United States Patent: 6,780,413
Issued: August 24, 2004
Inventors: Hott; Jonathan S. (Birmingham, MI); Youle; Richard J. (Bethesda, MD); Hallett; Mark (Bethesda, MD); Dalakas; Marinos C. (Bethesda, MD)
Assignee: The United States of America as represented by the Department of Health and (Washington, DC)
Appl. No.: 005512
Filed: November 7, 2001
Compositions and methods for treatment of focal muscle spasms. Immunotoxin conjugates comprise a toxin conjugated to an antibody reactive to a muscle specific antigen.
SUMMARY OF THE INVENTION
In one aspect the present invention is directed to a method of treating a focal muscle spasm. The method comprises the steps of administering, by intramuscular injection, a therapeutically effective dose of an immunotoxin conjugate to a muscle of the focal muscle spasm. The immunotoxin conjugate comprises an antibody conjugated to a toxin selected from the group consisting of: ricin and abrin, and the antibody is selectively reactive, under immunologically reactive conditions, to a nicotinic acetylcholine receptor (nAchR). In preferred embodiments the antibody is a monoclonal antibody. Typically, the mammalian acetylcholine receptor is a human acetylcholine receptor. In particularly preferred embodiments the toxin is ricin. Typically the focal muscle spasm is selected from the group consisting of: blepharospasm, cervical dystonia, hand dystonia, limb dystonia, hemifacial spasm, bruxism, strabismus, VI nerve palsy, spasmodic dysphonia, and oromandibular dystonia. In other embodiments a therapeutically effective amount of the immunotoxin conjugate is administered with a therapeutically effective amount of botulinum toxin, as an immunoconjugate or in unconjugated form.
In another aspect the present invention relates to a method of treating a focal muscle spasm. The method comprises the steps of administering, by intramuscular injection, a therapeutically effective dose of an immunotoxin conjugate to a muscle of the focal muscle spasm. The immunotoxin conjugate comprises an antibody conjugated to a galactose binding moiety and a toxin selected from the group consisting of: ricin-A and abrin-A, and the antibody is selectively reactive, under immunologically reactive conditions, to a nicotinic acetylcholine receptor (nAchR). In some embodiments the galactose binding moiety is selected from the group consisting of: ricin-B and abrin-B. In preferred embodiments the antibody is a monoclonal antibody. Typically, the mammalian acetylcholine receptor is a human acetylcholine receptor. In particularly preferred embodiments the toxin is ricin. Typically the focal muscle spasm is selected from the group consisting of: blepharospasm, cervical dystonia, hand dystonia, limb dystonia, hemifacial spasm, bruxism, strabismus, VI nerve palsy, spasmodic dysphonia, and oromandibular dystonia.
In another aspect the present invention relates to an immunotoxin conjugate, comprising an antibody conjugated to a toxin selected from the group consisting of: ricin and abrin, where the antibody is selectively reactive, under immunologically reactive conditions, to a mammalian nicotinic acetylcholine receptor. In preferred embodiments the antibody is a monoclonal antibody. Typically, the mammalian acetylcholine receptor is a human acetylcholine receptor. In particularly preferred embodiments the toxin is ricin.
DETAILED DESCRIPTION OF THE INVENTION
Intramuscular injection of botulinum toxin A (BTX) is often considered primary therapy of many disorders characterized by muscular spasms. The utility of BTX, however, is limited by its short duration of action, the possible development of resistance after repeated injections, and cross-reactivity with autonomic neurons. Surprisingly, we have determined an immunotoxin (ITX) engineered to damage skeletal muscle fibers selectively by chemically linking a monoclonal antibody against the nicotinic acetylcholine receptor to the toxin ricin was 20,000-fold more toxic to myotubes than myoblasts, consistent with the degree of acetylcholine receptor expression. In vivo, ITX produced destructive myopathic changes at a dose 300-fold less than the maximum tolerated dose. Assessment of rat muscle strength after unilateral gastrocnemius injections showed ITX was more effective and had a longer duration of action than BTX. Immunotoxins of the present invention have utility as a tissue culture selection agent against cells or tissues expressing nicotinic acetylcholine receptors (nAchR) Immunotoxins of the present invention also have utility in the treatment of involuntary muscle spasms. Patients repeatedly exposed to botulinum toxin for the treatment of muscle spasms frequently become resistant to its use. Consequently, surgical treatment is often indicated. Intramuscular injection of the immunotoxin conjugates of the present invention can delay or prevent the requirement for surgery.
Antibodies to Muscle Specific Antigens
Antibodies of the present invention are selectively reactive, under immunologically reactive conditions, to a muscle specific antigen. The term "muscle specific antigen" includes reference to those antigens whose presence is substantially limited to the membrane of muscle cells at the localized site at which the immunotoxin of the present invention is administered. Thus a muscle specific antigen may be present on non-muscle cells but is substantially inaccessible to the immunotoxins of the present invention due to the mode of administration. Preferably, however, muscle specific antigens are unique to muscle cells.
Muscle specific antigens are known in the art. For example, antibodies reactive to N-CAM (neuronal cell adhesion molecule) can and have been generated and are available commercially (Sigma Chemical Company, St. Louis, Mo.). Anti-N-CAM monoclonals bind to the CD56 differentiation antigen specifically expressed on regenerating or newly denervated muscle fibers (Couvalt and Sanes, Proc. Natl. Acad. Sci. USA (1985) 82:4544-4548; Cashman et al., Ann. Neurol. (1987) 21:481-489; IIIa I, Leon-Monzon M, Dalakas M C., Ann. Neurol. 1992; 31:46-52). Likewise, the muscle-specific antigen Leu-19 (Becton Dickinson) can be used to generate antibodies by standard immunological methods. Antibodies to other muscle specific antigens, such as monoclonal anti-dystrophin, are commercially available (Sigma).
In preferred embodiments the muscle specific antigen is a nicotinic acetylcholine receptor (nAchR). The nAch receptor and antibodies generated thereto are readily available from publicly accessible depositories. Cell line TE671 (equivalent to the RD cell line) expresses AchR for preparation of anti-human nAchR antibodies and is available from the ATCC under deposit number CRL 8805. See, U.S. Pat. Nos. 5,041,389, and 4,789,640, both incorporated herein by reference. Hybridomas producing a human IgG1 monoclonal antibody against nAchR is deposited with the Fermentation Research Institute of Japan under the accession number FERM BP-1798 (U.S. Pat. No. 5,192,684, incorporated herein by reference). Monoclonal antibodies to acetylcholine receptors are produced by hybridomas having accession number ATCC Nos.: HB 8987 (mAb 64), HB 189 (mAb 270), and TIB 175 (mAb 35), all of which are incorporated herein by reference.
Many methods of making antibodies are known to persons of ordinary skill. "Antibody" includes antigen binding forms of antibodies (e.g., Fab, F(ab)2). The term also refers to a polypeptide substantially encoded by an immunoglobulin gene or immunoglobulin genes, or fragments thereof which specifically bind and recognize an analyte (antigen). The recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon and mu constant region genes, as well as the myriad immunoglobulin variable region genes. Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.
An exemplary immunoglobulin (antibody) structural unit comprises a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light" (about 25 kD) and one "heavy" chain (about 50-70 kD). The N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The terms variable light chain (VL) and variable heavy chain (VH) refer to these light and heavy chains respectively.
Antibodies exist e.g., as intact immunoglobulins or as a number of well characterized fragments produced by digestion with various peptidases. Thus, for example, pepsin digests an antibody below the disulfide linkages in the hinge region to produce F(ab)'2, a dimer of Fab which itself is a light chain joined to VH -CH 1 by a disulfide bond. The F(ab)'2 may be reduced under mild conditions to break the disulfide linkage in the hinge region, thereby converting the F(ab)'2 dimer into an Fab' monomer. The Fab' monomer is essentially an Fab with part of the hinge region (see, Fundamental Immunology, Third Edition, W. E. Paul, ed., Raven Press, N.Y. 1993). While various antibody fragments are defined in terms of the digestion of an intact antibody, one of skill will appreciate that such fragments may be synthesized de novo either chemically or by utilizing recombinant DNA methodology. Thus, the term antibody, as used herein, also includes antibody fragments such as single chain Fv, chimeric antibodies (i.e., comprising constant and variable regions from different species), humanized antibodies (i.e., comprising a complementarity determining region (CDR) from a non-human source) and heteroconjugate antibodies (e.g., bispecific antibodies).
The following discussion is presented as a general overview of the techniques available; however, one of skill will recognize that many variations of the following methods are known.
A. Antibody Production
A number of immunogens are used to produce antibodies specifically reactive with a muscle specific antigen. A recombinant, synthetic, or native muscle specific antigen of 5 contiguous amino acids in length or greater from a muscle specific antigen is the preferred immunogen (antigen) for the production of monoclonal or polyclonal antibodies. The term "recombinant" when used with reference to a cell, or nucleic acid, or vector, includes reference to a cell, or nucleic acid, or vector, that has been modified by the introduction of a heterologous nucleic acid or the alteration of a native nucleic acid to a form not native to that cell, or that the cell is derived from a cell so modified. Thus, for example, recombinant cells express genes that are not found within the native (non-recombinant) form of the cell or express native genes that are otherwise abnormally expressed, under expressed or not expressed at all.
In a typical procedure, the muscle specific antigen is injected into an animal capable of producing antibodies. Methods of producing polyclonal antibodies are known to those of skill in the art. In brief, an immunogen (antigen), preferably a purified muscle specific antigen (e.g., nAchR), an muscle specific antigen coupled to an appropriate carrier (e.g., GST, keyhole limpet hemanocyanin, etc.), or an muscle specific antigen incorporated into an immunization vector such as a recombinant vaccinia virus (see, U.S. Pat. No. 4,722,848) is mixed with an adjuvant and animals are immunized with the mixture. The animal's immune response to the immunogen preparation is monitored by taking test bleeds and determining the titer of reactivity to the muscle specific antigen of interest. When appropriately high titers of antibody to the immunogen are obtained, blood is collected from the animal and antisera are prepared. Further fractionation of the antisera to enrich for antibodies reactive to the muscle specific antigen is performed where desired (see, e.g., Coligan (1991) Current Protocols in Immunology Wiley/Greene, NY; and Harlow and Lane (1989) Antibodies: A Laboratory Manual Cold Spring Harbor Press, N.Y.).
Antibodies, including binding fragments and single chain recombinant versions thereof, against predetermined fragments of muscle specific antigen are raised by immunizing animals, e.g., with conjugates of the fragments with carrier proteins as described above. Typically, the immunogen of interest is an muscle specific antigen of at least about 5 amino acids, more typically the muscle specific antigen is 10 amino acids in length, preferably, 15 amino acids in length and more preferably the muscle specific antigen is 20 amino acids in length or greater. The peptides are typically coupled to a carrier protein (e.g., as a fusion protein), or are recombinantly expressed in an immunization vector. Antigenic determinants on peptides to which antibodies bind are typically 3 to 10 amino acids in length.
Monoclonal antibodies are prepared from cells secreting the desired antibody. Monoclonals antibodies are screened for binding to an muscle specific antigen from which the immunogen was derived. Specific monoclonal and polyclonal antibodies will usually bind with a KD of at least about 0.1 mM, more usually at least about 50 .mu.M, and most preferably at least about 1 .mu.M or better.
In some instances, it is desirable to prepare monoclonal antibodies from various mammalian hosts, such as mice, rodents, primates, humans, etc. Description of techniques for preparing such monoclonal antibodies are found in, e.g., Stites et al. (eds.) Basic and Clinical Immunology (4th ed.) Lange Medical Publications, Los Altos, Calif., and references cited therein; Harlow and Lane, Supra; Goding (1986) Monoclonal Antibodies: Principles and Practice (2d ed.) Academic Press, New York, N.Y.; and Kohler and Milstein (1975) Nature 256: 495-497. Summarized briefly, this method proceeds by injecting an animal with an immunogen comprising an muscle specific antigen. The animal is then sacrificed and cells taken from its spleen, which are fused with myeloma cells. The result is a hybrid cell or "hybridoma" that is capable of reproducing in vitro. The population of hybridomas is then screened to isolate individual clones, each of which secrete a single antibody species to the immunogen. In this manner, the individual antibody species obtained are the products of immortalized and cloned single B cells from the immune animal generated in response to a specific site recognized on the immunogenic substance.
Alternative methods of immortalization include transfection with Epstein Barr Virus, oncogenes, or retroviruses, or other methods known in the art. Colonies arising from single immortalized cells are screened for production of antibodies of the desired specificity and affinity for the antigen, and yield of the monoclonal antibodies produced by such cells is enhanced by various techniques, including injection into the peritoneal cavity of a vertebrate (preferably mammalian) host. The muscle specific antigens and antibodies of the present invention are used with or without modification, and include chimeric antibodies such as humanized murine antibodies.
Other suitable techniques involve selection of libraries of recombinant antibodies in phage or similar vectors (see, e.g., Huse et al. (1989) Science 246: 1275-1281; and Ward, et al. (1989) Nature 341: 544-546; and Vaughan et al. (1996) Nature Biotechnology, 14: 309-314). Alternatively, high avidity human monoclonal antibodies can be obtained from transgenic mice comprising fragments of the unrearranged human heavy and light chain Ig loci (i.e., minilocus transgenic mice). Fishwild et al., Nature Biotech., 14:845-851 (1996).
Also, recombinant immunoglobulins may be produced. See, Cabilly, U.S. Pat. No. 4,816,567; and Queen et al. (1989) Proc. Nat'l Acad. Sci. USA 86: 10029-10033.
B. Human or Humanized (Chimeric) Antibody Production
The anti-muscle specific antigen antibodies of this invention can also be administered to a mammal (e.g., a human patient) for therapeutic purposes (e.g., as targeting molecules when conjugated or fused to effector molecules such as labels, cytotoxins, enzymes, growth factors, drugs, etc.). Antibodies administered to an organism other than the species in which they are raised are often immunogenic. Thus, for example, murine antibodies administered to a human often induce an immunologic response against the antibody (e.g., the human anti-mouse antibody (HAMA) response) on multiple administrations. The immunogenic properties of the antibody are reduced by altering portions, or all, of the antibody into characteristically human sequences thereby producing chimeric or human antibodies, respectively.
i) Humanized (Chimeric) Antibodies
Humanized (chimeric) antibodies are immunoglobulin molecules comprising a human and non-human portion. More specifically, the antigen combining region (or variable region) of a humanized chimeric antibody is derived from a non-human source (e.g., murine) and the constant region of the chimeric antibody (which confers biological effector function to the immunoglobulin) is derived from a human source. The humanized chimeric antibody should have the antigen binding specificity of the non-human antibody molecule and the effector function conferred by the human antibody molecule. A large number of methods of generating chimeric antibodies are well known to those of skill in the art (see, e.g., U.S. Pat. Nos. 5,502,167, 5,500,362, 5,491,088, 5,482,856, 5,472,693, 5,354,847, 5,292,867, 5,231,026, 5,204,244, 5,202,238, 5,169,939, 5,081,235, 5,075,431, and 4,975,369). Detailed methods for preparation of chimeric (humanized) antibodies can be found in U.S. Pat. No. 5,482,856.
ii) Human Antibodies
In another embodiment, this invention provides for fully human anti-muscle specific antigen antibodies. Human antibodies consist entirely of characteristically human polypeptide sequences. The human anti-muscle specific antigen antibodies of this invention can be produced in using a wide variety of methods (see, e.g., Larrick et al, U.S. Pat. No. 5,001,065, for review).
In preferred embodiments, the human anti-muscle specific antigen antibodies of the present invention are usually produced initially in trioma cells. Genes encoding the antibodies are then cloned and expressed in other cells, particularly, nonhuman mammalian cells. The general approach for producing human antibodies by trioma technology has been described by Ostberg et al. (1983), Hybridoma 2: 361-367, Ostberg, U.S. Pat. No. 4,634,664, and Engelman et al., U.S. Pat. No. 4,634,666. The antibody-producing cell lines obtained by this method are called triomas because they are descended from three cells; two human and one mouse. Triomas have been found to produce antibody more stably than ordinary hybridomas made from human cells.
The genes encoding the heavy and light chains of immunoglobulins secreted by trioma cell lines are cloned according to methods, including the polymerase chain reaction, known in the art (see, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor, N.Y., 1989; Berger & Kimmel, Methods in Enzymology, Vol. 152: Guide to Molecular Cloning Techniques, Academic Press, Inc., San Diego, Calif., 1987; Co et al. (1992) J. Immunol., 148: 1149). For example, genes encoding heavy and light chains are cloned from a trioma's genomic DNA or cDNA produced by reverse transcription of the trioma's RNA. Cloning is accomplished by conventional techniques including the use of PCR primers that hybridize to the sequences flanking or overlapping the genes, or segments of genes, to be cloned.
Formation of Immunotoxic Conjugates
Antibodies specifically reactive to muscle specific antigens are joined via covalent or non-covalent bond to a toxin selected from the group comprising: ricin, abrin, ricin-a, abrin-a, and botulinum toxin. Ricin, abrin, and subunits thereof as well as botulinum toxin A through F, are readily available from commercial sources (e.g, Sigma Chemical Company, St. Louis, Mo.). Methods of isolating ricin and abrin are also well known to those of ordinary skill in the art. See, e.g., Nicholson and Blaustein, J. Biochim. Biophys. Acta, 266:543 (1972); Tomita et al., Experientia, 28:84 (1972); Wei et al., J. Biol. Chem., 249:3061 (1974); Lin et al. Toxicon., 19:41 (1981); Olsnes et al. J. Biol. Chem. 249:803 (1974); Wei et al., J. Mol. Biol., 123:707 (1978); Lin and Li, Eur. J. Biochem., 105:453 (1980); Nicolson et al. Biochemistry, 13:196 (1974); and, Olsnes, Methods Enzymol. 50:330-335 (1978), all of which are incorporated herein by reference. The molecules may be attached by any of a number of means well-known to those of skill in the art. In some embodiments, the immunotoxic conjugates of the present invention are recombinantly expressed as single-chain fusion protein comprising both antibody and toxin. Typically the toxin will be conjugated, either directly or through a linker (spacer), to the ligand.
A "linker", as used herein, is a molecule that is used to join two molecules. The linker is capable of forming covalent bonds or high-affinity non-covalent bonds to both molecules. Suitable linkers are well known to those of ordinary skill in the art and include, but are not limited to, straight or branched-chain carbon linkers, heterocyclic carbon linkers, or peptide linkers. The linkers may be joined to the constituent amino acids through their side groups (e.g., through a disulfide linkage to cysteine).
The procedure for attaching a toxin to an antibody or other polypeptide targeting molecule will vary according to the chemical structure of the toxin. Antibodies contain a variety of functional groups; e.g., sulfhydryl (--S), carboxylic acid (COOH) or free amine (--NH2) groups, which are available for reaction with a suitable functional group on a toxin. Additionally, or alternatively, the antibody or toxin can be derivatized to expose or attach additional reactive functional groups. The derivatization may involve attachment of any of a number of linker molecules such as those available from Pierce Chemical Company, Rockford Ill.
A bifunctional linker having one functional group reactive with a group on the toxin, and another group reactive with an antibody, can be used to form a desired immunoconjugate. Alternatively, derivatization may involve chemical treatment of the toxin or antibody, e.g., glycol cleavage of the sugar moiety of a glycoprotein antibody with periodate to generate free aldehyde groups. The free aldehyde groups on the antibody may be reacted with free amine or hydrazine groups on the toxin to bind the toxin thereto. (See U.S. Pat. No. 4,671,958). Procedures for generation of free sulfhydryl groups on antibodies or antibody fragments, are also known (See U.S. Pat. No. 4,659,839).
Many procedures and linker molecules for attachment of various compounds including toxins are known. See, for example, European Patent Application No. 188,256; U.S. Pat. Nos. 4,671,958, 4,659,839, 4,414,148, 4,699,784; 4,680,338; 4,569,789; 4,589,071; and Borlinghaus et al. Cancer Res. 47: 4071-4075 (1987), which are incorporated herein by reference. In particular, production of various immunotoxin conjugates is well-known within the art and can be found, for example in "Monoclonal Antibody-Toxin Conjugates: Aiming the Magic Bullet," Thorpe et al., Monoclonal Antibodies in Clinical Medicine, Academic Press, pp. 168-190 (1982), Waldmann, Science, 252: 1657 (1991), U.S. Pat. Nos. 4,545,985 and 4,894,443 which are incorporated herein by reference. See also, e.g., Birch and Lennox, Monoclonal Antibodies: Principles and Applications, Chapter 4, Wiley-Liss, New York, N.Y. (1995); U.S. Pat. Nos. 5,218,112, 5,090,914; Hermanson, Bioconjugate Techniques, Academic Press, San Diego, Calif. (1996). In preferred embodiments, the linker molecule is m-Malimidobenzoyl-N-hydroxysuccinimideester (MBS) which can be used to prepare immunotoxin conjugates as described, for example, in Youle and Nevelle, Proc. Natl. Acad. Sci., 77(9):5483-5486 (1980).
In some circumstances, it is desirable to free the toxin from the antibody when the immunotoxic conjugate has reached its target site. Therefore, immunotoxic conjugates comprising linkages which are cleavable in the vicinity or within the target site may be used when the toxin is to be released at the target site. Cleaving of the linkage to release the agent from the ligand may be prompted by enzymatic activity or conditions to which the immunoconjugate is subjected either inside the target cell or in the vicinity of the target site. A number of different cleavable linkers are known to those of skill in the art. See U.S. Pat. Nos. 4,618,492; 4,542,225, and 4,625,014. SPDP is a reversible NHS-ester, pyridyl disulfide cross-linker used to conjugate amine-containing molecules to sulfhydryls. Another chemical modification reagent is 2-iminothiolane which reacts with amines and yields a sulfhydryl. Water soluble SPDP analogs, such as Sulfo-LC-SPDP (Pierce, Rockford, Ill.) are also available. SMPT is a reversible NHS-ester, pyridyl disulfide cross-linker developed to avoid cleavage in vivo prior to reaching the antigenic target. Additionally, the NHS-ester of SMPT is relatively stable in aqueous solutions.
Pharmaceutical Compositions and Method of Administration
Immunotoxic conjugates of the present invention are useful for the treatment of focal muscle spasms such as, but not limited to, blepharospasm, cervical dystonia, hand dystonia, limb dystonia, hemifacial spasm, bruxism, strabismus, VI nerve palsy, spasmodic dysphonia, and oromandibular dystonia. In preferred embodiments, the immunotoxin conjugate comprises ricin (RCA60). While not bound by theory, it is believed that the use of the galactose binding ricin B-chain helps prevent diffusion of the immunotoxin from the site of administration. Additionally, the B-chain increases the potency of the ricin A-chain toxin.
The formulations containing therapeutically effective amounts of the immunotoxin conjugates of the present invention are either sterile liquid solutions, liquid suspensions or lyophilized versions and optionally contain stabilizers or excipients. Lyophilized compositions are reconstituted with suitable diluents, e.g., water for injection, saline, 0.3% glycine and the like. The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, toxicity adjusting agents and the like, for example, sodium acetate, sodium chloride, potassium chloride, calcium chloride, and the like. Actual methods for preparing administrable compositions will be known or apparent to those skilled in the art and are described in more detail in such publications as Remington's Pharmaceutical Science, 19th ed., Mack Publishing Company, Easton, Pa. (1995).
The compositions for administration will commonly comprise a solution of the immunotoxin conjugate of the present invention dissolved in a pharmaceutically acceptable carrier, preferably an aqueous carrier. A variety of aqueous carriers can be used, e.g., buffered saline and the like. These solutions are sterile and generally free of undesirable matter. These compositions may be sterilized by conventional, well known sterilization techniques.
As will be readily understood by the clinician of ordinary skill in the art, the dose will be dependent upon the properties of the particular immunotoxin conjugate employed, e.g., its activity and biological half-life, the concentration of immunotoxin conjugate in the formulation, the site and rate of dosage, the clinical tolerance of the patient involved, the disease afflicting the patient, the severity of the disease, and the like.
Preferably, the pharmaceutical compositions containing the immunotoxin conjugates will be administered by intramuscular injection in a therapeutically effective dose ranging from about 1 ng to 200 ng depending upon the size of the muscle, the severity of the focal muscle spasm, and the specificity and toxicity of the conjugate. For example, for a ricin-anti-nAchR immunotoxin conjugate, an eye muscle will typically require between 5 and 20 ng of conjugate, and a vocal chord will generally require 1 to 2 ng. Preferably, the dose is administered at the site of the neuromuscular junctions of the muscle which is being treated. Those of skill will understand that the dose may be administered to the various neuromuscular junctions of the muscle whose activity one wishes to diminish, this being particularly preferred for muscles whose size allows for such a mode of administration. Therapeutically effective amounts of immunotoxin conjugates of the present invention can be administered alone, in combination, or in conjunction with therapeutically effective amounts of the unconjugated forms of the toxins (e.g., botulinum toxin).
Preferably, the dosage is administered once but may be applied periodically until either a therapeutic result is achieved or until side effects warrant discontinuation of therapy. Generally, the dose should be sufficient to treat or ameliorate symptoms or signs of focal muscle spasm without producing unacceptable toxicity to the patient. An effective amount of the compound is that which provides either subjective relief of a symptom(s) or an objectively identifiable improvement as noted by the clinician or other qualified observer.
Solutions comprising immunotoxin conjugates of the present invention will typically have a pH in the range of pH 5 to 9.5, preferably pH 6.5 to 7.5. The immunotoxin conjugates should be in a solution having a suitable pharmaceutically acceptable buffer such as phosphate, tris (hydroxymethyl) aminomethane-HCl, saline, or citrate and the like. Buffer concentrations should be in the range of 1 to 100 mM. The solution of antibody may also contain a salt, such as sodium chloride or potassium chloride in a concentration of 50 to 150 mM. An effective amount of a stabilizing agent such as an albumin, a globulin, a gelatin, a protamine or a salt of protamine can also be included to a solution comprising the immunotoxin conjugate of the present invention. In preferred embodiments the buffer is a saline solution of 0.9% comprising human serum albumin of 1 mg/ml.
Antibody or immunotoxin may also be administered via microspheres, liposomes or other microparticulate delivery systems placed in certain tissues including blood.
Claim 1 of 11 Claims
What is claimed is:
1. A method of treating a focal muscle spasm, comprising administering, by intramuscular injection, a therapeutically effective dose of an immunotoxin conjugate to a muscle of said focal muscle spasm, wherein said immunotoxin conjugate comprises an antibody conjugated to a cellular toxin selected from the group consisting of: ricin and abrin, wherein said antibody is selectively reactive, under immunologically reactive conditions, to a nicotinic acetylcholine receptor;
wherein said antibody of said immunotoxin conjugate binds to a nicotinic acetylcholine receptor of a muscle cell of said muscle, and said cellular toxin of said immunotoxin conjugate mediates the death of said muscle cell.