WO2011022721A1

WO2011022721A1 - Inhibitors of botulinum neurotoxins

Info

Publication number: WO2011022721A1
Application number: PCT/US2010/046314
Authority: WO
Inventors: Terry L. Bowlin; Norton P. Peet; Michelle M. Butler; Steven C. Cardinale; Bing Li; Ramdas Pai
Original assignee: Microbiotix, Inc
Priority date: 2009-08-21
Filing date: 2010-08-23
Publication date: 2011-02-24

Abstract

Organic compounds showing the ability to inhibit Botulinum neurotoxin activity are disclosed. The disclosed Botulinum neurotoxin inhibitor compounds are useful for treating, preventing, or reducing the intoxication of Botulinum neurotoxin, particularly Botulinum neurotoxin A and/or B.

Description

INHIBITORS OF BOTULINUM NEUROTOXINS

Cross-Reference to Priority Applications

This application claims priority to US Provisional Appln. No. 61/274,809 filed August 21, 2009, US Provisional Appln. No. 61/320,268 filed April 1, 2010, and Provisional Appln. No. 61/375,694 filed August 20, 2010, the contents of which are incorporated herein. Statement Regarding Federally Sponsored Research

The invention described herein was supported by National Institutes of Health grant no. 1 UOl AI070430. Accordingly, the United States Government has certain rights in the invention.

Field of the Invention

This invention is in the field of therapeutic drugs to inhibit the effects of Botulinum neurotoxin (BoNT). In particular, the invention provides low molecular weight inhibitors of Botulinum neurotoxin A and/or B. Background of the Invention

Botulinum neurotoxins are the most potent of the biological toxins, and due to their lethality are listed as category A (highest priority) bioterrorism agents by the Centers for Disease Control and Prevention (Arnon et al., JAMA, 285: 1059-70 (2001); Dembek et al., Disaster Med. Public Health Prep., 1: 122-34 (2007)). Botulinum neurotoxins are easily produced and may be delivered by the aerosol route (Burnett et al., Nat. Rev. Drug Discov., 4: 281-97 (2005); Paddle, B.M., J. Appl. Toxicol, 23: 139-70 (2003)). Consequently, these toxins represent a serious threat to both military personnel and civilians (Clarke, S.C., Br. J. Biomed. ScL, 62:40-6 (2005)).

Once inhaled into the lungs, Botulinum neurotoxins are taken up by the blood stream, target the peripheral cholinergic nerve endings, and cause death by interrupting autonomic nerve function (Rasetti-Escargueil et al., Toxicon., 53: 503-11 (2009)). The toxin is a two- chain polypeptide with a 100-kDa heavy chain joined by a disulphide bond to a 50-kDa light chain. This light chain is an enzyme (a protease) that attacks one of the fusion proteins (SNAP-25, syntaxin or synaptobrevin) at a neuromuscular junction, preventing vesicles from anchoring to the membrane to release acetylcholine. By inhibiting acetylcholine release, the toxin interferes with nerve impulses and causes the flaccid (sagging) paralysis of muscles in botulism. The zinc-dependent endopeptidase light chain (LC) portion of Botulinum neurotoxins impairs neuronal exocytosis through proteolysis of essential SNARE (soluble NSF- ethylmaleimide-sensitive factor attachment protein receptor) components of

neurotransmission (Simpson, L.L., Annu. Rev. Pharmacol. Toxicol., 44:167-93 (2004)).

There are seven Botulinum neurotoxin serotypes (A-G), which differ significantly in amino acid sequence, protein substrates, and substrate cleavage sites as well as in the duration of resulting paralysis (Dembek et al. (2007) op. cit.; Simpson, L.L. (2004) op. cit).

Botulinum neurotoxin A paralysis lasts the longest, typically 4-6 months, making it popular for both medicinal and cosmetic applications (Charles, P.D., Am. J. Health Syst. Pharm., 61 :S11-23 (2004)). Examination of several Botulinum neurotoxins in a rat cerebellar neuron model system revealed a rank order of half-lives: Botulinum neurotoxin A »31 days;

Botulinum neurotoxin C »25 days; Botulinum neurotoxin B ~10 days; Botulinum neurotoxin F ~2 days; and Botulinum neurotoxin E ~0.8 days (see, Foran et al., J. Biol. Chem., 278: 1363-71 (2003)). The duration of paralysis from Botulinum neurotoxin A coupled with its potency and the fact that several high resolution crystal structures are available (Silvaggi et al., Chem. Biol., 14: 533-542 (2007); Silvaggi et al., Biochem., 47: 5736-5745 (2008)) make it tractable and relevant for immediate drug discovery efforts. Of the seven types of Botulinum neurotoxin, only types A, B, E, and F cause illness in humans (U.S. Center for Disease Control, Botulism (2009) (see,

www.cdc.gov/nczved/dfbmd/disease_listing/botulism_gi.html)).

Currently, there are two primary Botulinum Antitoxins in development for treatment of botulism. Trivalent (A, B, E serotype) Botulinum Antitoxin is derived from equine sources utilizing whole antibodies. The second antitoxin is Heptavalent (A, B, C, D, E, F, G) Botulinum Antitoxin, which is derived from "despeciated" equine IgG antibodies, which have had the Fc portion cleaved off leaving the F(ab')2 antibody fragments. This is a less immunogenic antitoxin that is effective against all known strains of botulism where not contraindicated. On June 1, 2006 the United States Department of Health and Human Services awarded a $363 million contract with Cangene Corporation for 200,000 doses of Heptavalent Botulinum Antitoxin over five years for delivery into the Strategic National Stockpile beginning in 2007.

DynPort Vaccine Company LLC (DVC) is working on a government sponsored vaccine approach to develop a recombinant vaccine against Botulinum neurotoxin (BoNT) serotypes A and B. Like many products in DVCs advanced development pipeline, the rBV A/B vaccine candidate was conceived and initially developed at the U.S. Army Medical Research Institute of Infectious Diseases (USAMRIID). The responsibility for the advanced development of this vaccine was transferred to DVC in 2000, including nonclinical testing and production under current good manufacturing practices.

Although the foregoing passive and active immunization products have some promise, especially for mass inoculation, these measures cannot counter the toxic effects of Botulinum neurotoxins after they penetrate neurons (Willis et al., Angew Chem. Int. Ed. Engl., 47:8360-79 (2008). Currently, critical care mechanical ventilation is the only treatment option once neurons have been intoxicated and diaphragm muscles cease to function. However, the effects of internalized Botulinum neurotoxins can last for months (Eleopra et al., Neurosci. Lett, 256: 135-8 (1998); Meunier, F. A., MoI. Cell. NeuroscL, 22: 454-66 (2003)), making long-term mechanical ventilation impractical if even a limited number of individuals were simultaneously intoxicated.

Therefore, there is an urgent need to identify and develop low molecular weight non- peptidic inhibitors that will serve as both preventive medicines and post-exposure 'rescue' therapeutics. Potent inhibitors against Botulinum neurotoxins could be used advantageously during certain surgical procedures, natural outbreaks of botulism poisoning, or bio-terrorist attacks, and could be used either prophylactically to treat a potentially exposed population or therapeutically after exposure or infection.

Summary of the Invention

The invention addresses the above needs by providing new Botulinum neurotoxin inhibitor compounds of different chemotypes. To identify Botulinum neurotoxin inhibitor compounds described herein, a high-throughput screening assay based on fluorescence resonance energy transfer (FRET) was developed to detect proteolysis of a peptide substrate representing several amino acid residues spanning the site in the native protein substrate cleaved by the Botulinum neurotoxin during intoxication. The general assay method was used to screen a collection of over 70,000 molecules contained in a chemical compound library to identify Botulinum neurotoxin inhibitors. Libraries of thousands of discrete small molecule organic compounds and purified natural products were screened using this assay. The inhibitor compounds ("hits") from the high throughput primary FRET assay screen were further qualified through a series of secondary assays, including an HPLC-based assay. The specificity of selected Botulinum neurotoxin inhibitor compounds was further evaluated against various human metalloproteases and Bacillus anthracis Lethal Factor (as a counter screen to eliminate non-specific inhibitors), and cytotoxicity testing was performed for selected hit inhibitor compounds. Selected qualified, confirmed hits were further validated as exhibiting specificity for Botulinum neurotoxin A zinc-dependent endopeptidase light chain (BoNT/A LC) inhibition with respect to human metalloproteases and displayed activity in a neuronal assay for Botulinum intoxication.

Accordingly, a Botulinum neurotoxin inhibitor compound described herein inhibits or reduces Botulinum neurotoxin activity. Preferred Botulinum neurotoxin inhibitor compounds described herein inhibit or reduce Botulinum neurotoxin interruption of phrenic nerve function. Preferred Botulinum neurotoxin inhibitor compounds described herein inhibit and/or reduce the zinc-dependent endopeptidase light chain (LC) activity of Botulinum neurotoxin. Preferred inhibitor compounds of the present invention are Botulinum

neurotoxin A inhibitors; at least one of the compounds disclosed herein is shown to be an inhibitor of both Botulinum neurotoxin A and Botulinum neurotoxin B.

The present invention provides isolated Botulinum neurotoxin inhibitor compounds of the formulae A-R:

Compound designation: MSL-111012,

Compound name: N-(4-(5-chlorobenzo[d]oxazol-2-yl)phenyl)-2,3-dihydrobenzo[b]

[ 1 ,4]dioxine-6-carboxamide;

Compound designation MSL-113604,

Compound name: 3-chloro-4-methyl-N-(2-phenyl-2H-benzo[d][l,2,3]triazol-5-yl)benzamide;

Compound designation: MSL-130541,

Compound name: N-(3-acetylphenyl)-2-((5,6-dimethylthieno[2,3-d]pyrimidin-4-yl) thio)acetamide;

Compound designation: MSL-136988,

Compound name: 2-ethoxy-N-(6-sulfamoylbenzo[d]thiazol-2-yl)benzamide;

Compound designation: MSL-151862,

Compound name: 5-chloro-7-((pyridin-2-ylamino)(thiophen-3-yl)methyl)quinolin-8-ol;

Compound designation: MSL-066297,

Compound name: 5-chloro-7-(morpholino(pyridin-4-yl)methyl)quinolin-8-ol;

Compound designation: MSL-062516,

Compound name : 6-chloro-3 -(2-(cyclohex- 1 -en- 1 -yl)ethyl)-3 ,4-dihydro-2H-

[ 1 ,3]oxazino[5,6-h]quinoline;

Compound designation: MSL-059336,

Compound name: 5-(benzo[d]thiazol-2-yl)-2-((lE,3Z)-3-(5-(benzo[d]thiazol-2-yl)-3- ethyl- 1 -phenyl- 1 H-benzo [d]imidazol-2(3H)-ylidene)prop- 1 -en- 1 -yl)-3 - ethyl-1 -phenyl-lH-benzo[d]imidazol-3-ium iodide;

Compound designation: MSL-059327,

Compound name: (E)-3-butyl-2-((l-ethyl-6-methoxyquinolin-2(lH)-ylidene)methyl)-5- methoxybenzo[d]thiazol-3-ium acetate;

Compound designation: MSL- 145005,

Compound name : 1 -( 1 H-benzo [d]imidazol-2-yl)-3 -(4-methoxyphenyl)- 1 H-pyrazol-5 -ol;

Compound designation: MSL-111029,

Compound name: (E)-3-(4-(dimethylamino)phenyl)-2-(4-oxo-3,4-dihydroquinazolin-2- yl)acrylonitrile;

(L)

Compound designation: MSL-145815,

Compound name: 2-(lH-benzo[d]imidazol-2-yl)-3-(3-iodo-4-methoxyphenyl)

acrylonitrile;

Compound designation: MBX 1519,

Compound name: (E)-2-(lH-benzo[d]imidazol-2-yl)-3-(3-chloro-4-hydroxy-5- methoxyphenyl)acrylonitrile;

Compound designation: MBX 1520,

Compound name : (E)-2-( 1 H-benzo [d]imidazol-2-yl)-3 -(3 -bromo-4-hydroxy-5 - methoxyphenyl)acrylonitrile;

Compound designation: MBX 1553,

Compound name: (E)-3 -( [2,2'-bithiophen] -5 -yl)-2-( 1 H-benzo [d] imidazol-2-yl)acrylonitrile;

Compound designation: MBX 1638,

Compound name: (E)-2-(lH-benzo[d]imidazol-2-yl)-3-(biphenyl-4-yl)acrylonitrile;

(Q)

Compound designation: MBX 1722,

Compound name : (E)-3 -(4-( 1 H-imidazol- 1 -yl)phenyl)-2-( 1 H-benzo [d] imidazol-

2-yl)acrylonitrile;

Compound designation: MBX 1868,

Compound name: (E)-2-(lH-benzo[d]imidazol-2-yl)-3-(5-(furan-2-yl)thiophen-

2-yl)acrylonitrile,

and pharmaceutically acceptable salts thereof.

The present invention further provides isolated Botulinum neurotoxin inhibitor compounds of the formula J-I :

wherein

Y is O or NH;

R¹ is hydrogen, hydroxyl (-OH), lower (C_1-3) alkyl, lower (C_1-3) alkoxy (e.g., -OCH₃,

-OCH₂CH₃, -OC₃H₇), amino (-NH₂), or amido, i.e., -NHC(O)R, where R is phenyl or lower (C_1-3) alkyl;

R² is hydrogen, lower (C_1-3) alkyl, lower (C_1-3) alkoxy, cyano (-C≡N), cyanoalkyl (i.e., -(CH₂)_nC≡N, where n is 1, 2 or 3), a dialkyl ester radical of the formula

-(CH₂)_xC(=O)O(CH₂)_yCH₃ (where x is 1, 2 or 3 and y is 0, 1 or 2), phenyl (-C₆H₅), or C_1-3 alkylphenyl (i.e., -(CEb)_nC₆H₅, where n is 1-3);

R³ is hydrogen, lower (C_1-3) alkyl, lower (C_1-3) alkoxy, a dialkyl ester radical of the formula

-(CH₂)χC(=O)O(CH₂)_yCH₃ (where x is 1, 2 or 3 and y is 0, 1 or 2), phenyl (-C₆H₅), or phenyl substituted with halo (F, Cl, Br, I), with lower (C_1-3) alkyl, or with lower (C_1-3) alkoxy

(e.g., -OCH₃, -OCH₂CH₃, -OC₃H₇); or

R² and R³ together form a divalent hydrocarbon radical of 3-4 carbons, e.g., -(CH₂)_Z-, where z is 3 or 4, to form a five- or six-member ring fused with the pyrazole ring;

and pharmaceutically acceptable salts thereof.

Preferred hydroxypyrazolyl benzimidazoles will have the formula J-2:

wherein

R² is hydrogen, lower (C_1-3) alkyl, a dialkyl ester radical of the formula

-(CH₂)_xC(=O)O(CH₂)_yCH₃ (where x is 1, 2 or 3 and y is 0, 1 or 2), phenyl (-C₆H₅), or C_1-3 alkylphenyl (i.e., -(CH₂)_nC₆H₅, where n is 1-3);

R³ is hydrogen, C_1-3 alkyl, a dialkyl ester radical of the formula -(CH₂)_xC(=O)O(CH₂)_yCH₃

(where x is 1, 2 or 3 and y is 0, 1 or 2), phenyl (-C₆H₅), or phenyl substituted with halo (F,

Cl, Br, I), with lower (C_1-3) alkyl, or with lower (C_1-3) alkoxy (e.g., -OCH₃, -OCH₂CH₃, -OC₃H₇); or

and pharmaceutically acceptable salts thereof. Such compounds exhibit both BoNT/A and

BoNTVB inhibitory activity.

The present invention further provides isolated Botulinum neurotoxin inhibitor compounds of the formula L-I:

wherein

Ar is an aryl or heteroaryl ring of 4-6 members, which heteroaryl rings may have 1-3 heteroatoms independently selected from O, S, or N, and where Ar may be substituted or unsubstituted, with one or more substituents selected from lower (C_1-3) alkyl, lower (C_1-3) alkoxy, halo (F, Cl, Br, I), cyano (-C≡N), or nitro (-NO₂); and

n is 0 or 1 ;

and pharmaceutically acceptable salts thereof.

The foregoing compounds were identified as inhibitors of Botulinum neurotoxin A activity by assays showing specific inhibition of the zinc-dependent endopeptidase light chain (LC) activity of Botulinum neurotoxin. The light chain is an enzyme (a protease) that attacks one of the SNARE fusion proteins (e.g., SNAP-25, syntaxin or synaptobrevin, also known as vesicle-associated membrane protein, or VAMP-2) at a neuromuscular junction, preventing vesicles from anchoring to the membrane to release acetylcholine and thereby interfering with nerve impulses, causing paralysis of muscles.

Selected compounds were additionally tested for inhibition of Botulinum neurotoxin B activity by assays showing specific inhibition of the zinc-dependent endopeptidase light chain (LC) activity of Botulinum neurotoxin B. While the majority of compounds were specific for Botulinum neurotoxin A LC, at least one group of the compounds disclosed herein is shown to be an inhibitor of both Botulinum neurotoxin A and Botulinum neurotoxin B, indicating that the inhibitor compounds according to this invention may be effective inhibitors of multiple Botulinum neurotoxin species.

Botulinum neurotoxin inhibitory properties discovered for the compounds of the invention are set forth in the examples and Figures 1-5 infra. Inhibitor compounds were initially identified as inhibiting Botulinum neurotoxin A LC activity by at least 90% at a concentration of 83 μM using a 384- well FRET assay described infra with 24 μM SNAP-25 substrate and 1 nM Botulinum neurotoxin A LC in a volume of 25 μL. Initial hit compounds were re-confirmed as inhibiting Botulinum neurotoxin A LC activity by at least 70% at a concentration of 25 μM using a 96-well FRET assay described infra with 20 μM SNAP-25 substrate and 2 nM Botulinum neurotoxin A LC in a volume of 100 μL. A secondary HPLC assay was performed on most of the compounds to confirm BoNT/A inhibitory activity, and a counter-assay against B. anthracis Lethal Factor (BaLF) activity was performed to determine whether antitoxin activity of the BoNT/A inhibitor hits was specific for BoNT/A.

In a preferred embodiment, a Botulinum neurotoxin inhibitor compound useful in the compositions and methods described herein inhibits Botulinum neurotoxin A LC activity by greater than 70% at a 25 μM concentration as measured in a 96-well FRET assay such as described herein. Preferably, the Botulinum neurotoxin inhibitor compound inhibits

Botulinum neurotoxin A LC activity by more than 75%, 80%, 85%, 90%, or most preferably more than 95% at a 25 μM concentration.

hi a particularly preferred embodiment, a Botulinum neurotoxin inhibitor compound useful in the compositions and methods described herein has an ICs₀ of less than 100 μM, more preferably less than 75 μM, less than 50 μM, less than 40 μM, less than 25 μM, and most preferably less than 10 μM as measured in a standard assay for Botulinum neurotoxin inhibition such as the FRET or HPLC activity assays described herein. As between the FRET and HPLC assays, the HPLC assay described herein is considered more accurate, as it is less subject to fluorescence interference. Especially preferred compounds will also show a relatively low cytotoxicity toward human cells, such as a CC₅₀ value of greater than or equal to 25 μM (CC_5O≥ 25 μM) as measured in a standard cytotoxicity assay such as described herein or as employed in the pharmaceutical field for antitoxins. The ratio of BoNT inhibitory activity to the cytotoxicity is also a useful measure of inhibitor predicted therapeutic selectivity. Preferred compounds exhibit an IC₅o/CC₅o ratio of >2, more preferably >5, >10, most preferably >100. Such standard cytotoxicity assays may employ any mammalian cell typically employed in cytotoxicity assays for antitoxins, including but not limited to, Chinese hamster ovary (CHO) cells, Vero (African green monkey kidney) cells, HeLa cells, Hep-G2 (human hepatocellular carcinoma) cells, human embryonic kidney (HEK) 293 cells, 293T cells, 293FT cells (Invitrogen), BHK (newborn hamster kidney) cells, COS cells, and the like.

hi another embodiment, a Botulinum neurotoxin inhibitor compound useful in the compositions and methods described herein is selected from the group of inhibitor compounds consisting of:

(F)

(Q)

and pharmaceutically acceptable salts thereof.

Preferred Botulinum neurotoxin inhibitor compounds described herein include compounds of the formulae J-2 and L-I:

wherein

R² is hydrogen, lower (C_1-3) alkyl, a dialkyl ester radical of the formula

-(CH₂)_xC(=O)O(CH₂)_yCH₃ (where x is 1, 2 or 3 and y is 0, 1 or 2), phenyl (-C₆H₅), or C_1-3 alkylphenyl (i.e., -(CH₂)_nC₆H₅, where n is 1-3); R³ is hydrogen, C_1-3 alkyl, a dialkyl ester radical of the formula -(CH₂)_xC(=O)O(CH₂)_yCH₃

Cl, Br, I), with lower (C_1-3) alkyl, or with lower (C_1-3) alkoxy (e.g., -OCH₃, -OCH₂CH₃,

-OC₃H₇); or

and pharmaceutically acceptable salts thereof;

and

wherein

n is O or l;

and pharmaceutically acceptable salts thereof;

and combinations thereof.

The Botulinum neurotoxin inhibitor compounds described herein are useful as antitoxin agents and may be used to treat Clostridium botulinum infection, and more specifically, to inhibit or reduce Botulinum neurotoxin intoxication (poisoning), either prophylactically, i.e., when administered to an individual, group or population exposed to or contacted with the neurotoxins or threatened with exposure to such neurotoxins, or therapeutically after intoxication has occurred. Accordingly, an individual exposed to or intoxicated with a Botulinum neurotoxin, especially Botulinum neurotoxin types A and/or B, may be treated by administering to the individual in need an effective amount of a compound according to the invention, e.g., by administering one or more of the following compounds:

(Q)

compounds of the formula J-2:

wherein

R² is hydrogen, lower (C_1-3) alkyl, a dialkyl ester radical of the formula

R³ is hydrogen, lower (C_1-3) alkyl, a dialkyl ester radical of the formula

-(CH₂)_xC(=O)O(CH₂)_yCH₃ (where x is 1, 2 or 3 and y is 0, 1 or 2), phenyl (-C₆H₅), or phenyl substituted with halo (F, Cl, Br, I), with lower (C_1-3) alkyl, or with lower (C_1-3) alkoxy (e.g., -OCH₃, -OCH₂CH₃, -OC₃H₇); or

or

compounds of the formula L-I:

wherein

n is O or 1 ;

or pharmaceutically acceptable salts of any of the foregoing compounds or combinations thereof.

Use of one or more or a combination of the above compounds to treat Botulinum neurotoxin poisoning is contemplated herein. Especially, use of one or more or a

combination of the above compounds to inhibit Botulinum neurotoxin types A and/or B activity is contemplated herein. In particular, use of one or more or a combination of the above compounds for the inhibition of Botulinum neurotoxin type A activity is

advantageously carried out by following the teachings herein.

Use of one or more or a combination of the above compounds to prepare a

medicament for treating Botulinum neurotoxin intoxication (botulism) or, more particularly, a medicament for inhibiting Botulinum neurotoxin is contemplated herein. Especially, use of one or more or a combination of the above compounds for preparing a pharmaceutical composition to inhibit Botulinum neurotoxin type A and/or B is contemplated herein.

The present invention also provides pharmaceutical compositions containing one or more of the Botulinum neurotoxin inhibitor compounds disclosed herein and a

pharmaceutically acceptable carrier or excipient. The use of one or more of the Botulinum neurotoxin inhibitor compounds in the preparation of a medicament for combating Botulinum neurotoxin intoxication is disclosed.

In yet another embodiment, a composition comprising a Botulinum neurotoxin inhibitor or a combination of Botulinum neurotoxin inhibitors described herein may also comprise a second agent (second active ingredient, second active agent) that possesses a desired therapeutic or prophylactic activity other than that of direct Botulinum neurotoxin inhibition. Such a second active agent includes, but is not limited to, an antibiotic, an antibody or antibody fragment, an antiviral agent, an anticancer agent, an analgesic (e.g., a non-steroidal anti-inflammatory drug (NSAID), acetaminophen, an opioid, a COX-2 inhibitor), an immunostimulatory agent (e.g., a cytokine), a hormone (natural or synthetic), a central nervous system (CNS) stimulant, an antiemetic agent, an anti-histamine, an erythropoietin, a complement stimulating agent, a sedative, a muscle relaxant agent, an anesthetic agent, an anticonvulsive agent, an antidepressant, an antipsychotic agent, and combinations thereof.

Compositions comprising a Botulinum neurotoxin inhibitor described herein may be formulated for administration to an individual (human or other animal) by any of a variety of routes including, but not limited to, intravenous, intramuscular, subcutaneous, intra-arterial, parenteral, intraperitoneal, sublingual (under the tongue), buccal (cheek), oral (for swallowing), topical (epidermis), transdermal (absorption through skin and lower dermal layers to underlying vasculature), nasal (nasal mucosa), intrapulmonary (lungs), intrauterine, vaginal, intracervical, rectal, intraretinal, intraspinal, intrasynovial, intrathoracic, intrarenal, nasojejunal, and intraduodenal.

In other embodiments, Botulinum neurotoxin inhibitor compounds described herein are useful as toxin neutralizing agents, used individually or in combination with other Botulinum neurotoxin inhibitor compounds described herein or neutralizing agents known in the field.

Brief Description of the Drawings

Figure 1 is a graph showing a representative BoNT/A LC FRET assay kinetic trace of the SNAP-25 substrate cleavage over the 40 minute incubation period. Line A is native BoNT/A LC; line B is heat denatured BoNT/A LC Figure 2 is a graph showing a representative BoNT/A HPLC assay, with the top lines representing the programmed and actual gradient and the bottom line showing absorption at 365 nm of the HPLC effluent. The sharp peak in the bottom absorption line (at ~24 min.) represents the intact peptide while the peaks at ~8 min. and 10 min. represent the N-terrninal and C-terminal pieces of the peptide, respectively.

Figure 3 is a graph showing BoNT/A FRET-based IC₅₀ plots for the compound of formula L (MSL-145815, squares) and the compound of formula E (MSL-151862, diamonds).

Figures 4A-4C show results of a chick neuronal cell assay for BoNT/A inhibition. Fig. 4 A shows a dose response for screening compound L (MSL-145815) tested at 20 μM and 50 μM. Fig. 4B shows a dose response for screening compound E (MSL-151862) tested at 12.5 μM, 25 μM, 50 μM, and 100 μM. Fig. 4C shows a single dose result for compound MBX 1553 tested at 30 μM.

Figures 5 A, 5B, and 5C show inhibitor enzyme kinetics - linear transformation using Eadie-Hofstee plots. Figure 5 A shows a plot for hydroxyquinoline compound E (MSL- 151862) at selected concentrations: closed diamonds (♦), no inhibitor; closed squares (■), 3.125 μM; closed triangles (A), 6.25 μM; closed circles (•), 25 μM; asterisk (*), 50 μM. Figure 5B shows a plot for benzimidazole acrylonitrile compound L (MSL-145815) at selected concentrations: closed diamonds (♦), no inhibitor; closed squares (■), 6.25 μM; closed triangles (A), 12.5 μM; x sign (x), 25 μM; asterisk (*), 50 μM; closed circles (•), 75 μM; plus sign (+) 100 μM. Figure 5C shows a plot for benzimidazole acrylonitrile compound O (MBX 1553) at selected concentrations: closed diamonds (♦), no inhibitor; closed squares (■), 6.25 μM; closed triangles (A), 12.5 μM; closed circles (•), 25 μM;

asterisk (*), 50 μM.

Detailed Description of the Invention

The invention provides organic compounds that inhibit and/or reduce Botulinum neurotoxin activity. Botulinum neurotoxins are taken up by the blood stream, target the peripheral cholinergic nerve endings, and cause death by interrupting autonomic nerve function (Rasetti-Escargueil et al., 2009, op. cit.). Botulinum neurotoxin is a two-chain polypeptide with a 100-kDa heavy chain joined by a disulphide bond to a 50-kDa light chain. This zinc-dependent endopeptidase light chain (LC) portion of Botulinum neurotoxins impair neuronal exocytosis through proteolysis of essential SNARE (soluble NSF-ethylmaleimide- sensitive factor attachment protein receptor) components of neurotransmission (Simpson, L.L. 2004, op. cit).

In order that the invention may be more clearly understood, the following

abbreviations and terms are used as defined below.

Botulinum neurotoxin as defined herein refers to is a neurotoxic protein produced by the bacterium Clostridium botulinum, and specifically, the seven Botulinum neurotoxin serotypes (A-G).

Abbreviations for various substituents (side groups, radicals) of organic molecules are those commonly used in organic chemistry. Such abbreviations may include "shorthand" forms of such substituents. For example, "Ac" is an abbreviation for an acetyl group, "Ar" is an abbreviation for an aryl or heteroaryl group, and "halo" or "halogen" indicates a halogen radical (e.g., F, Cl, Br, I). "Me" and "Et" are abbreviations used to indicate methyl (CH₃-) and ethyl (CH₃CH₂-) groups, respectively; and "OMe" (or "MeO") and "OEt" (or "EtO") indicate methoxy (CH₃O-) and ethoxy (CH₃CH₂O-), respectively. Hydrogen atoms are not always shown in organic molecular structures or may be only selectively shown in some structures, as the presence and location of hydrogen atoms in organic molecular structures are understood and known by practitioners in the field of this invention. Likewise, carbon atoms are not always specifically abbreviated with "C" in structural formulae, as the presence and location of carbon atoms, e.g., connected by or at the end of bond-lines in structural diagrams, are known and understood by persons skilled in the art. Minutes are commonly abbreviated as "min"; hours are commonly abbreviated as "hr" or "h".

A composition or method described herein as "comprising" one or more named elements or steps is open-ended, meaning that the named elements or steps are essential, but other elements or steps may be added within the scope of the composition or method. To avoid prolixity, it is also understood that any composition or method described as

"comprising" (or which "comprises") one or more named elements or steps also describes the corresponding, more limited composition or method "consisting essentially of (or which "consists essentially of) the same named elements or steps, meaning that the composition or method includes the named essential elements or steps and may also include additional elements or steps that do not materially affect the basic and novel characteristic(s) of the composition or method. It is also understood that any composition or method described herein as "comprising" or "consisting essentially of one or more named elements or steps also describes the corresponding, more limited, and closed-ended composition or method "consisting of (or "consists of) the named elements or steps to the exclusion of any other unnamed element or step. In any composition or method disclosed herein, known or disclosed equivalents of any named essential element or step may be substituted for that element or step. It is also understood that an element or step "selected from the group consisting of refers to one or more of the elements or steps in the list that follows, including combinations of any two or more of the listed elements or steps.

The terms "Botulinum neurotoxin inhibitor compound" and "BoNT inhibitor" as used herein are interchangeable and denote compounds exhibiting the ability to specifically inhibit the toxic activity of at least one Botulinum neurotoxin serotype. The compounds disclosed herein were discovered by screening for activity with respect to Botulinum neurotoxin A (BoNT/ A). Some compounds were subsequently also discovered to have inhibitory activity with respect to Botulinum neurotoxin B (BoNT/B). Thus, compounds disclosed herein may also be referred to as "Botulinum neurotoxin A inhibitors" and/or "Botulinum neurotoxin B inhibitors" or "inhibitors of Botulinum neurotoxin A and/or B".

hi the context of therapeutic use of the Botulinum neurotoxin inhibitor compounds described herein, the terms "treatment", "to treat", or "treating" will refer to any use of the Botulinum neurotoxin inhibitor compounds calculated or intended to arrest, inhibit, prevent or reduce the activity of the Botulinum neurotoxin. Thus, treating an individual may be carried out after any diagnosis indicating possible Botulinum neurotoxin intoxication, i.e., whether an infection by Clostridium botulinum and/or intoxication by Botulinum neurotoxin has been confirmed or whether the possibility of infection/intoxication is only suspected, for example, after an individual's exposure to Clostridium botulinum or a neurotoxin thereof, or after ingestion of possibly spoiled food or inhalation of neurotoxm-carrying aerosols. The inhibitors of the present invention affect the activity of the Botulinum neurotoxin and thus block or decrease the effect of the neurotoxin resulting from intoxication. For this reason, it will be understood that elimination of any bacteria involved in neurotoxin intoxication, that is the presence of Clostridium botulinum, will be accomplished by the host's own immune system or immune effector cells or by antibacterial agents. Thus, it is contemplated that the compounds of the present invention may advantageously be combined with other active ingredients such as antibiotics, antibodies, antiviral agents, anticancer agents, analgesics (e.g., a non-steroidal anti-inflammatory drug (NSAID), acetaminophen, opioids, COX-2 inhibitors), immunostimulatory agents (e.g., cytokines or a synthetic immunostimulatory organic molecules), hormones (natural, synthetic, or semi-synthetic), central nervous system (CNS) stimulants, antiemetic agents, anti-histamines, erythropoietin, agents that activate complement, sedatives, muscle relaxants, anesthetic agents, anticonvulsive agents, antidepressants, antipsychotic agents, and combinations thereof, where the supplementary activities of such agents (i.e., in addition to direct inhibition of neurotoxin activity) are called for.

The meaning of other terms will be understood by the context as understood by the skilled practitioner in the art, including the fields of organic chemistry, pharmacology, and toxicology.

The invention provides specific organic compounds that inhibit Botulinum neurotoxin activity, particularly the activity of Botulinum neurotoxin serotype A. Inhibitors of

Botulinum neurotoxins ("hits") were initially identified by screening collections of organic molecules using assays showing specific inhibition of the zinc-dependent endopeptidase light chain (LC) activity of Botulinum neurotoxin A. Inhibitor compounds were initially identified as inhibiting Botulinum neurotoxin A LC activity by at least 90% at a concentration of 83 μM using a 384-well FRET assay with 24 μM SNAP-25 substrate and 1 nM Botulinum neurotoxin A LC in a volume of 25 μL. Initial hit compounds were re-confirmed as inhibiting Botulinum neurotoxin A LC activity by at least 70% at a concentration of 25 μM using a 96-well FRET assay with 20 μM SNAP-25 substrate and 2 nM Botulinum neurotoxin A LC in a volume of 100 μL.

Since compounds with intrinsic fluorescence quenching capability do not interfere with the activity measurements of an HPLC assay, an HPLC assay was used to verify the activity/potency of confirmed inhibitors derived from the fluorescence assays. An HPLC assay as described by Schmidt et al., in J. Protein Chem., 14: 703-8 (1995), using the same substrate and enzyme was performed, and the extent of hydrolysis of the peptide substrate was determined by HPLC separation of the products from the substrate, followed by measurement of the peak areas. The final confirmed hit rate, representing compounds that exhibited inhibition in the repeated FRET assay as well as in the HPLC assay, was 0.16%.

In some cases the HPLC assay results were not in close agreement with the FRET assay results for the identical compound. There are several sources for this variation. First, there are higher concentrations of enzyme and substrate in the HPLC assay versus the FRET assay, which raises the stringency of the assay. Second, the detergent NP-40 (nonyl phenoxypolyethoxylethanol) was used in the HPLC assay versus TWEEN®-20 nonionic surfactant in the FRET assay, which may affect the state of inhibitor aggregation,

subsequently changing potency. Finally, although strong quenchers were dropped from the screen, some inherent mild quenching activity for true hits may also be a source of variation.

In order to confirm that the compounds isolated as neurotoxin inhibitors were not general or non-specific of enzymatic activity, a counter assay was performed with selected compounds using B. anthracis Lethal Factor (BaLF), as described below. Compounds that showed equivalent or more potent inhibitory activity in the counter assay as well as the BoNT/A LC assays are regarded as non-specific inhibitors (that is, are not considered specific BoNT inhibitors).

Selected compounds were additionally tested for inhibition of Botulinum neurotoxin B activity by assays showing specific inhibition of the zinc-dependent endopeptidase light chain (LC) activity of Botulinum neurotoxin B. While the all of the confirmed hit compounds disclosed herein are inhibitors of Botulinum neurotoxin A, at least one group of the compounds disclosed herein (see formula J-I, supra) shows inhibitory activity with respect to both Botulinum neurotoxin A and Botulinum neurotoxin B, indicating that the inhibitor compounds according to this invention may be effective inhibitors of many

Botulinum neurotoxin species.

hi a preferred embodiment, a Botulinum neurotoxin inhibitor compound useful in the compositions and methods described herein inhibits Botulinum neurotoxin A LC activity by greater than 70% at a 25 μM concentration as measured in a neurotoxin light chain proteolysis assay such as the FRET assays described herein. Preferably, the Botulinum neurotoxin inhibitor compound inhibits Botulinum neurotoxin A LC activity by more than 75%, 80%, 85%, 90%, or most preferably more than 95% at a 25 μM concentration.

Organic compounds tested in the screening protocols described herein were considered to be Botulinum neurotoxin inhibitor compounds useful in the compositions and methods described herein where they exhibited an IC_5O of less than 100 μM as measured in a standard assay for Botulinum neurotoxin inhibition such as the FRET or HPLC activity assays described herein. As between the FRET and HPLC assays, the HPLC assay described herein is considered more accurate, as it is less subject to fluorescence interference. Many compounds exhibited IC₅₀ values less than 75 μM, less than 50 μM, less than 40 μM, less than 25 μM, and even less than 10 μM, and such values indicate an inhibitory potency that is high enough for the compounds to be considered for therapeutic uses.

Along with a suitable potency, compounds useful as therapeutics must not exhibit too great a cytoxicity. Several well-known cytoxicity assays are used in the pharmaceutical industry to give an indication of whether test compounds will be well tolerated by the intended patient population. Most assays involve determination of the concentration of a test compound leading to 50% reduction in cell viability (CC₅₀), which is tested by incubating test cells at a range of concentrations over a fixed period suitable for generating a signal indicative of the cell viability parameter (e.g., apoptosis, cell wall integrity, ATP quantitation, LDH-release, etc.) to generate a dose/response curve, from which a CC₅₀ value can be read. Selected inhibitor compounds isolated in the screening assays were tested in a 3-day HeLa cell cytotoxicity assay and a 3 -hour chick neuronal cell assay. Compounds were regarded as having acceptable cytotoxicity where the CC_5O value was 25 μM or greater. Such standard cytotoxicity assays may employ any mammalian cell typically employed in cytotoxicity assays for toxins, including but not limited to, Chinese hamster ovary (CHO) cells, Vero

(African green monkey kidney) cells, HeLa cells, Hep-G2 (human hepatocellular carcinoma) cells, human embryonic kidney (HEK) 293 cells, 293T cells, 293FT cells (Invitrogen), BHK (newborn hamster kidney) cells, COS cells, and the like. Neuronal cells are particularly relevant to neurotoxin cytotoxicity testing, and suitable test cells available include chick neuronal cells and rat neuronal cells.

The Botulinum neurotoxin inhibitor compounds described herein are organic compounds that can be either synthesized or ordered from suppliers such as ChemBridge Corporation (San Diego, CA, USA), Life Chemicals hie. (Burlington, ON, Canada),

ChemDiv Inc. (San Diego, CA, USA), Microsource Corporation (Gaylordsville, CT, USA), Timtec LLC (Newark, DE, USA) and other companies. Botulinum neurotoxin inhibitor compounds as described herein may also be synthesized using established chemistries, and suitable synthesis schemes for the compounds include the following: Scheme A. Synthetic scheme for synthesis of MSL-111012:

Scheme B. Synthetic scheme for synthesis of MSL-113604:

H,

MSL-113604 Scheme C. Synthetic scheme for synthesis of MSL-130541:

MSL-130541

Scheme D. Synthetic scheme for synthesis of MSL- 136988:

MSL-136988 Scheme E. Synthetic scheme for synthesis of MSL-151862:

MSL-151862

Scheme F. Synthetic scheme for synthesis of MSL-066297:

MSL-066297

Scheme G. Synthetic scheme for synthesis of MSL-062516:

Benzene, EtOH

MSL-062516

Scheme H. Synthetic scheme for synthesis of MSL-059336:

MSL-059336

Scheme I. Synthetic scheme for synthesis of MSL-059327:

Scheme J. Synthetic scheme for synthesis of MSL-145005:

MSL-145005

Scheme K. Synthetic scheme for synthesis of MSL-111029:

NaOAc, AcOH heating

Scheme L. Synthetic scheme for synthesis of MSL-145815:

MSL-145815 Synthesis of benzimidazole acrylonitriles

General synthetic scheme:

General synthetic procedure:

A mixture of 2-(lH-benzo[d]imidazol-2-yl)acetonitrile (0.5 g, 3.2 mmol, commercially available), an aldehyde (3.2 mmol), ammonium acetate (0.736 g, 9.5 mol) in 10 mL of glacial acetic acid was refluxed for 2 h and then cooled to room temperature. The precipitate thus formed was collected by filtration, washed with water thoroughly and dried in a vacuum oven at 50°C to obtain the desired benzimidazole acrylonitrile product.

Scheme M. Synthetic scheme for synthesis of MBX 1519:

MBX 1519

Scheme N. Synthetic scheme for synthesis of MBX 1520:

NaOAc, AcOH heating

MBX 1520

Scheme O. Synthetic scheme for synthesis of MBX 1553:

MBX 1553 Scheme P. Synthetic scheme for synthesis of MBX 1638:

Scheme Q. Synthetic scheme for synthesis of MBX 1722: NaOAc, AcOH

heating

MBX 1722

Scheme R. Synthetic scheme for synthesis of MBX 1868:

Unless otherwise indicated, it is understood that description of the use of a Botulinum neurotoxin inhibitor compound in a composition or method also encompasses embodiments wherein a combination of two or more Botulinum neurotoxin inhibitor compounds are employed as active ingredients providing Botulinum neurotoxin inhibitory activity in a composition or method of the invention.

Pharmaceutical compositions according to the invention comprise an isolated Botulinum neurotoxin inhibitor compound as described herein, or a pharmaceutically acceptable salt thereof, as the active ingredient and a pharmaceutically acceptable carrier (or "vehicle"), which may be a liquid, solid, or semi-solid compound. By "pharmaceutically acceptable" is meant that a compound or composition is not biologically, chemically, or in any other way, incompatible with body chemistry and metabolism and also does not adversely affect the Botulinum neurotoxin inhibitor or any other component that may be present in a composition in such a way that would compromise the desired therapeutic and/or preventative benefit to a patient. Pharmaceutically acceptable carriers useful in the invention include those that are known in the art of preparation of pharmaceutical compositions and include, without limitation, water, physiological pH buffers, physiologically compatible salt solutions (e.g., phosphate buffered saline), and isotonic solutions. Pharmaceutical compositions of the invention may also comprise one or more excipients, i.e., compounds or compositions that contribute or enhance a desirable property in a composition other than the active ingredient.

Various aspects of formulating pharmaceutical compositions, including examples of various excipients, dosages, dosage forms, modes of administration, and the like are known to those skilled in the art of preparing pharmaceutical compositions and are also available in standard pharmaceutical texts, such as Remington's Pharmaceutical Sciences, 18th edition, Alfonso R. Gennaro, ed. (Mack Publishing Co., Easton, PA 1990), Remington: The Science and Practice of Pharmacy, Volumes 1 & 2, 19th edition, Alfonso R. Gennaro, ed., (Mack Publishing Co., Easton, PA 1995), or other standard texts on preparation of pharmaceutical compositions.

Pharmaceutical compositions may be in any of a variety of dosage forms particularly suited for an intended mode of administration. Such dosage forms, include, but are not limited to, aqueous solutions, suspensions, syrups, elixirs, tablets, lozenges, pills, capsules, powders, films, suppositories, and powders, including inhalable formulations. Preferably, the pharmaceutical composition is in a unit dosage form suitable for single administration of a precise dosage, which may be a fraction or a multiple of a dose that is calculated to produce effective inhibition of Botulinum neurotoxin.

A composition comprising a Botulinum neurotoxin inhibitor compound (or combination of Botulinum neurotoxin inhibitors) described herein may optionally possess a second active ingredient (also referred to as "second agent", "second active agent") that provides one or more other desirable therapeutic or prophylactic activities other than

Botulinum neurotoxin inhibitory activity. Suitable second agents useful in compositions of the invention include, but without limitation, an antibiotic, an antibody, an antiviral agent, an anticancer agent, an analgesic (e.g., a non-steroidal anti-inflammatory drug (NSAJD), acetaminophen, an opioid, a COX-2 inhibitor), an immunostimulatory agent (e.g., a cytokine or a synthetic immunostimulatory organic molecule), a hormone (natural, synthetic, or semi- synthetic), a central nervous system (CNS) stimulant, an antiemetic agent, an anti-histamine, an erythropoietin, a complement stimulating agent, a sedative, a muscle relaxant agent, an anesthetic agent, an anticonvulsive agent, an antidepressant, an antipsychotic agent, pluralities of such agents, and combinations thereof.

Pharmaceutical compositions as described herein may be administered to humans and other animals in a manner similar to that used for other known therapeutic or prophylactic agents, and particularly as used for therapeutic antitoxins. The dosage to be administered to an individual and the mode of administration will depend on a variety of factors including the type and amount of toxin exposure or ingestion; age, weight, sex, and condition of the patient; and possibly genetic factors. Proper dosage and dosage form will ultimately be decided by an attending qualified physician or healthcare provider.

Pharmaceutically acceptable salts may be made of Botulinum neurotoxin inhibitor compounds described herein, and suitable such salts include those derived from

pharmaceutically acceptable inorganic and organic acids and bases. Examples of suitable acids include hydrochloric, hydrobromic, sulfuric, nitric, perchloric, fumaric, maleic, malic, pamoic, phosphoric, glycolic, lactic, salicylic, succinic, p-toluenesulfonic, tartaric, acetic, citric, methanesulfonic, formic, benzoic, malonic, naphthalene-2-sulfonic, tannic, carboxymethylcellulose, polylactic, polyglycolic, and benzenesulfonic acids.

The invention may also envision the "quaternization" of any basic nitrogen-containing groups of a compound described herein, provided such quaternization does not destroy the ability of the compound to inhibit Botulinum neurotoxin. Such quaternization may be especially desirable to enhance solubility. Any basic nitrogen can be quaternized with any of a variety of compounds, including but not limited to, lower (e.g., C₁-C₄) alkyl halides (e.g., methyl, ethyl, propyl and butyl chlorides, bromides, and iodides); dialkyl sulfates (e.g., dimethyl, diethyl, dibutyl and diamyl sulfates); long chain halides (e.g., decyl, lauryl, myristyl and stearyl chlorides, bromides and iodides); and aralkyl halides (e.g., benzyl and phenethyl bromides).

For solid compositions, conventional nontoxic solid carriers may be used including, but not limited to, mannitol, lactose, starch, magnesium stearate, sodium saccharin, talc, cellulose, glucose, sucrose, and magnesium carbonate.

Pharmaceutical compositions may be formulated for administration to a patient by any of a variety of parenteral and non-parenteral routes or modes. Such routes include, without limitation, intravenous, intramuscular, intra-articular, intraperitoneal, intracranial, paravertebral, periarticular, periostal, subcutaneous, intracutaneous, intrasynovial, intrasternal, intrathecal, intralesional, intratracheal, sublingual, pulmonary, topical, rectal, nasal, buccal, vaginal, or via an implanted reservoir. Implanted reservoirs may function by mechanical, osmotic, or other means. Generally and particularly when administration is via an intravenous, intra-arterial, or intramuscular route, a pharmaceutical composition may be given as a bolus, as two or more doses separated in time, or as a constant or non-linear flow infusion.

A pharmaceutical composition may be in the form of a sterile injectable preparation, e.g., as a sterile injectable aqueous solution or an oleaginous suspension. Such preparations may be formulated according to techniques known in the art using suitable dispersing or wetting agents (e.g., polyoxyethylene 20 sorbitan monooleate (also referred to as

"polysorbate 80"); TWEEN® 80 nonionic surfactant, ICI Americas, Inc., Bridgewater, New Jersey) and suspending agents. Among the acceptable vehicles and solvents that may be employed for injectable formulations are mannitol, water, Ringer's solution, isotonic sodium chloride solution, and a 1,3-butanediol solution. In addition, sterile, fixed oils maybe conventionally employed as a solvent or suspending medium. For this purpose, a bland fixed oil may be employed including synthetic mono- or diglycerides. Fatty acids, such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharmaceutically-acceptable oils, including olive oil or castor oil, especially in their polyoxyethylated versions.

A Botulinum neurotoxin inhibitor described herein may be formulated in any of a variety of orally administrable dosage forms including, but not limited to, capsules, tablets, caplets, pills, films, aqueous solutions, oleaginous suspensions, syrups, or elixirs. In the case of tablets for oral use, carriers, which are commonly used include lactose and corn starch. Lubricating agents, such as magnesium stearate, are also typically added. For oral administration in a capsule form, useful diluents include lactose and dried cornstarch.

Capsules, tablets, pills, films, lozenges, and caplets may be formulated for delayed or sustained release.

Tablets and other solid or semi-solid formulations may be prepared that rapidly disintegrate or dissolve in an individual's mouth. Such rapid disintegration or rapid dissolving formulations may eliminate or greatly reduce the use of exogenous water as a swallowing aid. Furthermore, rapid disintegration or rapid dissolve formulations are also particularly useful in treating individuals with swallowing difficulties. For such formulations, a small volume of saliva is usually sufficient to result in tablet disintegration in the oral cavity. The active ingredient (a Botulinum neurotoxin inhibitor described herein) can then be absorbed partially or entirely into the circulation from blood vessels underlying the oral mucosa (e.g., sublingual and/or buccal mucosa), or it can be swallowed as a solution to be absorbed from the gastrointestinal tract.

When aqueous suspensions are to be administered orally, whether for absorption by the oral mucosa or absorption via the gut (stomach and intestines), a composition comprising a Botulinum neurotoxin inhibitor may be advantageously combined with emulsifying and/or suspending agents. Such compositions may be in the form of a liquid, dissolvable film, dissolvable solid (e.g., lozenge), or semi-solid (chewable and digestible). If desired, such orally administrable compositions may also contain one or more other excipients, such as a sweetener, a flavoring agent, a taste-masking agent, a coloring agent, and combinations thereof.

The pharmaceutical compositions comprising a Botulinum neurotoxin inhibitor as described herein may also be formulated as suppositories for vaginal or rectal administration. Such compositions can be prepared by mixing a Botulinum neurotoxin inhibitor compound as described herein with a suitable, non-irritating excipient that is solid at room temperature but liquid at body temperature and, therefore, will melt in the appropriate body space to release the Botulinum neurotoxin inhibitor and any other desired component of the composition. Excipients that are particularly useful in such compositions include, but are not limited to, cocoa butter, beeswax, and polyethylene glycols.

Topical administration of a Botulinum neurotoxin inhibitor may be useful when the desired treatment involves areas or organs accessible by topical application, such as the epidermis, surface wounds, or areas made accessible during surgery. Carriers for topical administration of a Botulinum neurotoxin inhibitor described herein include, but are not limited to, mineral oil, liquid petroleum, white petroleum, propylene glycol, polyoxyethylene and polyoxypropylene compounds, emulsifying wax, and water. Alternatively, a topical composition comprising a Botulinum neurotoxin inhibitor as described herein may be formulated with a suitable lotion or cream that contains the inhibitor suspended or dissolved in a suitable carrier to promote absorption of the inhibitor by the upper dermal layers without significant penetration to the lower dermal layers and underlying vasculature. Carriers that are particularly suited for topical administration include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol, and water. A Botulinum neurotoxin inhibitor may also be formulated for topical application as a jelly, gel, or emollient. Topical administration may also be accomplished via a dermal patch.

Persons skilled in the field of topical and transdermal formulations are aware that selection and formulation of various ingredients, such as absorption enhancers, emollients, and other agents, can provide a composition that is particularly suited for topical

administration (i.e., staying predominantly on the surface or upper dermal layers with minimal or no absorption by lower dermal layers and underlying vasculature) or transdermal administration (absorption across the upper dermal layers and penetrating to the lower dermal layers and underlying vasculature).

Pharmaceutical compositions comprising a Botulinum neurotoxin inhibitor as described herein may be formulated for nasal administrations, in which case absorption may occur via the mucous membranes of the nasal passages or the lungs. Such modes of administration typically require that the composition be provided in the form of a powder, solution, or liquid suspension, which is then mixed with a gas (e.g., air, oxygen, nitrogen, or a combination thereof) so as to generate an aerosol or suspension of droplets or particles.

Inhalable powder compositions preferably employ a low or non-irritating powder carrier, such as melezitose (melicitose). Such compositions are prepared according to techniques well-known in the art of pharmaceutical formulation and may be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, and/or other solubilizing or dispersing agents known in the art.

Pharmaceutical compositions described herein may be packaged in a variety of ways appropriate to the dosage form and mode of administration. These include but are not limited to vials, bottles, cans, packets, ampoules, cartons, flexible containers, inhalers, and nebulizers. Such compositions may be packaged for single or multiple administrations from the same container. Kits may be provided comprising a composition, preferably as a dry powder or lyophilized form, comprising a Botulinum neurotoxin inhibitor and preferably an appropriate diluent, which is combined with the dry or lyophilized composition shortly before administration as explained in the accompanying instructions of use. Pharmaceutical composition may also be packaged in single use pre-filled syringes or in cartridges for auto- injectors and needleless jet injectors. Multi-use packaging may require the addition of antimicrobial agents such as phenol, benzyl alcohol, meta-cresol, methyl paraben, propyl paraben, benzalconium chloride, and benzethonium chloride, at concentrations that will prevent the growth of bacteria, fungi, and the like, but that are non-toxic when administered to a patient.

Consistent with good manufacturing practices, which are in current use in the pharmaceutical industry and which are well known to the skilled practitioner, all components contacting or comprising a pharmaceutical composition must be sterile and periodically tested for sterility in accordance with industry norms. Methods for sterilization include ultrafiltration, autoclaving, dry and wet heating, exposure to gases such as ethylene oxide, exposure to liquids, such as oxidizing agents, including sodium hypochlorite (bleach), and exposure to high energy electromagnetic radiation (e.g., ultraviolet light, x-rays, gamma rays, ionizing radiation). Choice of method of sterilization will be made by the skilled practitioner with the goal of effecting the most efficient sterilization that does not significantly alter a desired biological function of the Botulinum neurotoxin inhibitor or other component of the composition.

Additional embodiments and features of the invention will be apparent from the following non-limiting examples. Example 1 : BoNT/A LC FRET Initial Screening Assay

Chemical compound libraries were purchased from Chembridge Corporation (San Diego, CA), ChemDiv Incorporated (San Diego, CA), and from Microsource Corporation (Gaylordsville, CT). Peptide substrates were purchased from Peptides International

(Louisville, KY) (BoNT/ A-dacia/Dnp and BaLF-MCA/Dnp), List Biological Laboratories (Campbell, CA) (Vamptide® BoNT/B), and Biomol, International (Plymouth Meeting, PA) (Omni MMP fluorogenic substrate). Metalloproteases were purchased from List Biological Laboratories (Campbell, CA) (BoNT/A LC, BoNT/B LC, BaLF) and Biomol, International (Plymouth Meeting, PA) (MMP-I, MMP-2 and MMP-9). BoNT/A holotoxin was purchased from MetaBiologics, Inc. (Madison, WI). Chemical reagents were purchased from Sigma Aldrich (St. Louis, MO) and VWR (Bridgeport, NJ). BoNT/A LC FRET Screening Assay. 384- Well Format

The zinc-dependent endopeptidase light chain (LC) portions of Botulinum

neurotoxins impair neuronal exocytosis through proteolysis of essential SNARE (soluble NSF-ethylmaleimide-sensitive factor attachment protein receptor) components of

neurotransmission (Simpson, L.L., 2004, op. cit.).

To screen/monitor inhibition of BoNT/ A LC, a fluorescence resonance energy transfer (FRET) assay (see, Schmidt et al., Appl. Environ. Microbiol, 69: 297-303 (2003)) was modified to a 384- well format to increase throughput. The substrate consisted of a synthetic peptide representing residues 187 to 203 of the natural BoNT/A substrate, SNAP- 25. The peptide substrate was prepared using S-(N- [4-methyl-7-dimethylaminocoumarin-3- yl]carboxamidomethyl)cysteine (daciaC) as the fluorescence donor and N-(2,4- dinitrophenyl)lysine (dnpK) as the fluorescence acceptor, incorporated into the peptide, yielding the sequence SNRTRIDEAN[dnpK]RA[daciaC]RML (SEQ ID NO:1). BoNT/A LC cleaves the substrate between the dnpK and R residues, thereby eliminating the quenching of coumarin fluorescence by Dnp and producing a fluorescent signal at 485 nm following excitation at 398 nm.

The compound libraries were screened at 83 μM (final concentration) utilizing the 384-well assay format. Briefly, 24 μM SNAP-25 substrate (amino acids 187-203 of the SNAP-25 protein) of sequence SNRTRIDEAN[dnpK]RA[daciaC]RML (SEQ ID NO:1), was incubated at 37°C for 60 min. in the presence of buffer (50 mM HEPES-0.05% TWEEN® 20 nonionic surfactant [pH 7.4]) and 1 nM BoNT/A LC in a volume of 25 μL. Compounds at 83 μM (final concentration) were placed into opaque black 384-well microplates (Costar, Corning, NY), utilizing a SciClone ALH 3000 liquid handling robot (Caliper Life Sciences, Hopkinton, MA). The reactions were stopped by addition of acetic acid to 0.5%, and the fluorescence of the cleaved substrate was measured at 485 nm following excitation at 398 nm in a Victor²V (Perkin Elmer, Boston, MA) plate reader. Compounds that inhibited BoNT/A LC activity greater than 90% were considered as hits and were set aside to be further evaluated as discussed infra. Any compound that possessed intrinsic fluorescence was not analyzed further and was rejected to maintain screening efficiency.

Over 70,000 compounds were screened for inhibition of BoNT/ A LC using the

FRET-based assay, yielding a primary hit rate of 0.47%. Z¹ scores, a measure of assay robustness that takes into account the numerical spread between negative and positive controls, as well as standard deviations of each, were very favorable, averaging about 0.75. A score greater than 0.5 is indicative of an assay qualitatively suitable for screening {see, Zhang et al., J. Biomol. Screen, 4: 67-73 (1999)).- Example 2: BoNT/A LC FRET Assav-96 Well Format for Hit Confirmations, ICsn

Determinations, and Inhibitor Kinetic Analysis

Hit confirmations, IC_5O determinations, and kinetic analysis for inhibitor mechanism of action were carried out as described above but utilizing a 96-well FRET assay format.

Specifically, for hit confirmation, hit compounds from Example 1 (25 μM [final]), 20 μM SNAP-25 substrate (see FRET assay for sequence), and 2 nM of BoNT/A LC were incubated at 37⁰C for 40 min, which was the initial linear rate phase of catalytic activity (data not shown), in the presence of buffer (50 mM HEPES-0.05% TWEEN® 20 nonionic surfactant [pH 7.4]) in a volume of 100 μL. The reactions were stopped with 0.5% acetic acid prior to measuring the fluorescence of the cleaved substrate at 485 nm following excitation at 398 nm in a Molecular Devices (Sunnyvale, CA) plate reader. Any compound that inhibited BoNT/A LC activity greater than 70% at 25 μM was classified as a hit and set aside to be further evaluated as discussed infra.

For kinetic analysis of hit compounds from the 96-well FRET assay, substrate concentration was varied in the presence of increasing inhibitor concentrations, and the results were analyzed utilizing Eadie-Hofstee transformations (Atkins et al., Biochem. J, 149: 775-777 (1975)). The FRET-based 96-well assay displayed linear kinetics {see, Figure 1) at the SNAP-25 and enzyme concentrations used for hit confirmation (R² greater than 0.98). The K_m for the substrate ranged between 23-29 μM. Example 3: HPLC Secondary Assay to Confirm FRET ICsn Values

Subsequent to the FRET-based assay, HPLC assays using the same substrate and enzyme were conducted to confirm activity of selected confirmed hits observed in the FRET assays. Since compounds with intrinsic fluorescence quenching capability do not interfere with the activity measurements of the HPLC assay, an HPLC assay was used to verify the activity/potency of inhibitor compounds derived from the fluorescent LC assay. The extent of hydrolysis of the peptide substrate was determined by HPLC separation of the products from the substrate, followed by measurement of the peak areas. The HPLC assay was a modified version of a published protocol (Schmidt et al., J. Protein Chem., 14: 703-708 (1995)). Incubation conditions were the same as for the FRET assays of Examples 1 and 2 except that the enzyme concentration in the assay was 6 nM, the substrate concentration was 60 μM, and 0.05% NP-40 (nonyl phenoxypolyethoxylethanol) was used in place of TWEEN®-20 nonionic surfactant. Assays were incubated at 37°C for 60 minutes and stopped by addition of acetic acid to 0.5%. The extent of hydrolysis of the peptide substrate was determined by HPLC separation of the products from the substrate, followed by measurement of the peak areas (see, Figure 2). The samples were then analyzed by reverse-phase HPLC (Gilson, Middleton WI) (Alltima™ Cl 8 column, 150 x 4.6 mm, 5 μM from Grace, Deerfield, IL) with a gradient of 35% B to 40% B over 21 min, 100% B for 8 min. (solvent A: 0.1% trifluoroacetic acid (TFA), aqueous; solvent B: 0.1% TFA in 70% acetonitrile). The effluent was monitored at 365 nm and the resultant peaks quantified by integration utilizing Gilson Trilution® software.

The final confirmed hit rate, representing compounds that repeated in the FRET assay and displayed activity in the HPLC assay, was 0.16 %.

The screening-confirmed hits consisted of a variety of structures. Among these, hydroxyquinolines, benzimidazole acrylonitriles, arylamides, 2-(3-hydroxypyrazol-2- yl)benzindazoles, vinylbenzothiazoles, and vinylbenzimidazoles were represented. The IC₅₀ values for BoNT/A LC inhibition by the screening hits are shown in Table 1, below.

Example 4: BoNT/B LC Assay

Selected confirmed hits were evaluated for specificity by testing them for their relative potency of inhibition of BoNT/B LC. The FRET assay was performed using 10 μM Vamptide® peptide substrate (List Biological Laboratories; Campbell, CA), 20 mM HEPES- 0.05% TWEEN®-20 nonionic surfactant [pH 7.4] and 13 nM of BoNT/B LC and incubating at 37⁰C for 60 min. Inactivation of the enzyme was achieved by addition of acetic acid to 0.5%, and the fluorescent signal of the cleaved substrate was measured at 418 nm after excitation at 321 nm.

The concentration-dependence of inhibition (IC₅₀ values) of BoNT/B LC for the screening hits is shown in Table 1, infra. Example 5: Bacillus anthracis Lethal Factor (BaLF) Assay

Selected confirmed hits were evaluated for BoNT specificity by testing them for their relative potency of inhibition of a non-Botulinum toxin component, BaLF. A BaLF FRET assay was performed using 20 μM of a peptide substrate modified with N-ε-[(7- methoxycoumarin-4-yl)acetyl]-L-lysine (MCA-K) and N-ε-(2,4-dinitrophenyl)lysine (dnpK): MG4-KKVYPYPME[dnpK] amide (SEQ ID NO:2), 20 mM HEPES-0.05% TWEEN® 20 nonionic surfactant [pH 8.2] and 5.55 nM BaLF and incubating at 37⁰C for 30 min. as described previously (Panchal et al, Nat. Struct. MoI. Biol., 11 : 67-72 (2004). Inactivation of the enzyme was achieved by addition of acetic acid to 0.5% and the fluorescent signal of the cleaved substrate was measured at 395 nm after excitation at 324 nm.

The concentration-dependence of inhibition (IC₅₀ values) of B. anthracis LF (BaLF) for the screening hits is shown in Table 1, below.

As shown in Table 1, there were four arylamide hits with all four demonstrating specificity for BoNT/ A. The most potent compound (IC₅₀ = 10 μM) was identified as MSL- 130541 (Compound C). Three quinoline hit compounds were identified, with MSL-062516 (Compound G) being the most potent (IC₅₀ = 5.4), but it was not very specific, exhibiting inhibition of both BoNT/B LC and BaLF. The hydroxyquinoline MSL-151862 (Compound E) was chosen for further studies (see below) because of its potency and selectivity.

Quinolones were the only structural class of inhibitors identified in both the Chembridge and ChemDiv screening libraries used in the screens. Two benzimidazole acrylonitrile compounds were identified as hits, and MSL- 145815 (Compound L) was selected for further specificity studies (see below) based on its potency and selectivity. Figure 3 shows IC₅₀ plots for MSL-145815 (Compound L) and MSL-151862 (Compound E). The arylamide hits demonstrated specificity for BoNT/A; MSL-130541(Compound C) was the most potent in both the FRET and the HPLC assays (IC₅₀ = 10 μM and 27 μM, respectively). MSL-059336 (Compound H), MSL-059327 (Compound I) and MSL-145005 (Compound J) were each single hits with no other structurally related inhibitors; with MSL-059336 (Compound H) displaying the greatest potency in the BoNT/A FRET assay. However, this hit also showed activity in the BaLF FRET assay, indicating a comparatively lesser specificity for Botulinum neurotoxin inhibition, and was not studied further. The hydroxypyrazole chemotype (MSL- 145005) (Compound J) was amenable to medicinal chemistry efforts and was selected for further SAR study (see, Table 2 and 3 below).

Example 6: Human Matrix Metalloproteinase (MMP) 1, 2 and 9 Assays

To further assess selectivity, the 8-hydroxyquinoline inhibitor MSL-151862

(compound E) and the benzimidazole acrylonitrile, MSL-145815 (compound L) were tested for inhibition of a panel of metalloproteases consisting of BoNTYB LC, recombinant human matrix metalloprotease-1 (MMP-I), recombinant human matrix metalloprotease-2 (MMP-2), recombinant human matrix metalloprotease-9 (MMP-9), and BaLF. An MMP FRET assay was performed using 25 μM Omni MMP fluorogenic substrate, 50 mM MOPS-0.05% NP-40 (nonyl phenoxypolyethoxylethanol) [pH 6.0], and either MMP-I (38 nM), MMP-2 (19 nM), or MMP-9 (13 nM). The enzyme reactions were incubated at 37⁰C for 60 min. The fluorescent signal of the cleaved substrate was measured at 393 nm after excitation at 328 nm. Results are shown in Table 4 below.

Example 7: Zinc Chelation Assessments.

Since 8-hydroxyquinoline compounds are known chelators of zinc (Ding et al.,

Cancer Research, 65:3389-3395 (2005); Fraser and Creanor, Biochem Journal, 147:401-411 (1975), MSL-151862 (compound E) was examined to determine the effect of added zinc on its activity. MSL- 145815 (compound L) was also examined. The methodology used for zinc chelation assessment was based on a procedure previously described (Burnett et al., Biochem. Biophys. Res. Commun., 310: 84-93 (2003)) and modified as follows. Compounds were diluted in DMSO without zinc, DMSO solution containing 5 mM ZnCl₂, or DMSO solution containing 10 mM ZnCl₂, such that the final concentration OfZnCl₂ was 0, 2.5, or 5 mM, respectively. Likewise, N-hydroxy-2,4-dichlorocinnamide, a hydroxamate zinc non-chelator was analyzed for zinc chelation and as a control in the other assays of Table 4

("hydroxamate"). The compounds were incubated in the ZnCl₂ solutions for 15 minutes at room temperature (18-24°C) and then were diluted 100-fold to final concentrations of 0, 25, and 50 μM, respectively, with assay mix and assessed for potency utilizing the BoNT/A 96 well FRET assay. The final concentrations of zinc chloride in the assay inhibited BoNT/A activity minimally (i.e., 4-5% inhibition, data not shown). Compounds were classified as zinc chelators if they displayed a zinc concentration-dependent decrease in potency. Results are shown in Table 4 below.

Example 8: Determination of Mammalian Cytotoxicity

The 8-hydroxyquinoline inhibitor MSL-151862 (compound E) and the benzimidazole acrylonitrile, MSL- 145815 (compound L) were tested for cytotoxicity in human HeLa cells. Cytotoxicity of the compounds was measured by plating HeLa cells (ATCC# CCL-2) in 96- well plates (4 x 10³ cells per well) in the presence or absence of compounds that had been added as a DMSO stock (final concentration of 1%). The latter culture, and an identical control culture containing only DMSO, were incubated at 37⁰C for 72 hr in Minimal Essential Medium (Gibco/Invitrogen, Carlsbad, CA) supplemented with 10% fetal calf serum, and cell viability was tested with the vital stain MTS (see, Marshall et al., Growth Regul., 5: 69-84 (1995)) according to the manufacturer's instructions (Promega, Madison, WI). Cytotoxicity was quantified as the CC₅₀, the concentration of compound that inhibited 50% of conversion of MTS to formazan. Results are shown in Table 4 below.

As shown in Table 4, MSL- 151862 (compound E) demonstrated poor specificity for

BoNT/A compared to the human enzymes, MMP-I, 2 and 9. The compound was demonstrated to be a potent zinc chelator based on a zinc concentration-dependent decrease in potency in the BoNT/A LC FRET assay. Finally, MSL-151862 (compound E) was shown to have a CC₅₀ value (27.2 μM) that coincided roughly with the inhibitory constants for the two human enzymes, MMP-2 and -9 (IC₅₀ = 13 μM and 10 μM, respectively).

Interestingly, MSL-145815 (compound L) displayed considerably more specificity than did MSL-151862 (compound E), yielding IC₅₀ values greater than 100 μM for BoNT/B LC, MMP-I, MMP-2, and MMP-9, while some inhibition of BaLF was noted with an IC₅₀ of 74 μM (see, Table 4). Consistent with its specificity for BoNT/A, MSL-145815 (compound L) was also not a zinc chelator, since its activity in the BoNT/A LC FRET assay was not dependent on Zn⁺⁺ concentrations. The compound proved to be slightly more cytotoxic (CC₅₀ = 18.5 μM) than MSL-151862 (compound E), yielding a selectivity index of -2.6 (HeLa-CCso/BoNT/A-ICso).

Given its potential to be a potent and specific inhibitor, MSL-145815 (compound L) became the target of a preliminary SAR program. More than 60 (see, Table 5 for a representative selection) compounds were synthesized and one, MBX 1553 (compound O), appeared to exhibit improved selectivity with respect to other proteases tested (see, Tables 4 and 5). Like its parent compound (compound L), MBX 1553 (compound O) inhibited BoNT/A, did not inhibit BoNT/B, MMP-I, MMP-2 nor MMP-9 or demonstrate zinc chelation {see, Table 4). Unlike its parent, MBX 1553 (compound O) did not inhibit BaLF, and thus was a truly specific BoNT/A inhibitor, albeit with a somewhat greater cytotoxicity to human cells (CC₅₀ = 3.6 μM; see Tables 4 and 5).

Table 5

Example 9: Chick Neuronal Cell SNAP-25 Cleavage Assay

The representative hydroxyquinoline (MSL-151862) (compound E) and

benzimidazole acrylonitrile (MSL- 145815) (compound L) BoNT/A LC inhibitors, as well as the benzimidazole analog, MBX 1553 (compound O), were evaluated for activity in cultured cells intoxicated with BoNT/A. In the chick neuronal cell assay, primary motor neurons were preincubated with test compounds and then exposed to BoNT/A. The ability of BoNT/ A to cleave its substrate SNAP-25 was assessed using Western Blot analysis of cell extracts.

Embryonic chicken spinal motor neurons were obtained by incubating fertilized chicken eggs (SPAFAS, Charles River Laboratories, North Franklin, CT) for 6 days and removing the ventral spinal cords from the embryos (see, Burnett et al., J. Biol. Chem., 282: 5004-5014 (2007); Kuhn, T. B., Methods Cell Biol., 71: 67-87 (2003)). The dissociated cell population was enhanced for neuronal cells by briefly plating (to attach non-neuronal cells) and then treating with the mitotic inhibitor, 5-fiuorodeoxyuridine (to prevent the growth of dividing non-neuronal cells). Cells were then plated in 6-well tissue culture plates and incubated overnight at 37°C prior to intoxication.

Cells were preincubated in Leibovitz Ll 5 medium (Invitrogen; with N3 supplement and 10% fetal bovine serum) with inhibitor for 45 min., followed by a 3.5 hr. incubation with 5-10 nM BoNT/A holotoxin and inhibitor. Cells were rinsed with fresh growth medium, scraped, collected, washed with phosphate-buffered saline, lysed and assessed for protein content by Bradford assay prior to loading on a 12% Tris-glycine gel (Invitrogen). Gel contents were transferred to nitrocellulose and probed with SMI 81 mouse anti-SNAP-25 (Covance, Berkley, CA) as the primary antibody. A horseradish peroxidase-conjugated goat anti-mouse secondary antibody (Pierce) was used in combination with an ECL Western blotting detection system (Pierce) and densitometry was performed using a UN-SCAN-IT gel automated digitizing system (SiUc Scientific, Inc., Orem, UT).

MSL-145815 (compound L) was tested at 20 and 50 μM (see, Figure 4A), MSL- 151862 (compound E) was tested at 100, 50, 25, and 12.5 μM (Figure 4B), and MBX 1553 (compound O) was tested at a single concentration of 30 μM (Figure 4C). Structurally unrelated molecules NSC240898 and MBX 1130 having previously established, specific BoNT/A inhibitor activity were used (at 30 μM) as positive controls. As seen in Figures 4A and 4C, MSL-145815 (compound L) exhibited only slight protection in the assay at the concentrations tested. However, the analog MBX 1553 (compound O) inhibited BoNT/A intoxication of neuronal cells by approximately 58% at a concentration of 30 μM. Finally, the 8-hydroxyquinoline MSL-151862 (compound E) inhibited BoNT/A in the assay with an IC₅o of 60 μM. While all three compounds exhibited moderate cytotoxicity in a 3-day HeLa cytotoxicity assay, they were well-tolerated in the 3-hr neuronal cell BoNT/A assay, when incubated with neuronal cells in the absence of BoNT; upon visual inspection, cells appeared healthy after incubation with compound (data not shown).

Example 10: Kinetic Analysis of BoNT/A LC Inhibition

The kinetics of BoNT/ A LC inhibition by the three compounds, the 8- hydroxyquinoline compound MSL-151862 (compound E) and the two benzimidazole acrylonitriles compounds, MSL-145815 (compound L) and MBX 1553 (compound O), were examined in detail using Eadie-Hofstee analysis (Atkins et al., 1975, op. cit.) See Example 2 for further details. MSL-151862 (compound E), the hydroxyquinoline inhibitor, exhibited a

noncompetitive mechanism of inhibition {see, Figure 5A), as evidenced by a decrease in V_max (y intercept) and no change in K_m (line slope) with increasing inhibitor concentrations, thus yielding parallel lines. The benzimidazole acrylonitrile compounds, MSL-145815

(compound L) and MBX 1553 (compound O), also appeared to act by a noncompetitive mechanism since the Eadie-Hofstee plots revealed series of parallel lines, indicating no change in the K_m values and decreasing V_max values with increasing inhibitor concentrations (see, Figures 5B and 5C). The results observed for all three compounds indicate that excess peptide substrate cannot compete out the effects of inhibitor. Analyses of the data using both Lineweaver-Burk and Hanes-Woolf plots yielded the same results (i.e., interpreted data indicated noncompetitive inhibition, data not shown).

All publications, patent applications, patents, and other documents cited herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

Obvious variations to the disclosed compounds and alternative embodiments of the invention will be apparent to those skilled in the art in view of the foregoing disclosure. All such obvious variants and alternatives are considered to be within the scope of the invention as described herein.

Claims

Claims:

1. An isolated Botulinum neurotoxin inhibitor compound of the formula:

(L)

or a pharmaceutically acceptable salt thereof.

2. An isolated Botulinum neurotoxin inhibitor compound of the formula J- 1 :

wherein

Y is O or NH;

R¹ is hydrogen, hydroxyl, lower alkyl, lower alkoxy, amino, or the radical -NHC(=0)R, where R is phenyl or lower alkyl;

R² is hydrogen, lower alkyl, lower alkoxy, cyano, a cyanoalkyl radical of the formula -(CH₂)_nC≡N (where n is 1, 2 or 3), a dialkyl ester radical of the formula

-(CH₂)_xC(=O)O(CH₂)_yCH₃ (where x is 1, 2 or 3 and y is 0, 1 or 2), phenyl, or an alkylphenyl radical of the formula -(CH₂)_nC₆H₅, where n is 1, 2 or 3;

R³ is hydrogen, lower alkyl, lower alkoxy, a dialkyl ester radical of the formula

-(CH₂)_xC(=O)O(CH₂)_yCH₃ (where x is 1, 2 or 3 and y is 0, 1 or 2), phenyl, or phenyl substituted with halo, with lower alkyl, or with lower alkoxy; or

R² and R³ together form a divalent hydrocarbon radical of 3-4 carbons to form a five-member or six-member ring fused with the pyrazole ring of formula J-I ;

or a pharmaceutically acceptable salt thereof.

3. A compound according to Claim 2 of the formula J-2:

wherein

R² is hydrogen, lower alkyl, a dialkyl ester radical of the formula -(CH₂)_xC(=O)O(CH₂)_yCH -3 (where x is 1, 2 or 3 and y is 0, 1 or 2), phenyl, or C_1-3 alkylphenyl;

R³ is hydrogen, lower alkyl, a dialkyl ester radical of the formula -(CH₂)_xC(=O)O(CH₂)_yCH₃ (where x is 1, 2 or 3 and y is 0, 1 or 2), phenyl, or phenyl substituted with halo, with lower alkyl, or with lower alkoxy; or

R² and R³ together form a divalent hydrocarbon radical of 3-4 carbons, to form a five- member or six-member ring fused with the pyrazole ring of formula J-2;

or a pharmaceutically acceptable salt thereof.

4. An isolated Botulinum neurotoxin inhibitor compound of the formula L-I :

wherein Ar is an aryl or heteroaryl ring of 4-6 members, which heteroaryl ring may have 1-3 heteroatoms independently selected from O, S, or N, and where Ar may be unsubstituted or substituted with one or more substituents selected from lower alkyl, lower alkoxy, halo, cyano, or nitro;

n is 0 or 1 ;

or a pharmaceutically acceptable salt thereof.

5. A pharmaceutical composition comprising one or more Botulinum neurotoxin inhibitor compounds according to any one of Claims 1-4 and a pharmaceutically acceptable carrier or excipient.

6. Use of a compound according to any one of Claims 1 -4 for the inhibition of Botulinum neurotoxin activity.

7. Use of a pharmaceutical composition according to Claim 5 for the treatment of Botulinum neurotoxin intoxication.

8. The use according to Claim 6 or Claim 7, wherein said Botulinum neurotoxin is Botulinum neurotoxin A.

9. The use according to Claim 6 or Claim 7, wherein said Botulinum neurotoxin is Botulinum neurotoxin A and/or Botulinum neurotoxin B.

10. A method for treating an individual intoxicated by or exposed to Botulinum neurotoxin comprising administering to said individual an effective amount of a compound according to any one of Claims 1-4.

11. The method according to Claim 10, wherein said individual is human.

12. The method according to Claim 10, wherein said Botulinum neurotoxin is Botulinum neurotoxin A.

13. The method according to Claim 10, wherein said Botulinum neurotoxin is Botulinum neurotoxin A and/or Botulinum neurotoxin B.

14. The method according to Claim 10, further comprising administering an additional active ingredient selected from the group consisting of an antibiotic, an antibody, an antiviral agent, an anticancer agent, an analgesic, an immunostimulatory agent, a natural hormone, a synthetic or semi-synthetic hormone, a central nervous system stimulant, an antiemetic agent, an anti-histamine, an erythropoietin, a complement stimulating agent, a sedative, a muscle relaxant agent, an anesthetic agent, an anticonvulsive agent, an antidepressant, an

antipsychotic agent, and combinations thereof.