TREATMENT METHODS USING ATOXIC NEUROTOXIN DERIVATIVES

- New York University

The present invention relates to a treatment method. This method involves contacting a subject with an isolated, physiologically active, atoxic derivative of a Clostridial neurotoxin. Contacting is carried out to treat the subject. The derivative of a Clostridial neurotoxin does not possess a cargo attachment peptide sequence at its N-terminus.

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Description

This application is a continuation of U.S. patent application Ser. No. 14/166,434 filed Jan. 28, 2014, which claims the benefit of U.S. Provisional Patent Application Ser. No. 61/757,478, filed Jan. 28, 2013, which is hereby incorporated by reference in its entirety.

The subject matter of this application was made with support from the United States Government under National Institutes of Health grant RO1 A1093504. The United States Government has certain rights.

FIELD OF THE INVENTION

This invention relates to treatment methods using atoxic neurotoxin derivatives.

BACKGROUND OF THE INVENTION

The Clostridial neurotoxins are a family of structurally similar proteins that target the neuronal machinery for synaptic vesicle exocytosis. Produced by anaerobic bacteria of the Clostridium genus, botulinum neurotoxins (“BoNT”s, seven immunologically distinct subtypes, A-G) and Tetanus neurotoxin (“TeNT”) are the most poisonous substances known on a per-weight basis, with an LD50 in the range of 0.5-2.5 ng/kg when administered by intravenous or intramuscular routes (National Institute of Occupational Safety and Health, “Registry of Toxic Effects of Chemical Substances (R-TECS),” Cincinnati, Ohio: National Institute of Occupational Safety and Health (1996)). BoNTs target cholinergic nerves at their neuromuscular j unction, inhibiting acetylcholine release and causing peripheral neuromuscular blockade (Simpson, “Identification of the Major Steps in Botulinum Toxin Action,” Annu. Rev. Pharmacol. Toxicol. 44:167-193 (2004)).

A genetic engineering platform that enables rational design of therapeutic agents based on Clostridial toxin genes was described in U.S. Pat. No. 7,785,606 to Ichtchenko and Band. The genetic engineering scheme was based on a two-step approach. Gene constructs, expression systems, and purification schemes were designed that produce physiologically active, recombinant Clostridial neurotoxin derivatives. The recombinant toxin derivatives retained structural features important for developing therapeutic candidates, or useful biologic reagents. Using the genetic constructs and expression systems developed by this paradigm, selective point mutations were then introduced to create recombinant atoxic Clostridial neurotoxin derivatives.

Treatment methods that involve contacting a patient with isolated, physiologically active, toxic, Clostridial neurotoxin derivatives have been described in U.S. Pat. No. 7,785,606 to Band and Ichtchenko. Also, isolated, physiologically active, toxic and atoxic Clostridium botulinum neurotoxin derivatives that have an S6 peptide sequence fused to the N-terminus of the proteins to enable site-specific attachment of cargo using Sfp phosphopantetheinyl transferase have been described as suitable for treatment (U.S. Patent Application Publication No. 2011/0206616 to Ichtchenko and Band). However, methods that involve treatment with an atoxic derivative of a Clostridial neurotoxin lacking a cargo attachment sequence at its N-terminus, and having a much higher LD50 than a toxic derivative of a Clostridial neurotoxin or a wild type Clostridial neurotoxin, have not been described.

The present invention is directed to overcoming this and other limitations in the art.

SUMMARY OF THE INVENTION

The present invention relates to a treatment method. This method involves contacting a subject with an isolated, physiologically active, atoxic derivative of a Clostridial neurotoxin, said contacting being carried out to treat the subject, with the proviso that the neurotoxin derivative does not possess a cargo attachment peptide sequence at its N-terminus.

Genetic constructs and expression systems described herein are shown to produce a family of recombinant BoNT derivatives, with conformational and trafficking properties similar to the wild type BoNT toxins. These derivatives of Clostridial neurotoxins can be produced in toxic forms, having a toxicity comparable to that of the wild type toxin, or with mutations that reduce the metalloprotease activity responsible for toxicity (i.e., atoxic derivatives). The LD50 of the atoxic neurotoxin derivatives is much higher than that of the wild type toxin.

As described herein, the atoxic neurotoxin derivatives (see U.S. Pat. No. 7,785,606 to Ichtchenko et al., which is hereby incorporated by reference in its entirety) unexpectedly have in vivo activity similar to the wild type neurotoxins used for pharmaceutical purposes. Yet, atoxic neurotoxin derivatives described herein offer significant treatment benefits over current pharmaceutical preparations of wild type neurotoxins produced from cultures. In particular, the atoxic derivatives described herein are safer, providing distinct advantages for medical uses and production/manufacturing. The improved therapeutic index will enable application to conditions where the toxicity of wild type neurotoxins limit their use because of safety concerns.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-C are a comparative alignment of amino acid sequences of seven wild type botulinum neurotoxin serotypes, including Clostridium botulinum serotype A (wt BoNT A) (SEQ ID NO:1), Clostridium botulinum serotype B (wt BoNT B) (SEQ ID NO:2), Clostridium botulinum serotype C (wt BoNT C) (SEQ ID NO:3), Clostridium botulinum serotype D (wt BoNT D) (SEQ ID NO:4), Clostridium botulinum serotype E (wt BoNT E) (SEQ ID NO:5), Clostridium botulinum serotype F (wt BoNT F) (SEQ ID NO:6), and Clostridium botulinum serotype G (wt BoNT G) (SEQ ID NO:7). Gaps have been introduced to maximize homology. Amino acids identical in >50% of compared sequences are shown in black boxes. Amino acids constituting the active site of the catalytic domain of metalloprotease are marked by stars. Disulfide bridge between neurotoxin cysteine residues of the light and heavy chain are shown as a long horizontal bracket. The amino acid residues constituting the minimal catalytic domain of the light chain are hatched. The first amino acid of the C-terminal part of the protein heavy chain (N872 for BoNT A), is shown with a white arrow, as is the first amino acid considered to constitute the receptor-binding domain. Amino acids absent in the mature dichain BoNT A molecule along with the aligned amino acids of the other BoNT serotypes are boxed. A white arrow is also positioned at the first amino acid of the neurotoxins' light chain.

FIG. 2 is a photograph showing the results of in vivo studies performed by intramuscular injection into the lateral gastrocnemius with 0.5 μg/mouse of BoNT A/ad-0 (experimental) in 3 μl of 0.9% NaCl or by injecting 3 μl of 0.9% of NaCl without BoNT A/ad-0 (control). Muscle paralysis and digital abduction was recorded 48 hours after. The two upper panel photographs show control mice, with the arrow in the upper right photograph indicating the site of injection. The three lower panel photographs show experimental mice. Digital abduction muscle paralysis was only observed in mice injected with BoNT A/ad-O. Experimental, n=10. Control, n=5. Representative results are shown in the photographs in the three bottom panels.

 DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a treatment method. This method involves contacting a subject with an isolated, physiologically active, atoxic derivative of a Clostridial neurotoxin, said contacting being carried out to treat the subject, with the proviso that the neurotoxin derivative does not possess a cargo attachment peptide sequence at its N-terminus.

According to one embodiment, the derivative of a Clostridial neurotoxin of the present invention is a derivative of a Clostridium botulinum neurotoxin. Clostridium botulinum has multiple serotypes (A-G). Suitable derivatives of a Clostridial neurotoxin may be derivatives of any of the Clostridium botulinum serotypes. In addition, suitable derivatives of a Clostridial neurotoxin of the present invention may be derivatives of more than one Clostridium botulinum serotype. For example, it may be desirable to have a derivative of a Clostridial neurotoxin constructed of a light chain (LC) region from one Clostridium botulinum serotype (e.g., serotype A, BoNT A) and a heavy chain (HC) region from another Clostridium botulinum serotype (e.g., serotype B, BoNT B). Also, suitable derivatives of a Clostridial neurotoxin of the present invention include chimeras using other receptor ligands, e.g., epidermal growth factor (“EGF”) for LC delivery to cancer cells (see U.S. Patent Application Publication no. 2012/0064059 to Foster et al., which is hereby incorporated by reference in its entirety).

By “derivative” it is meant that the Clostridial neurotoxin is substantially similar to the wild type toxin, but has been modified slightly as described herein. For example, derivatives include Clostridial neurotoxins that are at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to a wild type neurotoxin.

Isolated derivatives of a Clostridial neurotoxin are physiologically active. This physiological activity includes, but is not limited to, toxin immunogenicity, trans- and intra-cellular trafficking, cell recognition and targeting, and paralytic activity. In one embodiment, the derivative of a Clostridal neurotoxin is a derivative of a full-length Clostridial neurotoxin.

The atoxic derivative of a Clostridial neurotoxin designated herein using the “ad-0” designation, does not have an S6 peptide sequence fused to the N-terminus of the neurotoxin derivative, as described in U.S. Patent Application Publication No. 2011/0206616 to Icthtchenko and Band, which is hereby incorporated by reference in its entirety.

The mechanism of cellular binding and internalization of Clostridial neurotoxins is still not completely understood. The C-terminal portion of the heavy chain of all Clostridial neurotoxins binds to gangliosides (sialic acid-containing glycolipids), with a preference for gangliosides of the Gib series (Montecucco et al., “Structure and Function of Tetanus and Botulinum Neurotoxins,” Q. Rev. Biophys. 28:423-472 (1995); Montecucco, “How Do Tetanus and Botulinum Toxins Bind to Neuronal Membranes?” TIBS 11:314-317 (1986); and Van Heyningen et al., “The Fixation of Tetanus Toxin by Ganglioside,” J. Gen. Microbiol. 24:107-119 (1961), which are hereby incorporated by reference in their entirety). The sequence responsible for ganglioside binding has been identified for the structurally similar TeNT molecule, and is located within the 34 C-terminal amino acid residues of its heavy chain. BoNT A, BoNT B, BoNT C, BoNT E, and BoNT F share a high degree of homology with TeNT in this region (FIG. 1) (Shapiro et al., “Identification of a Ganglioside Recognition Domain of Tetanus Toxin Using a Novel Ganglioside Photoaffinity Ligand,” J. Biol. Chem. 272:30380-30386 (1997), which is hereby incorporated by reference in its entirety). Multiple types of evidence suggest the existence of at least one additional component involved in the binding of Clostridial neurotoxins to neuronal membranes (Montecucco et al., “Structure and Function of Tetanus and Botulinum Neurotoxins,” Q. Rev. Biophys. 28:423-472 (1995); Montecucco, “How Do Tetanus and Botulinum Toxins Bind to Neuronal Membranes?” TIBS 11:314-317 (1986), which are hereby incorporated by reference in their entirety). In two reports (Nishiki et al., “The High-Affinity Binding of Clostridium Botulinum Type B Neurotoxin to Synaptotagmin II Associated with Gangliosides GT1b/GD1a,” FEBS Lett 378:253-257 (1996); Dong et al., “Synaptotagmins I and II Mediate Entry of Botulinum Neurotoxin B into Cells,” J. Cell Biol. 162:1293-1303 (2003), which are hereby incorporated by reference in their entirety), synaptotagmins were identified as possible candidates for the auxiliary BoNT B receptor, and synaptotagmins I and II were implicated as neuronal receptors for BoNT G (Rummel et al., “Synaptotagmins I and II Act as Nerve Cell Receptors for Botulinum Neurotoxin G,” J. Biol. Chem. 279:30865-30870 (2004), which is hereby incorporated by reference in its entirety). Dong et al., “SV2 is the Protein Receptor for Botulinum Neurotoxin A,” Science 312:592-596 (2006), which is hereby incorporated by reference in its entirety, showed that BoNT A enters neurons by binding to the synaptic vesicle protein SV2 (isoforms A, B, and C). However, despite the structural similarity in the putative receptor-binding domain of Clostridial neurotoxins, other toxin subtypes show no affinity for SV2 and instead may target synaptotagmins or synaptotagmin-related molecules. Lipid rafts (Herreros et al., “Lipid Rafts Act as Specialized Domains for Tetanus Toxin Binding and Internalization into Neurons,” Mol. Biol. Cell 12:2947-2960 (2001), which is hereby incorporated by reference in its entirety) have been implicated as a specialized domain involved in TeNT binding and internalization into neurons, but these domains are widely distributed on multiple cell types, and therefore cannot simply explain the high specificity of the toxins for neurons.

Clostridial neurotoxins are internalized through the presynaptic membrane by an energy-dependent mechanism (Montecucco et al., “Structure and Function of Tetanus and Botulinum Neurotoxins,” Q. Rev. Biophys. 28:423-472 (1995); Matteoli et al., “Synaptic Vesicle Endocytosis Mediates the Entry of Tetanus Neurotoxin into Hippocampal Neurons,” Proc. Natl. Acad. Sci. USA 93:13310-13315 (1996); and Mukherjee et al., “Endocytosis,” Physiol. Rev. 77:759-803 (1997), which are hereby incorporated by reference in their entirety), and rapidly appear in vesicles where they are at least partially protected from degradation (Dolly et al., “Acceptors for Botulinum Neurotoxin Reside on Motor Nerve Terminals and Mediate Its Internalization,” Nature 307:457-460 (1984); Critchley et al., “Fate of Tetanus Toxin Bound to the Surface of Primary Neurons in Culture: Evidence for Rapid Internalization,” J. Cell Biol. 100:1499-1507 (1985), which are hereby incorporated by reference in their entirety). The BoNT complex of light and heavy chains interacts with the endocytic vesicle membrane in a chaperone-like way, preventing aggregation and facilitating translocation of the light chain in a fashion similar to the protein conducting/translocating channels of smooth ER, mitochondria, and chloroplasts (Koriazova et al., “Translocation of Botulinum Neurotoxin Light Chain Protease through the Heavy Chain Channel,” Nat. Struct. Biol. 10:13-18 (2003), which is hereby incorporated by reference in its entirety). Acidification of the endosome is believed to induce pore formation, which allows translocation of the light chain to the cytosol upon reduction of the interchain disulfide bond (Hoch et al., “Channels Formed by Botulinum, Tetanus, and Diphtheria Toxins in Planar Lipid Bilayers: Relevance to Translocation of Proteins Across Membranes,” Proc. Natl. Acad. Sci. USA 82:1692-1696 (1985), which is hereby incorporated by reference in its entirety). Within the cytosol, the light chain displays a zinc-endopeptidase activity specific for protein components of the synaptic vesicle exocytosis apparatus. TeNT and BoNT B, BoNT D, BoNT F, and BoNT G recognize VAMP/synaptobrevin. This integral protein of the synaptic vesicle membrane is cleaved at a single peptide bond, which differs for each neurotoxin. BoNT A, BoNT C, and BoNT E recognize and cleave SNAP-25, a protein of the presynaptic membrane, at different sites within the carboxyl terminus segment. BoNT C also cleaves syntaxin, another protein of the nerve terminal plasmalemma (Montecucco et al., “Structure and Function of Tetanus and Botulinum Neurotoxins,” Q. Rev. Biophys. 28:423-472 (1995); Sutton et al., “Crystal Structure of a SNARE Complex Involved in Synaptic Exocytosis at 2.4 Å Resolution,” Nature 395:347-353 (1998), which are hereby incorporated by reference in their entirety). The cleavage of such components of the synaptic release machinery results in inhibition of acetylcholine release in motor neurons, ultimately leading to neuromuscular paralysis.

The isolated derivative of a Clostridial neurotoxin employed in the method of the present invention is physiologically active and atoxic. The endopeptidase activity responsible for Clostridial neurotoxin toxicity is believed to be associated with the presence of a HExxHxxH (SEQ ID NO:8) motif in the light chain, characteristic of metalloproteases (FIGS. 1A-C). Mutagenesis of BoNT A light chain, followed by microinjection of the corresponding mRNA into presynaptic cholinergic neurons of Aplysia californica, allowed the minimal essential domain responsible for toxicity to be identified (Kurazono et al., “Minimal Essential Domains Specifying Toxicity of the Light Chains of Tetanus Toxin and Botulinum Neurotoxin Type A,” J. Biol. Chem. 267:14721-14729 (1992), which is hereby incorporated by reference in its entirety). Site-directed mutagenesis of BoNT A light chain pinpointed the amino acid residues involved in Zn2+ coordination, and formation of the active metalloendoprotease core which cleaves SNAP-25 (Rigoni et al., “Site-Directed Mutagenesis Identifies Active-Site Residues of the Light Chain of Botulinum Neurotoxin Type A,” Biochem. Biophys. Res. Commun. 288:1231-1237 (2001), which is hereby incorporated by reference in its entirety). The three-dimensional structures of Clostridial neurotoxins and their derivatives confirmed the mutagenesis results, and detailed the spatial organization of the protein domains. For the BoNT A holotoxin, crystal structure was obtained to a resolution of 3.3 Å (Lacy et al., “Crystal Structure of Botulinum Neurotoxin Type A and Implications for Toxicity,” Nat. Struct. Biol. 5:898-902 (1998), which is hereby incorporated by reference in its entirety). The BoNT B holotoxin crystal structure was determined at 1.8 and 2.6 Å resolution (Swaminathan et al., “Structural Analysis of the Catalytic and Binding Sites of Clostridium Botulinum Neurotoxin B,” Nat. Struct. Biol. 7:693-699 (2000), which is hereby incorporated by reference in its entirety). Recently, a crystal structure for BoNT E catalytic domain was determined to 2.1 Å resolution (Agarwal et al., “Structural Analysis of Botulinum Neurotoxin Type E Catalytic Domain and Its Mutant Glu212>Gln Reveals the Pivotal Role of the Glu212 Carboxylate in the Catalytic Pathway,” Biochemistry 43:6637-6644 (2004), which is hereby incorporated by reference in its entirety). The later study provided multiple interesting structural details, and helps explain the complete loss of metalloendoproteolytic activity in the BoNT E LC E212>Q mutant. The availability of this detailed information on the relationship between the amino acid sequence and biological activities of Clostridial toxins enables the design of modified toxin genes with properties specifically altered for therapeutic goals.

Thus, in one embodiment, the physiologically active and atoxic derivative of a Clostridial neurotoxin has a metalloprotease disabling mutation. Specific metalloprotease disabling mutations are described in U.S. Pat. No. 7,785,606 to Ichthchenko and Band, which is hereby incorporated by reference in its entirety. Additional point mutations can be introduced to further modify the characteristics of the atoxic derivative, some of which are also described in U.S. Pat. No. 7,785,606 to Ichthchenko and Band, which is hereby incorporated by reference in its entirety.

The physiologically active and atoxic derivative of a Clostridial neurotoxin may also have a non-native motif (e.g., a SNARE motif) in the light chain region that is capable of inactivating light chain metalloprotease activity in a toxic Clostridial neurotoxin, or otherwise modifying the behavior of the derivative. The sequences of nine non-native motifs that may be substituted for alpha-helix domains are described in U.S. Pat. No. 7,785,606 to Ichtchenko and Band, which is hereby incorporated by reference in its entirety. Atoxic derivatives that incorporate sequences to target other cellular receptors are also possible (e.g., EGF or cancer cells) (see U.S. Patent Application Publication No. 2012/0064059 to Foster et al., which is hereby incorporated by reference in its entirety).

In one embodiment, the physiologically active and atoxic derivative of a Clostridial neurotoxin has an LD50 that is at least 1,000; 2,000; 5,000; 7,000; 9,000; 10,000; 20,000; 30,000; 40,000; 50,000; 60,000; 70,000; 80,000; 90,000; 100,000; or 500,000-fold higher than the LD50 of wild type Clostridial neurotoxin. The particular mode of administration may affect the LD50 of the derivative of a Clostridial neurotoxin.

In one embodiment, the derivative of a Clostridal neurotoxin of the present invention is produced by cleaving a propeptide. The propeptide is cleaved at the highly specific protease cleavage site to form a light and heavy chain, with molecular weights of approximately 50 kD and 100 kD, respectively. The light and heavy chain regions are linked by a disulfide bond.

In one embodiment, the propeptide is contacted with a highly specific protease (e.g., enterokinase or TEV protease) under conditions effective to enable cleavage at the intermediate region of the propeptide of the present invention. Preferably, the expressed propeptide has one or more disulfide bridges.

As discussed infra, Clostridial neurotoxins and their derivatives described herein are synthesized as single chain propeptides which are later activated by a specific proteolysis cleavage event, generating a dimer joined by a disulfide bond. These structural features can be illustrated using BoNT A as an example, and are generally applicable to all Clostridium botulinum serotypes. The mature BoNT A is composed of three functional domains of Mr 50,000, where the catalytic function responsible for toxicity is confined to the light chain (residues 1-437), the translocation activity is associated with the N-terminal half of the heavy chain (residues 448-872), and cell binding is associated with its C-terminal half (residues 873-1,295) (Johnson, “Clostridial Toxins as Therapeutic Agents: Benefits of Nature's Most Toxic Proteins,” Annu. Rev. Microbiol. 53:551-575 (1999); Montecucco et al., “Structure and Function of Tetanus and Botulinum Neurotoxins,” Q. Rev. Biophys. 28:423-472 (1995), which are hereby incorporated by reference in their entirety).

Optimized expression and recovery of recombinant neurotoxins for BoNT serotypes in a native and physiologically active state is achieved by the introduction of one or more alterations to the nucleotide sequences encoding the BoNT propeptides, as discussed infra. These mutations are designed to maximize yield of recombinant derivatives of a Clostridial neurotoxin, while retaining the native toxins' structure and biological activity.

Common structural features of the wild-type Clostridium botulinum neurotoxin propeptides are shown in FIGS. 1A-C. These structural features are illustrated using wt BoNT A propeptide as an example, and are generalized among all Clostridium botulinum serotypes. wt BoNT A propeptide has two chains, a light chain (“LC”) of Mr˜50,000 and a heavy chain (“HC”) of Mr˜100,000, linked by a disulfide bond between Cys429 and Cys453. As illustrated in FIGS. 1A-C, all seven BoNT serotype propeptides have a light chain region and a heavy chain region linked by a disulfide bond. Two essential Cys residues, one adjacent to the C-terminus of the light chain, and a second adjacent to the N-terminus of the heavy chain are present in all seven BoNT serotypes. These two Cys residues form the single disulfide bond holding the HC and LC polypeptides together in the mature neurotoxin. This disulfide bond enables the mature neurotoxin to accomplish its native physiological activities by permitting the HC and LC to carry out their respective biological roles in concert. The disulfide bond between HC and LC polypeptides in all seven serotypes is illustrated in FIG. 1A by the solid line joining the involved Cys residues. The outlined box in FIG. 1A illustrates the intermediate region defined by amino acid residues Lys438-Lys448 of wt BoNT A. This intermediate region identifies the amino acids eliminated during maturation of wt BoNT A, and believed to be excised by a protease endogenous to the host microorganism. This cleavage event, described infra, generates the biologically active BoNT HC-LC dimer. The outlined amino acid residues in FIGS. 1A-C, representing amino acid residues approximately in the 420 to 450 range for all seven BoNT serotypes, can be considered as a region “non-essential” to the toxins' physiological activity and, therefore, represents targets for directed mutagenesis in all seven BoNT serotypes.

All seven wt BoNT serotypes referred to herein contain Lys or Arg residues in the intermediate region defined by the box in FIG. 1A, which make the propeptides susceptible to activation by trypsin. Native BoNT A propeptide recovered from young bacterial cultures can be activated by trypsinolysis, with production of intact, S—S bound light and heavy chain. Though multiple additional trypsin-susceptible sites are present in the propeptides, they are resistant to proteolysis due to their spatial positions within the native toxin molecule (Dekleva et al., “Nicking of Single Chain Clostridium botulinum Type A Neurotoxin by an Endogenous Protease,” Biochem. Biophys. Res. Commun. 162:767-772 (1989); Lacy et al., “Crystal Structure of Botulinum Neurotoxin Type A and Implications for Toxicity,” Nat. Struct. Biol. 5:898-902 (1998), which are hereby incorporated by reference in their entirety). A second site in the native propeptide of several BoNT serotypes can be susceptible to trypsin cleavage when subjected to higher enzyme concentrations or incubation times (Chaddock et al., “Expression and Purification of Catalytically Active, Non-Toxic Endopeptidase Derivatives of Clostridium botulinum Toxin Type A,” Protein Expr. Purif. 25:219-228 (2002), which is hereby incorporated by reference in its entirety). This trypsin-susceptible site is located in the region adjacent to the toxin receptor binding domain. This region of the HC peptide is found to be exposed to solvent in BoNT serotypes for which information is available on their 3-D crystal structure (Lacy et al., “Crystal Structure of Botulinum Neurotoxin Type A and Implications for Toxicity,” Nat. Struct. Biol. 5:898-902 (1998); Swaminathan et al., “Structural Analysis of the Catalytic and Binding Sites of Clostridium botulinum Neurotoxin B,” Nat. Struct. Biol. 7:693-699 (2000), which are hereby incorporated by reference in their entirety).

In one embodiment, the propeptide has an intermediate region connecting the light and heavy chain regions which has a highly specific protease cleavage site and no low-specificity protease cleavage sites. For purposes of the present invention, a highly specific protease cleavage site has three or more specific adjacent amino acid residues that are recognized by the highly specific protease in order to permit cleavage (e.g., an enterokinase cleavage site or a TEV recognition sequence). In contrast, a low-specificity protease cleavage site has two or less adjacent amino acid residues that are recognized by a protease in order to enable cleavage (e.g., a trypsin cleavage site).

In all seven BoNT serotypes, the amino acid preceding the N-terminus of the heavy chain is a Lys or Arg residue which is susceptible to proteolysis with trypsin. This trypsin-susceptible site can be replaced with a five amino acid enterokinase cleavage site (i.e., DDDDK (SEQ ID NO:9)) upstream of the heavy chain's N-terminus. Alternatively, the trypsin-susceptible site can be replaced with a tobacco etch virus protease recognition (“TEV”) sequence. Use of a TEV sequence enables a one-step heterodimer-forming cleavage event. See U.S. Patent Application Publication No. 2011/0206616 to Ichtchenko et al., which is hereby incorporated by reference in its entirety. Either of these modifications enables standardization activation with specific enzymes. In serotypes A and C, additional Lys residues within this region may be mutated to either Gln or His, thereby eliminating additional trypsin-susceptible sites. Trypsin-susceptible recognition sequences also occur upstream of the heavy chain's receptor-binding domain in serotypes A, E, and F. This region's susceptibility to proteolysis is consistent with its exposure to solvent in the toxin's 3-D structure, as shown by X-ray crystallography analysis. Therefore, in serotypes A, E, and F, the susceptible residues are modified to Asn. These modifications enable standardization activation with either enterokinase or TEV.

Signal peptides and N-terminal affinity tags are also preferably introduced, as required, to enable secretion and recovery and to eliminate truncated variants. The affinity tags can be separated from the N-terminus and C-terminus of the neurotoxin by cleavage using the same specific proteases used to cleave the heavy and light chain.

In one embodiment, the derivative of a Clostridial neurotoxin is from a propeptide that has a metalloprotease disabling mutation. The amino acid residues constituting the minimal catalytic domain of the light chain of the propeptide are illustrated in FIG. 1A by hatching. Specific amino acid residues constituting the active site of the catalytic domain of the metalloprotease are marked by stars in FIG. 1A.

A variety of Clostridial neurotoxin propeptides with light chain regions containing non-native motifs (e.g., SNARE motif peptides) in place of surface alpha-helix domains can be created. These non-native motif bearing propeptides are generated by altering the nucleotide sequences of nucleic acids encoding the propeptides.

In one embodiment, the light and heavy chains of the propeptide are not truncated.

In one embodiment, the propeptide further comprises a signal peptide coupled to the light chain region, where the signal peptide is suitable to permit secreation of the propeptide from a eukaryotic cell to a medium. Suitable signal peptides are described in U.S. Pat. No. 7,785,606 to Ichtchenko and Band, which is hereby incorporated by reference in its entirety. A suitable signal peptide is a gp64 signal peptide.

The propeptide may also have an affinity tag located between the signal peptide and the light chain region and/or at the C-terminus of the propeptide. A suitable affinity tag is the hexahistidine affinity tag MPMLSAIVLYVLLAAAAHSAFAAMVHHHHHHSAS . . . (SEQ ID NO:10). Structural variations of suitable Clostridial neurotoxin propeptides that possess a cargo attachment peptide sequence are described in U.S. Patent Application Publication No. 2011/0206616 to Ichtchenko and Band, which is hereby incorporated by reference in its entirety. Propeptides that encode atoxic derivatives of a Clostridial neurotoxin suitable for use in the method of the present invention may have any of the structural features of the propeptides described in U.S. Patent Application Publication No. 2011/0206616 to Ichtchenko and Band, which is hereby incorporated by reference in its entirety, other than the cargo attachment peptide sequence at the N-terminus. As described in U.S. Patent Application Publication No. 2011/0206616 to Ichtchenko and Band, which is hereby incorporated by reference in its entirety, a single protease cleavage step can be used for activation and removal of affinity tags.

Isolated nucleic acid molecules that encode atoxic derivatives of a Clostridial neurotoxin suitable for use in the method of the present invention are described in U.S. Pat. No. 7,785,606 to Ichtchenko and Band, which is hereby incorporated by reference in its entirety.

In one embodiment, the nucleic acid molecule has a metalloprotease disabling mutation, as described supra.

In one embodiment, the derivative of a Clostridal neurotoxin is a recombinant protein. Expression systems having a nucleic acid molecule encoding an isolated, physiologically active, atoxic derivative of a Clostridial neurotoxin in a heterologous vector, and host cells having a heterologous nucleic acid molecule encoding an isolated, physiologically active, atoxic derivative of a Clostridial neurotoxin are described in U.S. Pat. No. 7,785,606 to Ichtchenko and Band, which is hereby incorporated by reference in its entirety.

Expressing a recombinant, physiologically active, atoxic derivative of a Clostridial neurotoxin is carried out by providing a nucleic acid construct having a nucleic acid molecule encoding a propeptide as described herein. The nucleic acid construct has a heterologous promoter operably linked to the nucleic acid molecule and a 3′ regulatory region operably linked to the nucleic acid molecule. The nucleic acid construct is then introduced into a host cell under conditions effective to express the physiologically active, atoxic derivative of a Clostridial neurotoxin.

In one embodiment, the expressed neurotoxin derivative is contacted with a highly specific protease under conditions effective to effect cleavage at the intermediate region. Preferably, the intermediate region of the propeptide is not cleaved by proteases endogenous to the expression system or the host cell.

Expression of a derivative of a Clostridial neurotoxin can be carried out by introducing a nucleic acid molecule encoding a propeptide into an expression system of choice using conventional recombinant technology. Generally, this involves inserting the nucleic acid molecule into an expression system to which the molecule is heterologous (i.e., not normally present). The introduction of a particular foreign or native gene into a mammalian host is facilitated by first introducing the gene sequence into a suitable nucleic acid vector. “Vector” is used herein to mean any genetic element, such as a plasmid, phage, transposon, cosmid, chromosome, virus, virion, etc., which is capable of replication when associated with the proper control elements and which is capable of transferring gene sequences between cells. Thus, the term includes cloning and expression vectors, as well as viral vectors. The heterologous nucleic acid molecule is inserted into the expression system or vector in proper sense (5′→3′) orientation and correct reading frame. The vector contains the necessary elements for the transcription and translation of the inserted Clostridial neurotoxin propeptide-coding sequences.

U.S. Pat. No. 4,237,224 to Cohen and Boyer, which is hereby incorporated by reference in its entirety, describes the production of expression systems in the form of recombinant plasmids using restriction enzyme cleavage and ligation with DNA ligase. These recombinant plasmids are then introduced by means of transformation and replicated in unicellular cultures including prokaryotic organisms and eukaryotic cells grown in culture.

Recombinant genes may also be introduced into viruses, including vaccinia virus, adenovirus, and retroviruses, including lentivirus. Recombinant viruses can be generated by transfection of plasmids into cells infected with virus.

Suitable vectors include, but are not limited to, the following viral vectors such as lambda vector system gt11, gt WES.tB, Charon 4, and plasmid vectors such as pBR322, pBR325, pACYC177, pACYC184, pUC8, pUC9, pUC18, pUC19, pLG339, pR290, pKC37, pKC101, SV 40, pBluescript II SK+/− or KS+/−(see “Stratagene Cloning Systems” Catalog (1993) from Stratagene, La Jolla, Calif., which is hereby incorporated by reference in its entirety), pQE, pIH821, pGEX, pFastBac series (Invitrogen), pET series (see F. W. Studier et. al., “Use of T7 RNA Polymerase to Direct Expression of Cloned Genes,” Gene Expression Technology Vol. 185 (1990), which is hereby incorporated by reference in its entirety), and any derivatives thereof. Recombinant molecules can be introduced into cells via transformation, particularly transduction, conjugation, mobilization, or electroporation. The DNA sequences are cloned into the vector using standard cloning procedures in the art, as described by Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Springs Laboratory, Cold Springs Harbor, N.Y. (1989), which is hereby incorporated by reference in its entirety.

A variety of host-vector systems may be utilized to express the propeptide-encoding sequence in a cell. Primarily, the vector system must be compatible with the host cell used. Host-vector systems include but are not limited to the following: bacteria transformed with bacteriophage DNA, plasmid DNA, or cosmid DNA; microorganisms such as yeast containing yeast vectors; mammalian cell systems infected with virus (e.g., vaccinia virus, adenovirus, etc.); insect cell systems infected with virus (e.g., baculovirus); and plant cells infected by bacteria. The expression elements of these vectors vary in their strength and specificities. Depending upon the host-vector system utilized, any one of a number of suitable transcription and translation elements can be used.

Different genetic signals and processing events control many levels of gene expression (e.g., DNA transcription and messenger RNA (“mRNA”) translation).

Transcription of DNA is dependent upon the presence of a promoter which is a DNA sequence that directs the binding of RNA polymerase and thereby promotes mRNA synthesis. The DNA sequences of eukaryotic promoters differ from those of prokaryotic promoters. Furthermore, eukaryotic promoters and accompanying genetic signals may not be recognized in or may not function in a prokaryotic system, and, further, prokaryotic promoters are not recognized and do not function in eukaryotic cells.

Similarly, translation of mRNA in prokaryotes depends upon the presence of the proper prokaryotic signals which differ from those of eukaryotes. Efficient translation of mRNA in prokaryotes requires a ribosome binding site called the Shine-Dalgarno (“SD”) sequence on the mRNA. This sequence is a short nucleotide sequence of mRNA that is located before the start codon, usually AUG, which encodes the amino-terminal methionine of the protein. The SD sequences are complementary to the 3′-end of the 16S rRNA (ribosomal RNA) and probably promote binding of mRNA to ribosomes by duplexing with the rRNA to allow correct positioning of the ribosome. For a review on maximizing gene expression see Roberts and Lauer, Methods in Enzymology 68:473 (1979), which is hereby incorporated by reference in its entirety.

Promoters vary in their “strength” (i.e., their ability to promote transcription). For the purposes of expressing a cloned gene, it is desirable to use strong promoters in order to obtain a high level of transcription and, hence, expression of the gene. Depending upon the host cell system utilized, any one of a number of suitable promoters may be used. For instance, when cloning in E. coli, its bacteriophages, or plasmids, promoters such as the PH promoter, T7 phage promoter, lac promoter, trp promoter, recA promoter, ribosomal RNA promoter, the PR and PL promoters of coliphage lambda and others, including but not limited, to lacUV5, ompF, bla, lpp, and the like, may be used to direct high levels of transcription of adjacent DNA segments. Additionally, a hybrid trp-lacUV5 (tac) promoter or other E. coli promoters produced by recombinant DNA or other synthetic DNA techniques may be used to provide for transcription of the inserted gene.

Bacterial host cell strains and expression vectors may be chosen which inhibit the action of the promoter unless specifically induced. In certain operons, the addition of specific inducers is necessary for efficient transcription of the inserted DNA. For example, the lac operon is induced by the addition of lactose or IPTG (isopropylthio-beta-D-galactoside). A variety of other operons, such as trp, pro, etc., are under different controls.

Specific initiation signals are also required for efficient gene transcription and translation in prokaryotic cells. These transcription and translation initiation signals may vary in “strength” as measured by the quantity of gene specific messenger RNA and protein synthesized, respectively. The DNA expression vector, which contains a promoter, may also contain any combination of various “strong” transcription and/or translation initiation signals. For instance, efficient translation in E. coli requires a Shine-Dalgarno (“SD”) sequence about 7-9 bases 5′ to the initiation codon (ATG) to provide a ribosome binding site. Thus, any SD-ATG combination that can be utilized by host cell ribosomes may be employed. Such combinations include but are not limited to the SD-ATG combination from the cro gene or the N gene of coliphage lambda, or from the E. coli tryptophan E, D, C, B or A genes. Additionally, any SD-ATG combination produced by recombinant DNA or other techniques involving incorporation of synthetic nucleotides may be used.

Depending on the vector system and host utilized, any number of suitable transcription and/or translation elements, including constitutive, inducible, and repressible promoters, as well as minimal 5′ promoter elements may be used.

The nucleic acid, a promoter molecule of choice, a suitable 3′ regulatory region, and if desired, a reporter gene, are incorporated into a vector-expression system of choice to prepare a nucleic acid construct using standard cloning procedures known in the art, such as described by Sambrook et al., Molecular Cloning: A Laboratory Manual, Third Edition, Cold Spring Harbor: Cold Spring Harbor Laboratory Press, New York (2001), which is hereby incorporated by reference in its entirety.

The nucleic acid molecule encoding a derivative of a Clostridial neurotoxin is inserted into a vector in the sense (i.e., 5′→3′) direction, such that the open reading frame is properly oriented for the expression of the encoded propeptide under the control of a promoter of choice. Single or multiple nucleic acids may be ligated into an appropriate vector in this way, under the control of a suitable promoter, to prepare a nucleic acid construct.

Once the isolated nucleic acid molecule encoding the propeptide has been inserted into an expression vector, it is ready to be incorporated into a host cell. Recombinant molecules can be introduced into cells via transformation, particularly transduction, conjugation, lipofection, protoplast fusion, mobilization, particle bombardment, or electroporation. The DNA sequences are incorporated into the host cell using standard cloning procedures known in the art, as described by Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Springs Laboratory, Cold Springs Harbor, N.Y. (1989), which is hereby incorporated by reference in its entirety. Suitable hosts include, but are not limited to, bacteria, virus, yeast, fungi, mammalian cells, insect cells, plant cells, and the like. Preferable host cells of the present invention include, but are not limited to, Escherichia coli, insect cells, and Pichia pastoris cells.

Typically, an antibiotic or other compound useful for selective growth of the transformed cells only is added as a supplement to the media. The compound to be used will be dictated by the selectable marker element present in the plasmid with which the host cell was transformed. Suitable genes are those which confer resistance to gentamycin, G418, hygromycin, puromycin, streptomycin, spectinomycin, tetracycline, chloramphenicol, and the like. Similarly, “reporter genes” which encode enzymes providing for production of an identifiable compound, or other markers which indicate relevant information regarding the outcome of gene delivery, are suitable. For example, various luminescent or phosphorescent reporter genes are also appropriate, such that the presence of the heterologous gene may be ascertained visually.

In carrying out the method of the present invention, contacting a subject with the isolated, physiologically active, atoxic derivative of a Clostridal neurotoxin can be carried out by administering the isolated derivative of a Clostridial neurotoxin to a subject inhalationally, parenterally, for example, subcutaneously, intravenously, intramuscularly, intraperitoneally, by intranasal instillation, or by application to mucous membranes, such as, that of the nose, throat, and bronchial tubes. The neurotoxin derivative may be administered alone or with suitable pharmaceutical carriers, and can be in solid or liquid form such as, tablets, capsules, powders, solutions, suspensions, or emulsions.

The neurotoxin derivative may be orally administered, for example, with an inert diluent, or with an assimilable edible carrier, or may be enclosed in hard or soft shell capsules, or may be compressed into tablets, or may be incorporated directly with the food of the diet. For oral therapeutic administration, the neurotoxin derivative may be incorporated with excipients and used in the form of tablets, capsules, elixirs, suspensions, syrups, and the like. In one embodiment, the formulation includes hemagglutinin proteins similar to those produced by Clostridium species to protect the neurotoxin in the gastrointestinal tract. Such compositions and preparations should contain at least 0.1% of active compound. The percentage of the compound in these compositions may, of course, be varied and may conveniently be between about 2% to about 60% of the weight of the unit. The amount of active compound in such therapeutically useful compositions is such that a suitable dosage will be obtained.

The tablets, capsules, and the like may also contain a binder such as gum tragacanth, acacia, corn starch, or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid; a lubricant such as magnesium stearate; and a sweetening agent such as sucrose, lactose, or saccharin. When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier, such as a fatty oil.

Various other materials may be present as coatings or to modify the physical form of the dosage unit. For instance, tablets may be coated with shellac, sugar, or both. A syrup may contain, in addition to active ingredient, sucrose as a sweetening agent, methyl and propylparabens as preservatives, a dye, and flavoring such as cherry or orange flavor.

The neurotoxin derivative may also be administered parenterally. Solutions or suspensions can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof in oils. Illustrative oils are those of petroleum, animal, vegetable, or synthetic origin, for example, peanut oil, soybean oil, or mineral oil. In general, water, saline, aqueous dextrose and related sugar solution, and glycols such as, propylene glycol or polyethylene glycol, are preferred liquid carriers, particularly for injectable solutions. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid to the extent that syringability is possible. It must be stable under the conditions of manufacture and storage and can be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), vegetable oils, hyaluronic acid, and suitable mixtures thereof.

The neurotoxin derivative may also be administered directly to the airways in the form of an aerosol. For use as aerosols, the neurotoxin derivative in solution or suspension may be packaged in a pressurized aerosol container together with suitable propellants, for example, hydrocarbon propellants like propane, butane, or isobutane with conventional adjuvants. The neurotoxin derivative also may be administered in a non-pressurized form such as in a nebulizer or atomizer.

BoNTs pass across epithelial surfaces without being destroyed or causing local toxicity. Passage across epithelia is believed to occur by specific binding and transcytosis. The ability of intact BoNT A to pass though pulmonary epithelia and resist proteolytic inactivation was demonstrated in rat primary alveolar epithelial cells and in immortalized human pulmonary adenocarcinoma (Calu-3) cells. The rate of transport was greater in the apical-to-basolateral direction than in the basolateral-to-apical direction, and it was blocked by serotype-specific toxin antibodies (Park et al., “Inhalational Poisoning by Botulinum Toxin and Inhalation Vaccination with Its Heavy-Chain Component,” Infect. Immun. 71:1147-1154 (2003), which is hereby incorporated by reference in its entirety).

Targeting the CNS may require intra-thecal or intra-ventricular administration. Administration may occur directly to the CNS. Alternatively, administration to the CNS may involve retrograde transport from peripheral neurons (motor neurons, nociceptors) to spinal ganglia (see Caleo et al., “A Reappraisal of the Central Effects of Botulinum Neurotoxin Type A: By What Mechanism?” Journal of Neurochemistry 109:15-24 (2009), which is hereby incorporated by reference in its entirety).

Derivatives of a Clostridial neurotoxin of the present invention can be used to augment the endogenous pharmaceutical activity of wild type Clostridial neurotoxins (e.g., BOTOX®), e.g., as a combination therapy.

Derivatives of a Clostridial neurotoxin can be administered as a conjugate with a pharmaceutically acceptable water-soluble polymer moiety. By way of example, a polyethylene glycol conjugate is useful to increase the circulating half-life of the treatment compound, and to reduce the immunogenicity of the molecule. Specific PEG conjugates are described in U.S. Patent Application Publ. No. 2006/0074200 to Daugs et al., which is hereby incorporated by reference in its entirety. Other conjugates include HA, which are described in U.S. Pat. No. 7,879,341 to Taylor and U.S. Patent Application Publication No. 2012/0141532 to Blanda et al., each of which is hereby incorporated by reference in its entirety. Liquid forms, including liposome-encapsulated formulations, are illustrated by injectable solutions and suspensions. Exemplary solid forms include capsules, tablets, and controlled-release forms, such as a miniosmotic pump or an implant. Other dosage forms can be devised by those skilled in the art, as shown, for example, by Ansel and Popovich, Pharmaceutical Dosage Forms and Drug Delivery Systems, 5th Edition (Lea & Febiger 1990), Gennaro (ed.), Remington's Pharmaceutical Sciences, 19th Edition (Mack Publishing Company 1995), and by Ranade and Hollinger, Drug Delivery Systems (CRC Press 1996), each of which is hereby incorporated by reference in its entirety.

According to one embodiment, by treatment it is meant dermatologic or aesthetic treatment (see e.g., Carruthers et al., “Botulinum Toxin A in the Mid and Lower Face and Neck,” Dermatol. Clin. 22:151-158 (2004); Lang, “History and Uses of BOTOX (Botulinum Toxin Type A),” Lippincotts Case Manag. 9:109-112 (2004); Naumann et al., “Safety of Botulinum Toxin Type A: A Systematic Review and Meta-Analysis,” Curr. Med. Res. Opin. 20:981-990 (2004); Vartanian et al., “Facial Rejuvenation Using Botulinum Toxin A: Review and Updates,” Facial Plast. Surg. 20:11-19 (2004), which are hereby incorporated by reference in their entirety) as well as therapeutic treatment (see e.g., Bentsianov et al., “Noncosmetic Uses of Botulinum Toxin,” Clin. Dermatol. 22:82-88 (2004); Carruthers et al., “Botox: Beyond Wrinkles,” Clin. Dermatol. 22:89-93 (2004); Jankovic, “Botulinum Toxin In Clinical Practice,” J. Neurol. Neurosurg. Psychiatry 75:951-957 (2004); Klein, “The Therapeutic Potential of Botulinum Toxin,” Dermatol. Surg. 30:452-455 (2004); Schurch, “The Role of Botulinum Toxin in Neurology,” Drugs Today (Banc) 40:205-212 (2004), which are hereby incorporated by reference in their entirety).

Subjects to be treated pursuant to the method of the present invention include, without limitation, human and non-human primates, or other animals such as dog, cat, horse, cow, goat, sheep, rabbit, or rodent (e.g., mouse or rat).

Preferred treatment methods of the present invention include, but are not limited to, dermatologic or aesthetic treatment, gastroenterologic treatment, genitourinaric treatment, neurologic treatment, oncological treatment, and/or the treatment of any condition characterized by synaptopathology (see, e.g., Brose et al., “Synaptopathies: Dysfunction of Synaptic Function,” Biochem. Soc. Trans. 38:443-444 (2010); Yu & Lu, “Synapses and Dendritic Spines as Pathogenic Targets in Alzherimer's Disease,” Neural Plasticity 2012:1-8 (2012); Siskova et al., “Reactive Hypertrophy of Synaptic Varicosities Within the Hippocampus of Prion-Infected Mice,” Biochem Soc. Trans. 38:471-475 (2010); Warner et al., “TorsinA and DYT1 Dystonia: A Synaptopathy?” Biochem. Soc. Trans. 38:452-456 (2010); Rozas et al., “Presynaptic Dysfunction in Huntington's Disease,” Biochem Soc. Trans. 38:488-492 (2010); and Jones, “Errant Ensembles: Dysfunctional Neuronal Network Dynamics in Schizophrenia,” Biochem. Soc. Trans. 38:516-521 (2010), which are hereby incorporated by reference in their entirety). Treatment of a condition characterized by synaptopathology may involve the neuromodulation of the synapse by the neurotoxin derivative.

Dermatologic or aesthetic treatment includes, but is not limited to, treatment for Rhtyiddess (wrinkles) (Sadick et al., “Comparison of Botulinum Toxins A and B in the Treatment of Facial Rhytides,” Dermatol. Clin. 22:221-226 (2004), which is hereby incorporated by reference in its entirety), including glabellar (Carruthers et al., “Botulinum Toxin type A for the Treatment of Glabellar Rhytides,” Dermatol. Clin. 22:137-144 (2004); Ozsoy et al., “Two-Plane Injection of Botulinum Exotoxin A in Glabellar Frown Lines,” Aesthetic Plast. Surg. 28:114-115 (2004); which are hereby incorporated by reference in their entirety), neck lines (Brandt et al., “Botulinum Toxin for the Treatment of Neck Lines and Neck Bands,” Dermatol. Clin. 22:159-166 (2004), which is hereby incorporated by reference in its entirety), crows feet (Levy et al., “Botulinum Toxin A: A 9-Month Clinical and 3D In Vivo Profilometric Crow's Feet Wrinkle Formation Study,” J. Cosmet. Laser Ther. 6:16-20 (2004), which is hereby incorporated by reference in its entirety), and brow contour (Chen et al., “Altering Brow Contour with Botulinum Toxin,” Facial Plast. Surg. Clin. North Am. 11:457-464 (2003), which is hereby incorporated by reference in its entirety). Other dermatologic treatment includes treatment for hypertrophic masateer muscles (Ahn et al., “Botulinum Toxin for Masseter Reduction in Asian Patients,” Arch. Facial Plast. Surg. 6:188-191 (2004), which is hereby incorporated by reference in its entirety) and focal hyperhydrosis (Glogau, “Treatment of Hyperhidrosis with Botulinum Toxin,” Dermatol. Clin. 22:177-185, vii (2004), which is hereby incorporated by reference in its entirety), including axillary (“Botulinum Toxin (Botox) for Axillary Hyperhidrosis,” Med Lett. Drugs Ther. 46:76 (2004), which is hereby incorporated by reference in its entirety) and genital (Lee et al., “A Case of Foul Genital Odor Treated with Botulinum Toxin A,” Dermatol. Surg. 30:1233-1235 (2004), which is hereby incorporated by reference in its entirety).

Gastroentologic treatment includes, but is not limited to, treatment for esophageal motility disorders (Achem, “Treatment of Spastic Esophageal Motility Disorders,” Gastroenterol. Clin. North Am. 33:107-124 (2004), which is hereby incorporated by reference in its entirety), pharyngeal-esophageal spasm (Bayles et al., “Operative Prevention and Management of Voice-Limiting Pharyngoesophageal Spasm,” Otolaryngol. Clin. North Am. 37:547-558 (2004); Chao et al., “Management of Pharyngoesophageal Spasm with Botox,” Otolaryngol. Clin. North Am. 37:559-566 (2004), which are hereby incorporated by reference in their entirety), and anal fissure (Brisinda et al., “Botulinum Neurotoxin to Treat Chronic Anal Fissure: Results of a Randomized ‘Botox vs. Dysport’ Controlled Trial,” Ailment Pharmacol. Ther. 19:695-701 (2004); Jost et al., “Botulinum Toxin A in Anal Fissure: Why Does it Work?” Dis. Colon Rectum 47:257-258 (2004), which are hereby incorporated by reference in their entirety).

Genitourinaric treatment includes, but is not limited to, treatment for neurogenic dysfunction of the urinary tract (“Botulinic Toxin in Patients with Neurogenic Dysfunction of the Lower Urinary Tracts,” Urologia July-August:44-48 (2004); Giannantoni et al., “Intravesical Resiniferatoxin Versus Botulinum-A Toxin Injections for Neurogenic Detrusor Overactivity: A Prospective Randomized Study,” J. Urol. 172:240-243 (2004); Reitz et al., “Intravesical Therapy Options for Neurogenic Detrusor Overactivity,” Spinal Cord 42:267-272 (2004), which are hereby incorporated by reference in their entirety), overactive bladder (Cruz, “Mechanisms Involved in New Therapies for Overactive Bladder,” Urology 63:65-73 (2004), which is hereby incorporated by reference in its entirety), and neuromodulation of urinary urge incontinence (Abrams, “The Role of Neuromodulation in the Management of Urinary Urge Incontinence,” BJU Int. 93:1116 (2004), which is hereby incorporated by reference in its entirety).

Neurologic treatment includes, but is not limited to, treatment for tourettes syndrome (Porta et al., “Treatment of Phonic Tics in Patients with Tourette's Syndrome Using Botulinum Toxin Type A,” Neurol. Sci. 24:420-423 (2004), which is hereby incorporated by reference in its entirety) and focal muscle spasticity or dystonias (MacKinnon et al., “Corticospinal Excitability Accompanying Ballistic Wrist Movements in Primary Dystonia,” Mov. Disord. 19:273-284 (2004), which is hereby incorporated by reference in its entirety), including, but not limited to, treatment for cervical dystonia (Haussermann et al., “Long-Term Follow-Up of Cervical Dystonia Patients Treated with Botulinum Toxin A,” Mov. Disord. 19:303-308 (2004), which is hereby incorporated by reference in its entirety), primary blepharospasm (Defazio et al., “Primary Blepharospasm: Diagnosis and Management,” Drugs 64:237-244 (2004), which is hereby incorporated by reference in its entirety), hemifacial spasm, post-stroke (Bakheit, “Optimising the Methods of Evaluation of the Effectiveness of Botulinum Toxin Treatment of Post-Stroke Muscle Spasticity,” J. Neurol. Neurosurg. Psychiatry 75:665-666 (2004), which is hereby incorporated by reference in its entirety), spasmodic dysphonia (Bender et al., “Speech Intelligibility in Severe Adductor Spasmodic Dysphonia,” J Speech Lang. Hear Res. 47:21-32 (2004), which is hereby incorporated by reference in its entirety), facial nerve disorders (Finn, “Botulinum Toxin Type A: Fine-Tuning Treatment of Facial Nerve Injury,” J Drugs Dermatol. 3:133-137 (2004), which is hereby incorporated by reference in its entirety), and Rasmussen syndrome (Lozsadi et al., “Botulinum Toxin A Improves Involuntary Limb Movements in Rasmussen Syndrome,” Neurology 62:1233-1234 (2004), which is hereby incorporated by reference in its entirety). Other neurologic treatments include treatment for amputation pain (Kern et al., “Effects of Botulinum Toxin Type B on Stump Pain and Involuntary Movements of the Stump,” Am. J. Phys. Med. Rehabil. 83:396-399 (2004), which is hereby incorporated by reference in its entirety), voice tremor (Adler et al., “Botulinum Toxin Type A for Treating Voice Tremor,” Arch. Neurol. 61:1416-1420 (2004), which is hereby incorporated by reference in its entirety), crocodile tear syndrome (Kyrmizakis et al., “The Use of Botulinum Toxin Type A in the Treatment of Frey and Crocodile Tears Syndrome,” J. Oral Maxillofac. Surg. 62:840-844 (2004), which is hereby incorporated by reference in its entirety), marginal mandibular nerve paralysis, pain control, and anti-nociceptive effects (Cui et al., “Subcutaneous Administration of Botulinum Toxin A Reduces Formalin-Induced Pain,” Pain 107:125-133 (2004) and U.S. Patent Application Publication No. 2012/0064059 to Foster et al., which are hereby incorporated by reference in its entirety), including but not limited to pain after mastectomy (Layeeque et al., “Botulinum Toxin Infiltration for Pain Control After Mastectomy and Expander Reconstruction,” Ann. Surg. 240:608-613 (2004), which is hereby incorporated by reference in its entirety) and chest pain of esophageal origin (Schumulson et al., “Current and Future Treatment of Chest Pain of Presumed Esophageal Origin,” Gastroenterol. Clin. North Am. 33:93-105 (2004), which is hereby incorporated by reference in its entirety). Another neurologic treatment amenable to the methods of the present invention is headache (Blumenfeld et al., “Botulinum Neurotoxin for the Treatment of Migraine and Other Primary Headache Disorders,” Dermatol. Clin. 22:167-175 (2004), which is hereby incorporated by reference in its entirety).

The method of the present invention is also suitable for treatment of cerebral palsy (Balkrishnan et al., “Longitudinal Examination of Health Outcomes Associated with Botulinum Toxin Use in Children with Cerebral Palsy,” J. Surg. Orthop. Adv. 13:76-80 (2004); Berweck et al., “Use of Botulinum Toxin in Pediatric Spasticity (Cerebral Palsy),” Mov. Disord. 19:S162-S167 (2004); Pidcock, “The Emerging Role of Therapeutic Botulinum Toxin in the Treatment of Cerebral Palsy,” J. Pediatr. 145:S33-S35 (2004), which are hereby incorporated by reference in their entirety), hip adductor muscle dysfunction in multiple sclerosis (Wissel et al., “Botulinum Toxin Treatment of Hip Adductor Spasticity in Multiple Sclerosis,” Wien Klin Wochesnchr 4:20-24 (2001), which is hereby incorporated by reference in its entirety), neurogenic pain and inflammation, including arthritis, iatrogenic parotid sialocele (Capaccio et al., “Diagnosis and Therapeutic Management of Iatrogenic Parotid Sialocele,” Ann. Otol. Rhinol. Laryngol. 113:562-564 (2004), which is hereby incorporated by reference in its entirety), and chronic TMJ pain and displacement (Aquilina et al., “Reduction of a Chronic Bilateral Temporomandibular Joint Dislocation with Intermaxillary Fixation and Botulinum Toxin A,” Br. J. Oral Maxillofac. Surg. 42:272-273 (2004), which is hereby incorporated by reference in its entirety). Other conditions that can be treated by local controlled delivery of pharmaceutically active neurotoxin derivatives include intra-articular administration for the treatment of arthritic conditions (Mahowald et al., “Long Term Effects of Intra-Articular BoNT A for Refractory Joint Pain,” Annual Meeting of the American College of Rheumatology (2004), which is hereby incorporated by reference in its entirety), and local administration for the treatment of joint contracture (Russman et al., “Cerebral Palsy: A Rational Approach to a Treatment Protocol, and the Role of Botulinum Toxin in Treatment,” Muscle Nerve Suppl. 6: S181-S193 (1997); Pucinelli et al., “Botulinic Toxin for the Rehabilitation of Osteoarthritis Fixed-Flexion Knee Deformity,” Annual Meeting of the Osteoarthitis Research Society International (2004), which are hereby incorporated by reference in their entirety). The methods of the present invention are also suitable for the treatment of pain associated with various conditions characterized by the sensitization of nociceptors and their associated clinical syndromes, as described in Bach-Rojecky et al., “Antinociceptive Effect of Botulinum Toxin Type A In Rat Model of Carrageenan and Capsaicin Induced Pain,” Croat. Med. J. 46:201-208 (2005); Aoki, “Evidence for Antinociceptive Activity of Botulinum Toxin Type A in Pain Management,” Headache 43 Suppl 1:59-15 (2003); Kramer et al., “Botulinum Toxin A Reduces Neurogenic Flare But Has Almost No Effect on Pain and Hyperalgesia in Human Skin,” J. Neurol. 250:188-193 (2003); Blersch et al., “Botulinum Toxin A and the Cutaneous Nociception in Humans: A Prospective, Double-Blind, Placebo-Controlled, Randomized Study,” J. Neurol. Sci. 205:59-63 (2002), which are hereby incorporated by reference in its entirety.

The neurotoxin derivatives may be customized to optimize therapeutic properties (See e.g., Chaddock et al., “Retargeted Clostridial Endopeptidases: Inhibition of Nociceptive Neurotransmitter Release In Vitro, and Antinociceptive Activity in In Vivo Models of Pain,” Mov. Disord. 8:S42-S47 (2004); Finn, “Botulinum Toxin Type A: Fine-Tuning Treatment of Facial Nerve Injury,” J. Drugs Dermatol. 3:133-137 (2004); Eleopra et al., “Different Types of Botulinum Toxin in Humans,” Mov. Disord. 8:S53-S59 (2004); Flynn, “Myobloc,” Dermatol. Clin. 22:207-211 (2004); and Sampaio et al., “Clinical Comparability of Marketed Formulations of Botulinum Toxin,” Mov. Disord. 8:S129-S136 (2004), which are hereby incorporated by reference in their entirety).

The derivative of a Clostridial neurotoxin may also be used, pursuant to the treatment method of the present invention, to treat diseases influenced by activity-dependent changes in synaptic structure (e.g., synaptopathologies) or hyperactivity of synapse forming apparatus (e.g., tubulin polymerization), and conditions associated with the proliferation of microtubules. For example, Alzheimer's Disease, Parkinson's Disease, and neuronal cancers (of both neural and glial origin). Other conditions that may be treated by the method of the present invention include conditions where the synaptic complex is a disease target.

In one embodiment, neurotoxin derivatives of the present invention accumulate within neuronal cytosol in higher amounts than wild-type Clostridial neurotoxin.

EXAMPLES Example 1 In-Vivo Pharmaceutical Activity Experiments for BoNT A/Ad-0

Material and Methods

An atoxic derivative of Clostridium botulinum serotype A (“BoNT A/ad”), as described in U.S. Pat. No. 7,785,606 to Ichtchenko and Band (which is hereby incorporated by reference in its entirety), was expressed as described. Since this neurotoxin derivative is atoxic and does not possess a cargo attachment peptide sequence at its N-terminus, it was designated “BoNT A/ad-0,” where “ad-0” means atoxic derivative with no cargo site (0), as described herein. BoNT A/ad-0 was purified to electrophoretic homogeneity and activated by specific protease cleavage as described in Band et al., “Recombinant Derivatives of Botulinum Neurotoxin A Engingeered for Trafficking Studies and Neuronal Delivery,” Protein Expression & Purification 71:62 (2010), which is hereby incorporated by reference in its entirety. The purified protein was prepared as a stock at a concentration of 10 mg/ml in PBS containing 40% glycerol for stabilization. The studies described below, evaluate the recombinant molecule's toxicity and pharmacologic activity.

Animals

Mice: female Balb/C mice, 5 to 7 weeks old; weight around 19+/−3 grams.

Digit Abduction Score (DAS) Assay

A modification of the classic mouse Digit Abduction Scoring (“DAS”) assay was used to determine local pharmacologic activity in muscle, measured by muscle weakening effectiveness, as described in Aoki, “Preclinical Update on BOTOX® (Botulinum Toxin Type A)-Purified Neurotoxin Complex Relative to Other Botulinum Neurotoxin Preparations,” European Journal of Neurology (1999), which is hereby incorporated by reference in its entirety. In the DAS Assay, mice are suspended by their tails briefly to elicit a characteristic startle response in which the animal extends its hind limbs and abducts its hind digits. The DAS assay is especially useful to compare the muscle weakening effectiveness of different BoNT preparations (Aoki, “Preclinical Update on BOTOX® (Botulinum Toxin Type A)-Purified Neurotoxin Complex Relative to Other Botulinum Neurotoxin Preparations,” European Journal of Neurology (1999) and Aoki, “A Comparison of the Safety Margins of Botulinum Neurotoxin Serotypes A, B, and F In Mice,” Toxicon 39:1815-1820 (2001), which are hereby incorporated by reference in their entirety).

This test was utilized to define pharmacological activity of BoNT A/ad-0 in mice. Mice were scored as having a positive DAS response when they were unable to fully extend all digits on the injected leg. A negative score is given to mice that spread the toes of the injected leg comparable to that of the non-injected leg.

Female Balb/C mice were given unilateral gastrocnemius intramuscular injections with the concentration described in a volume of 3 μl of 0.9% NaCl using a 25 μl Hamilton syringe. Muscle weakness was assessed from day 1 until 5 days post injection by suspending the mice in order to elicit a characteristic startle response and observing whether the toes on the injected leg were spreading compared to the non injected leg.

Measuring Paralysis

Definitive paralysis is described using two independent variables. First, the inability to use the injected leg to walk (paralysis); and second, the inability to spread the toes on the injected leg (digital abduction).

Results: Toxicity, LD50

The BoNT A/ad-0 preparation described above was used for the following toxicity study. The study was designed to approximate the standard murine LD50 test for wild type BoNT A (“wt BoNT A”).

A total of 30 female mice were used in this study. Each mouse was injected intraperitoneally with the indicated dose of BoNT A/ad-0 in 200 μl of PBS (Table 1), and observed for 24 hours.

Doses ranging from 0.5 μg/mouse to 2 μg/mouse, based on the LD50 published by Pellett et al., “Neuronal Targeting, Internalization, and Biological Activity of a Recombinant Atoxic Derivative of Botulinum Neurotoxin A,” Biochemical & Biophysical Research Communications 405(4):673-677 (2011), which is hereby incorporated by reference in its entirety), using BoNT A/ad (1.2 μg per mouse or 50 μg/kg body weight. The LD50 for BoNT A/ad-0 was found to be very similar to that for BoNT A/ad (Table 1). Briefly, 50% or 5 out of 10 mice injected with a dose of 50 μg/kg body weight showed symptoms of botulism intoxication by 36 hours. All mice injected with a dose of 2 μg, which is approximately 83.3 μg/kg body weight, expired within 48 hours. From this study it is concluded that 50 μg/kg body weight is the approximate LD50 of BoNT A/ad-0.

TABLE 1 Results of Toxicity (LD50) Study for BoNT A/ad-0 Injected Dose No. Mice Dead Survive   2 μg 10 10 0 1.2 μg 10 5 5   1 μg 5 1 4 0.5 μg 5 0 5

The LD50 of wt BoNT A is approximately 0.5 ng/kg (Aoki, “A Comparison of the Safety Margins of Botulinum Neurotoxin Serotypes A, B, and F In Mice,” Toxicon 39:1815-1820 (2001), which is hereby incorporated by reference in its entirety), or 100,000-fold lower than that of BoNT A/ad-O. Because of this toxicity, the effectiveness of wt BoNT A at extremely low doses, and the variability in potency for BoNTs produced from a wild type bacterial source, pharmacological doses of wt BoNT A are generally specified in terms of “activity units,” with 1 mouse LD50 of wt BoNT A considered to be 1 activity unit, or approximately 0.5 ng/kg of wt BoNT A (Aoki, “A Comparison of the Safety Margins of Botulinum Neurotoxin Serotypes A, B, and F In Mice,” Toxicon 39:1815-1820 (2001), which is hereby incorporated by reference in its entirety). This takes into account concentration variations in the level of active toxin between preparations and manufacturers. Harmonized standards across producers remain undefined. This is due to both different manufacturing methods and batch-to-batch variation, but is also related to marketing claims. The final pharmaceutical preparations are formulated with albumin (BOTOX) and/or lactose (Dysport®). From the LD50 results described here, it can be concluded that 1 LD50 Unit (1 U) of BoNT A/ad-0 corresponds to a dose of approximately 50 μg/kg, or approximately 1.2 μg per mouse.

Results: Muscle Paralysis Study/DAS Assay for Pharmacologic Activity In Vivo

BoNT A/ad-0 described above was tested in the murine DAS to determine if BoNT A/ad-0 possesses pharmacological activity at doses significantly below its LD50, and whether it displays typical dose-response activity. Mice were injected in the gastrocnemius muscle with 3 μl of BoNT A/ad-0 in 0.9% NaCl using a 25 μl Hamilton Syringe. The doses administered are expressed as the μg administered per mouse, or units of BoNT A/ad-0 activity administered per mouse (Table 2).

Two observations are noted to categorize a mouse as positive for muscle paralysis induced by administration of BoNT A/ad-O. First, by the inability of the mouse to use the injected leg to walk (muscle paralysis). Second, by observing whether the digits on the injected leg appeared collapsed (digital abduction). Definite muscle paralysis was initially observed and recorded 24 hours after the initial administration. Mice were daily evaluated for definitive muscle paralysis for a maximum of 5 days.

The results of this pharmacologic study of BoNT A/ad-0 are shown in Table 2 and FIG. 2. Mice administered doses ranging from 0.008 LD50 units (0.01 μg) to 0.42 LD50 units (0.5 μg) of BoNT A/ad-0 showed definitive muscle paralysis and digital abduction (FIG. 2 and Table 2), without any signs of mortality. In fact, 4 out of 5 animals injected with 0.01 μg presented with muscle paralysis and some degree of digital abduction (Table 2), indicating that the ED50 for BoNT A/ad-0, the lowest dose at which 50% of the injected animals demonstrate the intended pharmacologic activity, is 0.01 μg or lower, which corresponds to 0.008 LD50 units or lower. All mice that presented paralysis on day 1 continued to present paralysis to the end of the study, day 5. No signs of systemic toxicity were observed in any of the mice in this study.

These data confirm that BoNT A/ad-0 has similar pharmaceutical properties compared to wt BoNT A, albeit with a dose-response profile, a significantly increased range of safe therapeutic activity and, therefore, an improved therapeutic index, and an improved safety margin. This comparison of BoNT A/ad-0 to pharmaceutical preparations of wt BoNT is illustrated in Table 3, and contrasted to the data reported by Aoki, “A Comparison of the Safety Margins of Botulinum Neurotoxin Serotypes A, B, and F In Mice,” Toxicon 39:1815-1820 (2001), which is hereby incorporated by reference in its entirety. For instance, Aoki, “A Comparison of the Safety Margins of Botulinum Neurotoxin Serotypes A, B, and F In Mice,” Toxicon 39:1815-1820 (2001), which is hereby incorporated by reference in its entirety, reported that the safety margin for BOTOX® is about 13.9+/−1.7 and for Dysport® 7.6+/−0.9. Here it is shown that at the lowest dose of BoNT A/ad-0 studied, 0.01 μg, definite paralysisis was observed in 4/5 mice. This dose can be considered a conservative estimate of the ED50. Therefore, for BoNT A/ad-0, the safety margin is approximately 120, or expressed differently, approximately 10-fold better that that for BOTOX® or Dysport® (Table 3).

TABLE 2 Results of Pharmacologic Study of BoNT A/ad-0 No. with Dose LD50 No. Definitive No. Injected per Mouse Units Mice Paralysis Dead 0 (placebo) 0 9 0 0 0.01 μg  0.008 5 4 0 0.1 μg 0.08 5 5 0 0.5 μg 0.42 10 10 0   1 μg 0.83 5 5 0 1.2 μg 1 5 2 3 1.5 μg 1.25 5 1 4

Naive mice were administered BoNT A/ad-0 in the left gastrocnemius via intramuscular injection with 3 μl containing the indicated mass or units of BoNT A/ad-O.

TABLE 3 LD50 and ED50 of BoNT A/ad-0 LD50 = ~1.2 μg ED50 = ~0.01 ug (ED50 = 0.01 μg or lower) LD50/ED50 = safety margin = ~120

If expressed as units, the ED50 of BoNT A/ad-0 is 0.008 LD50 units, or lower.

Comparison to Prior Studies and Conclusions

Prior studies have found that mutations introduced into the light chain of recombinant BoNT A/ad (a molecule containing a cargo attachment peptide as described in U.S. Patent Application Publication No. 2011/0206616 to Ichtchenko and Band, which is hereby incorporated by reference in its entirety) increased the LD50 of the toxin by 100,000-fold. In particular, injections of 0.5 μg (n=25) or 1 μg (n=15) of BoNT A/ad (in the absence of any therapeutic agent) were made into the tibialis muscle two months prior to administration of the repeat dose to each animal. The repeat dose, consisting of 3 μl containing the indicated quantitites of BoNT A/ad, 1 μg (n=18) or 2 μg (n=20), were similarly injected into the tibialis muscle. These data (Table 4 and Table 5) suggest that immune resistance to BoNT A/ad is not developing with repeat treatment.

TABLE 4 BoNT A/ad Induces Paralysis No. with Definitive No. Dead (within Dose No. Mice Paralysis 48 hrs) 0 (placebo) 21 0 0 0.5 μg 38 34 0   1 μg 15 12 1 1.2 μg 10 5 5

1.2 μg is the apparent LD50 for intramuscular injections of BoNT A/ad estimated from this experiment.

TABLE 5 Paralytic Effect After Re-injection of BoNT A/ad No. with Definitive No. Dead (within Repeat Dose No. Mice Paralysis 48 hrs) 1 μg 18 17 0 2 μg 20 15 dead, with 3 appearing sick. 2 mice appeared normal at 48 hrs.

In the present study it was found that the LD50 of BoNT A/ad-0, which has identical toxin-disabling mutations as BoNT A/ad, is likewise elevated ˜100,000-fold relative to wt BoNT A. But surprisingly, it was observed that BoNT A/ad-0 still possessed pharmacologic activity similar to that observed for wt BoNT A, and that a therapeutic agent need not be delivered via the cargo site of BoNT/A ad to render it therapeutic. By comparing the dose-response of BoNT A/ad-0 to that reported for pharmaceutical preparations of wt BoNT A, it can be concluded that BoNT A/ad-0 can be used for pharmaceutical treatments in the same way as wt BoNTs, but with significantly reduced danger of systemic toxicity, and thus significant improved safety advantages for clinical use.

Although the invention has been described in detail for the purposes of illustration, it is understood that such detail is solely for that purpose, and variations can be made therein by those skilled in the art without departing from the spirit and scope of the invention which is defined by the following claims.

Claims

1. A treatment method comprising:

contacting a subject with an isolated, physiologically active, atoxic derivative of a Clostridial neurotoxin, said contacting being carried out to treat the subject, with the proviso that the derivative of a Clostridial neurotoxin does not possess a cargo attachment peptide sequence at its N-terminus.
Patent History
Publication number: 20160296608
Type: Application
Filed: Apr 15, 2016
Publication Date: Oct 13, 2016
Applicant: New York University (New York, NY)
Inventors: Edwin J. Vazquez-Cintron (New York, NY), Konstantin Ichtchenko (Brooklyn, NY), Philip A. Band (West Orange, NJ)
Application Number: 15/130,100
Classifications
International Classification: A61K 38/48 (20060101); A61K 9/00 (20060101);