Drug – bio-affecting and body treating compositions – Antigen – epitope – or other immunospecific immunoeffector
Reexamination Certificate
1997-05-01
2001-03-20
Minnifield, Nita (Department: 1645)
Drug, bio-affecting and body treating compositions
Antigen, epitope, or other immunospecific immunoeffector
C424S234100, C424S235100, C424S236100, C424S239100, C424S247100, C424S183100, C424S178100, C424S179100, C424S164100, C424S167100, C424S832000, C530S300000, C530S350000
Reexamination Certificate
active
06203794
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates generally to the field of receptor-targeted biochemical delivery systems. More specifically, this invention relates to the use of modified polypeptide toxins as vehicles for delivering chemical compounds to cells bearing toxin receptors.
BACKGROUND OF THE INVENTION
Tetanus toxin (TeTx) and botulinum toxin (BoNT) are potent neurotoxins that induce paralysis by mechanisms that involve the inhibition of neurotransmitter release. These Clostridial neurotoxins are initially produced as single-chain proteins of ~150 kDa. Proteolytic cleavage then generates an active dichain molecule having a ~100 kDa heavy (H) and a ~50 kDa light (L) chain that are linked by a single interchain disulfide bond. The H chain contains domains which contribute to the binding of the toxin to neuronal cell surface receptors and which facilitate translocation of the L chain into cells. The L chain is responsible for blocking neurotransmitter release.
The mechanisms of toxin action have recently been clarified. The TeTx-L chain is a zinc-dependent protease specific for the vesicle-associated protein called synaptobrevin or vesicle-associated membrane protein (VAMP). The cleavage of VAMP by the TeTx-L chain inhibits neurotransmitter release by preventing the docking/fusion of transmitter-containing vesicles and the presynaptic membrane.
While a single isoform of TeTx is produced by
Clostridium tetani
, seven serologically distinct isoforms of BoNT are produced by
Clostridia botulinum
. These seven botulinum toxin species are designated as BoNT/A-G. Like tetanus toxin, the botulinum type B neurotoxin is a zinc-dependent protease. In
EMBO J.
12:4821 (1993), Blasi et al. proposed that the botulinum neurotoxin serotypes have evolved distinct substrate specificities while retaining a common protease activity. Botulinum neurotoxins B, D, F and G also cleave VAMP or a closely related isoform. In contrast, BoNT/A and BoNT/E cleave a synaptosome associated protein of molecular weight 25 kDa (SNAP-25). Finally, BoNT/C has been shown to cleave syntaxin. In addition to these target proteins, TeTx and BoNT/B have been reported to cleave Cellubrevin. Thus, the intraneuronal targets of the Clostridial toxins universally participate in the process of neurotransmitter release.
All of the Clostridial neurotoxins apparently bind different cell surface receptors and proteolyze cellular components that are required for neurotransmitter release. TeTx exerts its effect in the spinal cord and lower brain stem by reducing the activity of inhibitory neurons. The seven isoforms of BoNT all produce a flaccid paralysis. Mechanistically, the botulinum toxins selectively inhibit peripheral cholinergic nerve terminals which are predominantly found at neuromuscular junctions.
Certain zinc-dependent endoproteases contain the conserved amino acid sequence HExxH. In thermolysin, zinc binding is achieved via His
142
and His
148
within this motif, together with Glu
166
; the fourth ligand is water. Comparison of tetanus L chain with thermolysin and other zinc endoproteases revealed the presence of the same consensus motif. Conceivably then, Glu
234
of TeTx-L chain might correspond to the critical Glu
145
residue in thermolysin.
The role of Glu
234
within this motif in the L chain of TeTx has been studied using site-directed mutagenesis and an in vitro assay for the proteolysis of cellubrevin. In
Nature
364:346 (1993), McMahon et al. demonstrated that cellubrevin was not cleaved when COS cells were cotransfected with mutant L chain (Glu
234
substituted by Gln) and cellubrevin DNA constructs.
SUMMARY OF THE INVENTION
One aspect of the present invention relates of a chemical conjugate for treating a nerve cell related disorder. This conjugate includes an active or inactive botulinum or tetanus toxin having specificity for a target nerve cell. The toxin is conjugated to a drug or other bioactive molecule without affecting the toxin's ability to enter the target nerve cell. Thus, one aspect of the present invention relates to a chemical conjugate for treating a nerve cell related disorder. The chemical conjugate includes an inactive Clostridial neurotoxin having specificity for a target nerve cell, and a drug or other bioactive molecule attached to the neurotoxin. The neurotoxin retains its ability to enter the target nerve cell. The Clostridial neurotoxin can be any of a variety of such toxins, including tetanus toxin, botulinum toxin A, botulinum toxin B, botulinum toxin C, botulinum toxin D, botulinum toxin E, botulinum toxin F and botulinum toxin G. Inactivation of the Clostridial neurotoxin can be accomplished by an amino acid change in its light chain. Thus, for example, the inactivated Clostridial neurotoxin can be tetanus toxin having a modification of Glu
234
, a botulinum toxin A having a modification at His
227
and/or Glu
224
, or a botulinum toxin other than botulinum toxin A having a modification at a site corresponding to His
227
and/or Glu
224
of botulinum toxin A.
Another aspect of the invention involves the chemical conjugate as described above, for use in the treatment of a neuromuscular dysfunction in a mammal, such as a dysfunction relating to uncontrollable muscle spasms.
The invention also includes the use of the chemical conjugate described above in the preparation of a medicament for treatment of a neuromuscular dysfunction, such as a dysfunction relating to uncontrollable muscle spasms in a mammal.
In a particular aspect of the invention, the drug in the chemical conjugate is an active ingredient for treatment of botulism or tetanus. This aspect of the invention can be used to treat botulism or tetanus in a mammal, and can thus be used in the preparation of a medicament for such treatment.
Another aspect of the invention relates to the use of an inactive Clostridial neurotoxin in the preparation of a medicament for treatment of botulinum toxin poisoning. In this aspect, the inactive Clostridial neurotoxin can be used alone without conjugation to another drug.
An additional aspect of the present invention relates to the use of chemical conjugate that includes an active clostridial neurotoxin and a drug. Such a conjugate is used in the preparation of a medicament for treatment of focal dystonias, spasticities due to stroke or traumatic brain or spinal cord injury, blepharospasm, strabismus, cerebral palsy or back pain due to muscle spasms.
In still another aspect, the invention relates to a method of treating a neuromuscular dysfunction in a mammal. This method includes the steps of preparing a pharmaceutically active solution which includes a Clostridial neurotoxin linked to a drug, and introducing an effective quantity of the pharmaceutically active solution into a mammal. The neurotoxin can be any of a variety of such toxins, including tetanus toxin, botulinum toxin A, botulinum toxin B, botulinum toxin C, botulinum toxin D, botulinum toxin E, botulinum toxin F and botulinum toxin G. The neurotoxin can be inactivated by an amino acid change in its light chain. In one embodiment, the drug inhibits neurotransmitter release, and in another the drug inhibits the activity of synaptobrevin. In a preferred application of the method, the method is used to treat a neuromuscular dysfunction relating to uncontrollable muscle spasms.
Further aspects of the present invention will be apparent to one having ordinary skill in the art upon reference to the ensuing detailed description.
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Aoki Kei Roger
Dolly James Oliver
Garst Michael Elwood
Wheeler Larry Allen
Allergan Sales Inc.
Baran Robert J.
Fisher Carlos A.
Minnifield Nita
Voet Martin A.
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