions are regenerated, full function of the muscle returns along with the spasmodic contractions or hyperstimulation symptomatic of the disease.
Botulinum toxin treatment is also known to be associated with a number of side effects. Such side effects include transient fatigue, dysphagia, neck weakness, hoarseness and localized pain. In addition, many patients who preliminarily respond to botulinum toxin therapy subsequently become non-responsive to the treatment. Accordingly, for many patients the botulinum injections fail to provide satisfactory long term treatment of the condition. Thus there is a need to develop additional chemodenervating agents that do not have the foregoing deficiencies of botulinum toxin.
SUMMARY OF THE INVENTION
The invention provides compositions and methods for nonpermanent and localized inhibition of neuronal cell function. It has now been discovered that &agr;-bungarotoxin and modified &agr;-bungarotoxin molecules are useful for reducing neurotransmitter effects at neuromuscular junctions, thereby inducing the temporary paralysis of muscles. These effects are useful for the treatment of aberrant muscle contraction, inter alia in the cosmetic treatment of facial wrinkles, in strabismus, blepharospasm, various dystonias and other conditions having neuromuscular components.
According to one aspect of the invention, methods of enhancing relaxation or slackening of cutaneous tissue are provided. The methods include locally administering to a cutaneous tissue an isolated native or modified &agr;-bungarotoxin molecule in an amount effective to enhance denervation of the muscle or group of muscles present subcutaneous to the cutaneous tissue to enhance relaxation or slackening of the cutaneous tissue. In some embodiments, the &agr;-bungarotoxin is administered subcutaneously.
Preferably the relaxation or slackening of the cutaneous tissue results in lessening of wrinkles or fine lines of the skin. In some embodiments the methods further include co-administering an anti-wrinkle agent selected from the group consisting of hydroxy acids and retinoids. In preferred embodiments the hydroxy acid is selected from the group consisting of &agr;-hydroxy acids and &bgr;-hydroxy acids, which can be linear, branched or cyclic and saturated or unsaturated and the retinoid is selected from the group consisting of retinoic acid, retinol and retinol esters.
In certain embodiments, the &agr;-bungarotoxin molecule is an isolated native &agr;-bungarotoxin molecule or a fragment thereof, preferably one that includes SEQ ID NO:2. On other embodiments, the &agr;-bungarotoxin molecule is an isolated modified &agr;-bungarotoxin molecule or a fragment thereof. Preferred modified &agr;-bungarotoxin molecules include those having at least one amino acid substitution selected from the group consisting of a substitution at amino acid 38 and a substitution at amino acid 42 of SEQ ID NO:2.
According to another aspect of the invention, methods of treating spasm or involuntary contraction in a muscle or a group of muscles in a subject are provided. The methods include administering to a muscle or a group of muscles in a subject in need of such treatment an isolated native or modified &agr;-bungarotoxin molecule, in an amount effective to inhibit spasm or involuntary contraction in the musc

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