WO2017120345A1 - Compositions and methods for treating peripheral neuropathy - Google Patents

Abstract

As described below, the present invention features methods for identifying agents that reduce or eliminate the neurotoxic effect of paclitaxel, and methods of using such agents for the treatment of peripheral neuropathy (e.g., paclitaxel-induced peripheral neuropathy, diabetic neuropathy) and wound healing.

Description

COMPOSITIONS AND METHODS FOR TREATING PERIPHERAL

NEUROPATHY

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit of the following U.S. Provisional Application No. : 62/276,140, filed January 7, 2016, the entire contents of which are incorporated herein by reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing, which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on December 30, 2016, is named 167699.010201 Sequence

Listing_ST25.txt and is 4,341 bytes in size.

STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY

SPONSORED RESEARCH

This work was supported by the following grants from the National Institutes of Health Grant Nos: P20GM104318 and P20GM10342. The government has certain rights in the invention.

BACKGROUND OF THE INVENTION

Paclitaxel is a microtubule-stabilizing chemotherapeutic agent that is widely used in cancer treatment and in a number of curative and palliative regimens. Despite its beneficial effects on cancer, paclitaxel also damages healthy tissues, most prominently the peripheral sensory nervous system. The mechanisms leading to paclitaxel-induced peripheral neuropathy, which affects -40% of chemotherapy patients, remain elusive and therapies that prevent or alleviate this condition are not available.

Paclitaxel-induced peripheral neuropathy affects primarily the intra-epidermal sensory axons and to a lesser extent motor and autonomous nerves. Sometimes the symptoms of neuropathy (e.g., tingling, numbness, acute or chronic pain) are so severe that chemotherapy must be terminated. Moreover, paclitaxel-induced peripheral neuropathy is sometimes irreversible and often symptoms persist over extended periods. Accordingly, methods for preventing or treating paclitaxel-induced peripheral neuropathy are urgently required. SUMMARY OF THE INVENTION

As described below, the present invention features methods for identifying agents that reduce or eliminate the neurotoxic effect of paclitaxel, and methods of using such agents for the treatment of peripheral neuropathies (e.g., paclitaxel induced peripheral neuropathy, diabetic neuropathy) or for enhancing wound-healing.

In one aspect, the invention features a method of inhibiting toxicity in a cell, the method involving contacting a cell with an MMP-13 inhibitor in the presence of a toxin (e.g., paclitaxel, cisplatin, carboplatin, and oxaliplatin, docetaxel, cabazitaxel, epothilones, ixabepilone, vinblastine, vincristine, vinorelbine, etoposide, thalidomide, lenalidomide, pomalidomide, bortezomib, carfilzomib, and eribulin), thereby inhibiting toxicity in the cell. In one embodiment, the cell is a neuron or an epithelial cell. In another embodiment, the neuron is a sensory neuron, motor neuron, autonomous neuron, and the epithelial cell is a keratinocyte, fibroblast, Langerhans cell, or leukocyte. In another embodiment, the toxin is associated with cell damage and/or a loss of cell function.

In another aspect, the invention features a method of inhibiting axonal degeneration in a neuron, the method involving contacting a cell with an MMP-13 inhibitor in the presence of paclitaxel, cisplatin, carboplatin, and oxaliplatin, docetaxel, cabazitaxel, epothilones, ixabepilone, vinblastine, vincristine, vinorelbine, etoposide, thalidomide, lenalidomide, pomalidomide, bortezomib, carfilzomib, or eribulin, thereby inhibiting axonal degeneration in the neuron.

In another aspect, the invention features a method of restoring function to a sensory neuron, the method involving contacting the neuron with an MMP-13 inhibitor, wherein the neuron is in the presence of or was previously contacted with paclitaxel or another neurotoxic agent.

In another aspect, the invention features a method of treating or preventing neuropathy (e.g., paclitaxel-induced peripheral neuropathy, diabetic neuropathy) or a symptom thereof in a subject, the method involving administering to the subject an effective amount of an MMP-13 inhibitor before, during or after administration of paclitaxel or another chemotherapeutic agent having neurotoxic activity. In one embodiment, the method reduces numbness, paresthesia, and/or hyperalgesia in the subject. In another embodiment, the method increases sensation in the subject. In another aspect, the invention features a method of promoting wound healing in a subject having a peripheral neuropathy, the method involving administering to the subject an effective amount of an MMP-13 inhibitor.

In another aspect, the invention features a method of inhibiting MMP-13 activity in a subject being treated with paclitaxel or another chemotherapeutic agent, the method involving administering to the subject an effective amount of an MMP-13 inhibitor before, during or after administration of paclitaxel or another chemotherapeutic agent having neurotoxic activity.

In another aspect, the invention features a combination therapeutic composition containing an MMP-13 inhibitor and paclitaxel or another chemotherapeutic agent, wherein the MMP-13 inhibitor and the paclitaxel or other chemotherapeutic agent are each present in a separate container or are co-formulated.

In various embodiments of any of the above aspects, the neuron is in vitro or in vivo. In various embodiments of any of the above aspects, the neuronal function is touch sensation, axon debris clearance, or axon regeneration. In various embodiments of any of the above aspects, the MMP-13 inhibitor is DB04760 or CL-82198.

Other features and advantages of the invention will be apparent from the detailed description, and from the claims.

Definitions

Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person of ordinary skill in the art to which this invention belongs. The following references provide one of skill with a general definition of many of the terms used in this invention: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991). As used herein, the following terms have the meanings ascribed to them below, unless specified otherwise.

By "MMP-13 polypeptide" is meant a matrix metalloproteinase or fragment thereof having type II collagen cleaving activity and having at least about 85%, 90%, or 95% amino acid sequence identity to the protein listed at NCBI Accession No. NP_002418. An exemplary sequence is provided below:

1 mhpgvlaafl flswthcral plpsggdedd lseedlqfae rylrsyyhpt nlagilkena 61 assmterlre mqsffglevt gklddntldv mkkprcgvpd vgeynvfprt lkwskmnlty 121 rivnytpdmt hsevekafkk afkvwsdvtp lnftrlhdgi adimisfgik ehgdfypfdg 181 psgllahafp pgpnyggdah fdddetwtss skgynlflva ahefghslgl dhskdpgalm 241 fpiytytgks hfmlpdddvq giqslygpgd edpnpkhpkt pdkcdpslsl daitslrget 301 mifkdrffwr lhpqqvdael fltksfwpel pnridaayeh pshdlififr grkfwalngy 361 dilegypkki selglpkevk kisaavhfed tgktllfsgn qvwryddtnh imdkdyprli 421 eedfpgigdk vdavyekngy iyffngpiqf eysiwsnriv rvmpansilw c

(SEQ ID NO: 1)

By "agent" is meant a peptide, nucleic acid molecule, or small compound.

By "ameliorate" is meant decrease, suppress, attenuate, diminish, arrest, or stabilize the development or progression of a disease or the accelerated recovery. In one embodiment, the disease is a condition associated with the neurotoxic effects of an agent.

By "alteration" is meant a change (increase or decrease).

By "analog" is meant a molecule that is not identical, but has analogous functional or structural features to a reference molecule.

In this disclosure, "comprises," "comprising," "containing" and "having" and the like can have the meaning ascribed to them in U.S. Patent law and can mean " includes,"

"including," and the like; "consisting essentially of or "consists essentially" likewise has the meaning ascribed in U.S. Patent law and the term is open-ended, allowing for the presence of more than that which is recited so long as basic or novel characteristics of that which is recited is not changed by the presence of more than that which is recited, but excludes prior art embodiments.

"Detect" refers to identifying the presence, absence or amount of the analyte to be detected.

By "disease" is meant any condition or disorder that damages or interferes with the normal function of a cell, tissue, or organ. Examples of diseases include peripheral neuropathy (e.g., paclitaxel induced peripheral neuropathy, diabetic neuropathy), and wound healing defects, including but not limited to impaired cell migration

By "effective amount" is meant the amount of an agent required to ameliorate the symptoms of a disease relative to an untreated patient. In particular embodiments, an effective amount of an MMP-13 inhibitor (e.g., CL82198, DB04760) is the amount required to treat, prevent, or reduce a symptom of neuropathy. The effective amount of an active compound(s) used to practice the present invention for therapeutic treatment of a disease varies depending upon the manner of administration, the age, body weight, and general health of the subject. Ultimately, the attending physician or veterinarian will decide the appropriate amount and dosage regimen. Such amount is referred to as an "effective" amount. The invention provides a number of targets that are useful for the development of highly specific drugs to treat or a disorder characterized by the methods delineated herein. In addition, the methods of the invention provide a facile means to identify therapies that are safe for use in subjects. In addition, the methods of the invention provide a route for analyzing virtually any number of compounds for effects on a disease described herein with high-volume throughput, high sensitivity, and low complexity.

By "inhibitory nucleic acid" is meant a double-stranded RNA, siRNA, shRNA, or antisense RNA, or a portion thereof, or a mimetic thereof, that when administered to a mammalian cell results in a decrease (e.g., by 10%, 25%, 50%, 75%, or even 90-100%) in the expression of a target gene (e.g., MMP-13). Typically, a nucleic acid inhibitor comprises at least a portion of a target nucleic acid molecule, or an ortholog thereof, or comprises at least a portion of the complementary strand of a target nucleic acid molecule. For example, an inhibitory nucleic acid molecule comprises at least a portion of any or all of the nucleic acids delineated herein.

The terms "isolated," "purified," or "biologically pure" refer to material that is free to varying degrees from components which normally accompany it as found in its native state. "Isolate" denotes a degree of separation from original source or surroundings. "Purify" denotes a degree of separation that is higher than isolation. A "purified" or "biologically pure" protein is sufficiently free of other materials such that any impurities do not materially affect the biological properties of the protein or cause other adverse consequences. That is, a nucleic acid or peptide of this invention is purified if it is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized. Purity and homogeneity are typically determined using analytical chemistry techniques, for example, polyacrylamide gel electrophoresis or high performance liquid chromatography. The term "purified" can denote that a nucleic acid or protein gives rise to essentially one band in an electrophoretic gel. For a protein that can be subjected to modifications, for example, phosphorylation or glycosylation, different modifications may give rise to different isolated proteins, which can be separately purified.

By "marker" is meant any protein or polynucleotide having an alteration in expression level or activity that is associated with a disease or disorder.

By "reduces" is meant a negative alteration of at least 10%, 25%, 50%, 75%, or

100%. By "reference" is meant a standard or control condition. For example, an untreated control subject.

By "subject" is meant a mammal, including, but not limited to, a human or non- human mammal, such as a rodent, bovine, equine, canine, ovine, or feline.

Ranges provided herein are understood to be shorthand for all of the values within the range. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.

As used herein, the terms "treat," treating," "treatment," and the like refer to reducing or ameliorating a disorder and/or symptoms associated therewith. It will be appreciated that, although not precluded, treating a disorder or condition does not require that the disorder, condition or symptoms associated therewith be completely eliminated.

Unless specifically stated or obvious from context, as used herein, the term "or" is understood to be inclusive. Unless specifically stated or obvious from context, as used herein, the terms "a", "an", and "the" are understood to be singular or plural.

Unless specifically stated or obvious from context, as used herein, the term "about" is understood as within a range of normal tolerance in the art, for example within 2 standard deviations of the mean. About can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear from context, all numerical values provided herein are modified by the term about.

The recitation of a listing of chemical groups in any definition of a variable herein includes definitions of that variable as any single group or combination of listed groups. The recitation of an embodiment for a variable or aspect herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.

Any compositions or methods provided herein can be combined with one or more of any of the other compositions and methods provided herein.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 A provides a schematic showing the experimental design for induction and assessment of peripheral neuropathy in adult zebrafish by daily injections of 10μΜ paclitaxel on four consecutive days, followed by 10-day recovery. FIG. IB provides a series of micrographs showing anti-acetylated-tubulin staining of axons one day after the last injection. Fine cutaneous nerve endings are present in vehicle control (top panel) but not in paclitaxel-treated (bottom panel) fish. Vehicle axons were partially traced. Scale bars: ΙΟΟμιη

FIG. 1C provides a graph showing selective nerve fiber loss in distal but not proximal caudal fin (n=5, 5-6 fish per group).

FIG. ID provides a graph showing that touch response is assessed prior to each daily injection and during a recovery period reveals a delayed response on day 4, which does not reverse during a 10-day recovery phase (n=5, 5-6 fish per group).

FIG. IE provides a graph showing that lhr swimming distances are not significantly different between vehicle and paclitaxel-treated fish (n=2, 5 fish per group). p*<0.05, p**<0.01, p****<0.0001; Abbreviations: Pre-inj : pre-injection day, D: Day, FL:

Fluorescence; rec: recovery

In sum, the panels of FIG. 1 show that paclitaxel induces sensory axon degeneration and loss of touch response in adult zebrafish.

FIG. 2A provides a scheme of larval paclitaxel (22μΜ) incubation and assessment of neuropathy.

FIG. 2B provides micrographs of a Tg(isl2b:GFP) zebrafish strain used to analyze axon degeneration in (FIGs. 2C, 2D, 2E, 21). Scale bar: 200μιη

FIG. 2C and FIG. 2D provide micrographs showing axon branches in caudal fins of vehicle (FIG. 2C) and paclitaxel (FIG. 2D)-treated larvae after 24, 72 and 96hrs (FIGs. 2C- 2D, panels C- D"). Scale bars: 20μιη Bright field images of fin morphology after 96hr treatment (FIGs. 2C-2D, panels C", D"'). Scale bars: 50μιη

FIG. 2E provides a graph showing that axon branch density is significantly reduced in paclitaxel-treated larvae after 96hrs (n=3, 5-7 larvae per group).

FIG. 2F provides a graph showing that a higher number of touch stimuli is required to evoke a response in paclitaxel-treated larvae (n=3, 10-15 larvae per group).

FIG. 2G provides in the upper panel: lhr traces of individual vehicle and paclitaxel- treated larvae in each well. FIG. 2G provides a graph in the bottom panel: , quantification shows no significant difference (n=2, 8 fish per group).

FIG. 2H provides a scheme of microinjections on 3 consecutive days with 10μΜ paclitaxel, and concomitant axon and behavioral analyses. FIG. 21 provides a graph showing axon branch density is significantly reduced after 3 injections and rapidly recovers. Note that the axon branch density at 11 dpf has also decreased in controls, as the RB neuron population diminishes (n=3, 8 fish per group).

FIG. 2J provides a graph showing that touch response is transiently delayed after the third injection and restored during recovery (n=3, 5 fish per group). p*<0.05, p**<0.01, p***<0.001, p****<0.0001. Abbreviations: dpf: days post fertilization.

In sum, the panels of FIG. 2 show that paclitaxel induces neurotoxicity in larval zebrafish. (A)

FIG. 3 A provides a scheme of caudal fin phenotypes observed within 3 hours after pacli-taxel injection in presence or absence of mechanical stress.

FIG. 3B shows altered fin morphology (arrows) 4hrs after paclitaxel injection (inset shows vehicle-injected controls). Scale bar: 200μιη.

FIG. 3C shows disheveled fin-fold (arrows, inset box) and skin injury (arrow, outside image) after 24hrs. Inset shows higher magnification of inset boxed region. Scale bars:

200μιη

FIG. 3D provides a graph that shows increased injury formation 24hrs after paclitaxel injection, which is exacerbated by mechanical stress (MS) (n=3, 5 larvae per group).

FIG. 3E provides a series of scanning electron micrographs of distal caudal fins following 3hr incubation in vehicle (inset) or paclitaxel. Paclitaxel animal with micro-tear (arrow) indicates brittle skin. Scale bars: ΙΟμιη.

FIG. 3F provides a series of graphs that show the percent of animals with micro-tears (left) and average number of micro-tears per animal (right, as shown in FIG. 3E) is both increased after 3hrs paclitaxel treatment.

FIG. 3G shows increased ROS/ H₂O₂ detection with pentafluorobenzenesulfonyl- fluorescein in the caudal fin of paclitaxel-treated, stressed animals, not seen in the injury site, or in stressed vehicle controls (in- Scale bars: 50μιη.).

FIG. 3H provides a series of micrographs showing NF-κΒ reporter activity with and without H₂O₂ scavengers in vehicle and paclitaxel treated stressed and unstressed larvae (n=3, 3-6 fish per group, p*<0.05, p***<0.001).

FIG. 31 provides a graph that shows NF-κΒ reporter activity with and without H₂0₂ scavengers in vehicle and paclitaxel-treated stressed and unstressed larvae (n=3, 3-6 fish per group, p*<0.05, p***<0.001). FIG. 3J provides a graph that shows tubulin tracker (ΙΟμΜ) fluorescence in caudal fins peaks around 3hrs post-injection (hpinj) and around 5hpinj in RB neuron cell bodies, shown in K, L (n=3, 4 fish per group).

FIG. 3K provides a series of micrographs showing tubulin tracker (white) in caudal fin within 3hrs following injection. Scale bar: ΙΟΟμιη.

FIG. 3L provides a series of micrographs showing tubulin tracker is present in large (boxed) and small (leftarrow) diameter RB neuron cell bodies at 5hpinj . Note: not all neurons accumulate tubulin tracker (right arrow). Scale bar: ΙΟμιη.

FIG. 3M provides a series of micrographs showing that tubulin tracker does not co- localize with cutaneous axons at 3hpinj (white bracket indicates axons (upper punctate layer) above tubulin tracker-positive basal layer). Scale bar: ΙΟμιη.

FIG. 3N provides a series of micrographs showing that tubulin tracker co-localizes with basal keratinocytes at 3hpinj . Scale bar: ΙΟμιη. Abbreviations: Pctx: paclitaxel, hpinj : hours post injection, MS: mechanically stressed, DPI: Diphenyleneiodonium, APC: apocynin In sum, panels of FIG. 3 show that paclitaxel-induced epithelial damage precedes cutaneous axon degeneration.

FIG. 4A provides a series of micrographs. Cutaneous axons of a single RB neuron innervating the caudal fin were traced for 12hrs following fin amputation. Larvae were incubated for 3hrs either in vehicle solution (0.5% DMSO/Ringers) (A) or paclitaxel (22μΜ) FIG. 4B provides a series of micrographs. Insets show higher magnification of boxed regions (arrowheads depict axon debris lost in vehicle but not paclitaxel-treated animals). Tracks in last panel depict branch growth over time.

FIG. 4C provides a graph showing the quantification of mean axon branch growth over 12hrs in larvae incubated in vehicle, paclitaxel (22μΜ), and paclitaxel plus either DB04760 or CL-82198 (10μΜ each)(n=3, 3-4 fish per group).

FIG. 4D provides a graph showing a comparison of mean axon growth and retraction in injured vehicle and paclitaxel-treated animals over 12hrs (n=2, 3-4 fish and 15 axons per group).

FIG. 4E provides a graph showing quantification of axon debris clearance (n=2, 3-4 fish and 5-7 axons per group).

FIG. 4F provides a scheme of compound screening assay. FIG. 4G provides chemical structures of the MMP-13 inhibitors CL-82198:N- [4- (4- morpholinyl)butyl]- 2- benzofurancarboxamide, and DB04760: N4,N6-bis[(4-fluoro-3- methylphenyl)methyl] pyrimidine-4,6-dicarboxamide.

FIG. 4H provides a series of micrographs showing that axon regeneration is partially restored with ΙΟμΜ DB04760 (arrowheads mark diminishing axon debris). Scale bar: 50μιη.

FIG. 41 provides a graph showing a comparison of axon branch density following 96hr treatment (n=2, D5 fish per group).

FIG. 4J provides a graph showing a touch response in fin and head region after 96hr treatment (n=4, 5 fish per group).

FIG. 4K provides a graph showing the percentage of animals with improved touch response upon co-administration of paclitaxel and either CL-82198 or DB04760. p*<0.05, p**<0.01, p***<0.001 Abbreviations: hpa: hours post amputation, Pctx: paclitaxel

In sum, the panels of FIG. 4 show paclitaxel-induced neurotoxicity is reduced upon MMP-13 inhibition.

FIG. 5 A provides a graph showing a quantitative real-time PCR shows increased mmpl3a expression in uninjured and injured animals treated with 22μΜ paclitaxel for 3hrs (15 pooled larvae per group).

FIG. 5B provies a Western analysis showing a higher abundance of the 54kDa isoform in uninjured and injured animals treated with paclitaxel for 3hrs (10 pooled larvae per group).

FIG. 5C provides a graph quantifying the 48 kDa band shown in FIG. 5B.

FIG. 5D provides a graph quantifying the 54 kDa band shown in FIG. 5B.

FIG. 5E provides micrographs. In FIG. 5C-5E, paclitaxel/vehicle ratios for normalized 48kDa (FIG. 5C) and 54kDa (FIG. 5D) bands in uninjured and injured animals (n=2, 10 pooled larvae). Dashed lines demarcate control levels. (FIG. 5E, panels E-F")

Immunofluorescence staining of MMP-13 in vehicle control (FIG. 5E, panel E) is increased at the wound margin after amputation (FIG. 5E, panel E'), and ubiquitous following 3hr paclitaxel treatment (FIG. 5E, panels F, F'). Immunofluorescence staining of larvae transiently injected with krt4:dsRed in the absence of primary MMP-13 antibody (FIG. 5E, panels E", F"). Scale bar: 50μιη.

Mosaic keratinocyte-specific expression following krt4:dsRed injection and MMP-13 staining shows co-localization. DAPI stains nuclei. (FIG. 5E, panel H) 3D rendering of one keratinocyte and MMP-13 staining shows co-localization. (I) 3D rendering of axons and MMP-13 staining shows lack of co-localization (arrows). (FIG. 5E, panels J-K") MMP-13 staining (J, K) and axons stained with acetylated tubulin (FIG. 5E, panels J", K") shows lack of co-localization in vehicle (FIG. 5E, panel J') and paclitaxel (FIG. 5E, panel K') incubated larvae. (FIG. 5E, panels L-L") MMP-13 staining ( arrows) is present in the deeper basal keratinocyte layer below axons (arrowheads)( FIG. 5E, panels L, L") but not in the superficial periderm (FIG. 5E, panel L'). p*<0.05, p**<0.01, p***<0.001. Abbreviations: veh: vehicle, Pctx: paclitaxel, Uninj : uninjured, Inj : injured, AB: antibody, a.u. : arbitrary units

In sum, FIG. 5A-5E show that Paclitaxel augments MMP-13 expression.

FIG. 6A provides a series of micrographs.

FIG. 6B provides a series of micrographs.

FIG. 6C provides a series of micrographs.

FIGs. 6A-6C show that epithelial defects induced by paclitaxel are rescued upon MMP-13 inhibition. (FIGs. 6A-6C)Temporal sequence of HyPer oxidation in Tg(krt4:Gal4 tdTomato 5xUAS HyPer) larva prior to and after addition of 0.01% exogenous H₂0₂ at 30min, visualized as 488/405nm emission ratio. Vehicle (0.5% DMSO) controls show some oxidation following H₂0₂ addition (FIG. 6A), which is increased after 3hrs paclitaxel incubation (FIG. 6B) and rescued when CL-82198 is co-administered (FIG. 6C). Scale bars: ΙΟΟμιη.

FIG. 6D provides a graph showing quantification of HyPer oxidation (n=2, 4-5 fish per group; paclitaxel vs. paclitaxel+CL-82198: p*=0.03).

FIG. 6E provides a graph showing the percentage of larvae with skin damage following injection of either wildtype mmpl3a or mmpl3aA373 mRNA into 1-cell stage embryos and mechanical stress at 2dpf (n=3 biological replicates, 15 larvae per group).

FIG. 6F provides a graph showing the rescue of skin damage following

pharmacological inhibition of MMP-13 and mechanical stress at 2dpf (n=3, 9-10 larvae per group).

FIG. 6G provide a graph showing the percent damaged animals for experimental groups.

FIG. 6H provides scanning electron microscopy images of larvae incubated for 3hrs in paclitaxel+CL-82198 (FIG. 6G) and 96hrs in vehicle (panel H), paclitaxel (panel FT) or paclitaxel+CL-82198 (panel H") shows improved skin morphology with CL-82198. Scale bars: 25μιη (FIG. 6G, FIG. 6H, panels H, FT) and 5μιη (FIG. 6H, panel FT) p*<0.05, p**<0.01 Abbreviations: Pctx: paclitaxel FIG. 7A provides a series of micrographs showing the rapid repair of a puncture wound (dashed circles) in a vehicle-treated transgenic Tg(tp63 :CAAX-GFP) zebrafish larva.

FIG. 7B provides a series of micrographs showing the puncture wound is retained in a larva incubated in 22μΜ paclitaxel.

FIG. 7C provides a graph showing quantification of wound diameter over 12hrs shows rescue of wound repair with MMP-13 inhibitors (n=2, 4-5 larvae per group).

FIG. 7D provides a series of micrographs showing the scratch wounding of HEK01 human keratinocytes shows ROS/ H₂O₂ formation at the scratch wound margin of vehicle (arrows) but not paclitaxel-treated cells (panels D', D").

FIG. 7E provides a series of micrographs showing the (panels E, E') ROS/H₂0₂ formation is present in paclitaxel-treated HEK01 cells after 12hrs.

FIG. 7F provides a series of micrographs showing a sealed scratch in vehicle (panel F) but not paclitaxel-treated wells (panel F') after 24hrs.

FIG. 7G provides a graph showing ROS/ H₂0₂fluorescence intensity measured from the scratch margin lhr post scratch (hps).

FIG. 7H provides a graph showing HEK01 scratch wound gap size over time (n=5; p***<0.001).

FIG. 71 provides a graph showing HEK01 gap closure distance at 12hps following CL- 82198 administration.

FIG. 7J provides a graph showing HEK01 gap closure distance at 12hps following

CL- 82198 administration. (J) HEK01 gap closure distance at 18hps following DB04760 administration.

FIG. 7K provdes still images (Eosin) of HEK01 cells from (J). Insets on the right of each image show higher magnifications of highlighted boxes. Control cells at the scratch margin show decreased cell-cell adhesion and formation of lamellipodia at the leading (migratory) edge, which is absent in paclitaxel-treated cells and rescued with DB04760. DB04760 alone increases the number of cells with lamellipodia formation. Scale bar: 20μπι.

FIG. 7L provides a model for paclitaxel-induced peripheral neuropathy. Paclitaxel damages epithelial keratinocytes by upregulating MMP-13, leading to loss of skin integrity and axon damage. p*<0.05, p**<0.01, p***<0.001 Abbreviations: mpp: minutes post puncture; hpp: hours post puncture; hps: hours post scratch; SC: scratch, a.u.: arbitrary units, Pctx: paclitaxel, CL: CL-82198, DB: DB04760.

In sum, the panels of FIG. 7 show that paclitaxel impairs skin wound repair. FIG. 8A provides a graph showing that paclitaxel reduces axon branch density, and that this reduction is rescued by treatment with the MMP-13 inhibitor CL82198.

FIG. 8B provides a graph showing the number of stimuli needed to induce a response.

FIG. 8C provides a graph showing that paclitaxel reduces axon branch density, and that this reduction is rescued by treatment with the MMP-13 inhibitor DB04760.

FIG. 8D provides a graph showing the number of stimuli needed to induce a response.

FIG. 8E provides a series of micrographs showing MMP-13 and tubulin

immunofluorescence in control and paclitaxel treated fish.

FIG. 9 A provides a schematic showing mmpl3a cDNA in wild-type and mmpl3a comprising a 709 base pair deletion.

FIG. 9B provides a schematic showing domains of MMP13 protein and the position of a frame shift.

FIG. 9C provides a series of micrographs showing fins treated with vehicle vs.

paclitaxel and the MMP-13 inhibitor.

FIGs 10-14 provide a series of electron micrographs of skin. As shown in FIGs. 10-

14 the following abbreviations are used to denote structural features: P=periderm

keratinocyte, B=basal keratinocyte, N=nucleus, M=mesenchyme, BM=basement membrane.

FIGs. 10A-10D provide a series of electron micrographs showing larval zebrafish skin following 4-day treatment with paclitaxel and MMP-13 inhibitors.

FIG. 10A, panels A- A", top to bottom, respectively, show the ultrastructure of

DMSO vehicle-treated control skin. Mitochondria (white arrow), junctions (black

arrowhead), basement membrane (arrowheads) and sensory axons embedded between the periderm and basal keratinocyte layers (black arrow in bottom panel A") are distinctive.

FIG. 10B panels B-B", top to bottom, respectively, show the ultrastructure of paclitaxel-treated skin. Large gaps are visible within the skin (top panel B, bottom panel B"). Mitochondrial cisternae can no longer be clearly distinguished (arrows in middle panel B'), the basement membrane is partially degraded (white arrowheads, middle panel B') and tight junctions are less distinct (black arrowhead in bottom panel B"). Remaining sensory axons are enlarged and exist in large empty spaces between two cell layers (black arrow in bottom panel B").

FIG. IOC panels C-C", top to bottom respectively, show the ultrastructure of skin treated with paclitaxel and DB04760. Organelle structures and basement membrane were significantly improved when DB04760 was co-administered. FIG. 10D, panels D-D", top to bottom respectively, show the ultrastructure of skin treated with paclitaxel and CL-82198. Organelle structures and basement membrane were significantly improved when CL-82198 was co-administered.

FIG. 11 provides a series of electron micrographs showing the ultrastructure of DMSO vehicle-treated control skin. Mitochondria with cisternae (white arrow in left panel A), basement membrane (arrowheads in middle panel B), junctions (black arrowhead in right panel C) and sensory axons embedded between the periderm and basal keratinocyte layers (black arrow in C) are clearly visible.

FIG. 12 provides a series of electron micrographs showing the ultrastructure of paclitaxel-treated skin. Large gaps are visible within the skin (left panel A and right panel C). Mitochondrial cisternae can no longer be clearly distinguished (arrows in middle panel B), the basement membrane is partially degraded (white arrowheads, middle panel B) and tight junctions are less distinct (black arrowhead in right panel C). Remaining sensory axons were enlarged and exist in large empty spaces between two cell layers (black arrow in right panel C).

FIG.13 provides a series of electron micrographs showing the ultrastructure of skin treated with paclitaxel and DB04760 (panels A-C, left to right respectively). Organelle structures and basement membrane were significantly improved when DB04760 was coadministered.

FIG. 14 provides a series of electron micrographs showing the ultrastructure of skin treated with paclitaxel and CL-82198 (panels A-C, left to right respectively). Organelle structures and basement membrane were significantly improved when CL-82198 is coadministered. DETAILED DESCRIPTION OF THE INVENTION

The invention features methods for identifying agents (e.g., agents that inhibit MMP- 13) useful for treating peripheral neuropathy (e.g., paclitaxel-induced peripheral neuropathy, diabetic neuropathy), and the use of such agents for the treatment of peripheral

neuropathy(e.g., paclitaxel-induced peripheral neuropathy, diabetic neuropathy) and wound healing.

The invention is based, at least in part, on the discovery that zebrafish can be used an in vivo model for peripheral neuropathy and wound healing. Both adult and larval zebrafish displayed signs of paclitaxel neurotoxicity, including sensory axon degeneration and the loss of touch response in the distal caudal fin. Intriguingly, studies in zebrafish larvae showed that paclitaxel rapidly promotes epithelial damage and decreased mechanical stress resistance of the skin prior to induction of axon degeneration. Moreover, injured paclitaxel-treated zebrafish skin and scratch-wounded human keratinocytes (HEK001) display reduced healing capacity. Epithelial damage correlated with rapid accumulation of fluorescein-conjugated paclitaxel in epidermal basal keratinocytes, but not axons, and upregulation of matrix metalloproteinase 13 (MMP-13, collagenase 3) in the skin. Pharmacological inhibition of MMP-13, in contrast, largely rescued paclitaxel-induced epithelial damage and neurotoxicity, whereas MMP-13 overexpression in zebrafish embryos rendered the skin vulnerable to injury under mechanical stress conditions. Without wishing to be bound to theory, the results detailed herein suggest that the epidermis plays a role in paclitaxel neurotoxicity, and that zebrafish can be used to identify candidate agents for therapeutic interventions.

Paclitaxel

Paclitaxel is a microtubule-stabilizing chemotherapeutic agent that is widely used in the treatment of common cancers, including breast, ovarian, and lung cancer. Despite its promising anticancerous properties, paclitaxel also damages healthy tissues, most

prominently peripheral axons of somatosensory neurons. Paclitaxel-induced peripheral neuropathy initiates in the distal extremities and presents as neuropathic pain syndrome, temperature-sensitivity and paresthesia (tingling and numbness). Nerve biopsies from patients suggest that axon degeneration is the primary manifestation of this condition, followed by secondary demyelination and nerve fiber loss in severely affected patients. Certain drugs have been shown in vitro and in vivo to protect against paclitaxel-induced nerve damage, including acetyl-L-carnitine, erythropoietin, alpha-lipoic acid, olesoxime, amifostine, nerve growth factor, and glutamate. However, so far these agents have either not successfully passed clinical trials or merely alleviate symptoms such as pain, but do not prevent them. Thus, a better understanding of the underlying causes of paclitaxel-induced peripheral neuropathy are necessary and may help identify new candidate drugs with which to treat this condition.

A widely accepted mechanism for paclitaxel neurotoxicity is the "dying back" of distal nerve endings, which has been attributed to aberrant axonal transport and cytoplasmic flow, as well as mitochondrial defects, both shown in vivo and in vitro. In vitro studies further demonstrated that paclitaxel induces axon degeneration upon direct application to either axons or cell bodies, and thus a general thought is that paclitaxel induced axon damage is neuron-autonomous. The specificity of paclitaxel-induced axon degeneration, which initiates in the intra-epidermal A and C fibers innervating the glabrous skin of palm and sole, suggests that environmental factors could play a critical role. The palms and soles are more frequently injured and exposed to biomechanical stresses, which could contribute to paclitaxel neurotoxicity. Cutaneous axons, for example, have been shown to be receptive to mechanical stress through binding via integrin receptors to the extracellular matrix (ECM). Keratinocytes also interact with axons after injury. For example, the data indicatedin larval zebrafish that keratinocytes promote axon regeneration by secretion of hydrogen peroxide (H₂0₂) into the wound. Therefore, perturbations of the intricate interactions between epidermal keratinocytes and axons by paclitaxel treatment could promote axon degeneration. Intriguingly, epithelial cells are highly susceptible to paclitaxel-induced damage, evident by its efficacy in the treatment of carcinomas as well as its damaging effects on human skin. Further in vitro studies demonstrated that paclitaxel induces caspase 3 and 8-dependent apoptosis in a human keratinocyte line (HaCaT) and alters microtubule behavior in cultured mouse keratinocytes, leading to retraction of microtubules from the plasma membrane. Thus, paclitaxel induced keratinocyte damage could contribute to neurotoxicity; yet no studies to date have examined this possibility. An in vivo zebrafish model was established to study paclitaxel' s neurotoxic effects in live animals. These studies demonstrated that paclitaxel disrupts the crosstalk between cutaneous axons and keratinocytes by inducing keratinocyte-specific upregulation of matrix-metalloproteinase 13 (MMP-13, collagenase 3). Increased MMP-13 activity promotes epidermal damage and neurotoxicity, which can be rescued upon pharmacological inhibition of MMP- 13, making it a novel therapeutic candidate.

Accordingly, the invention provides agents having MMP-13 inhibitory activity, such as CL-82198 and DB04760, and methods of using them for the treatment of neuropathies associated with a chemotherapeutic agent, such as paclitaxel. In addition to these agents, the invention provides methods of identifying other agents that are useful for the treatment of neuropathy.

Screening Methods

Using a zebrafish model of neuropathy, Applicants have found that the toxic effects of chemotherapeutic compounds are ameliorated when they are administered in combination with MMP-13 inhibitor. The invention provides a simple means for identifying agents (including nucleic acids, peptides, small molecule inhibitors, and mimetics) that are capable of protecting or restoring neuronal function when administered in combination with a chemotherapeutic (e.g., paclitaxel).

In general, candidate agents are identified by assaying those that restore neuronal function, reduce axonal degeneration, or that otherwise inhibit MMP-13 as described in the Examples. In one embodiment, the efficacy of a candidate agent is dependent upon its ability to selectively inhibit MMP-13. Such inhibition can be readily assayed using any number of standard assays, such as Chen et al.: Structure-Based Design of a Novel, Potent, and Selective Inhibitor for MMP-13 Utilizing NMR Spectroscopy and Computer- Aided Molecular Design, J. Am. Chem. Soc, 2000, 122 (40), pp 9648-9654, which is incorporated herein by reference. For example, a candidate agent may be tested in vitro for its ability to modulate MMP-13 activity and then tested in vivo on zebrafish as described herein.

Potential MMP-13 inhibitors include organic molecules, peptides, peptide mimetics, polypeptides, nucleic acid ligands, aptamers, and antibodies that bind to a MMP-13 polypeptide and reduce its activity. Agents isolated by this approach may be used, for example, as therapeutics to treat or prevent neuropathy.

Test Compounds and Extracts

In general, MMP-13 inhibitors (e.g., agents that selectively reduce the activity of a

MMP-13 polypeptide) are identified from large libraries of natural product or synthetic (or semi -synthetic) extracts or chemical libraries or from polypeptide or nucleic acid libraries, according to methods known in the art. Those of ordinary skill in the field of drug discovery and development will understand that the precise source of test extracts or compounds is not critical to the screening procedure(s) of the invention. Agents used in screens may include known those known as therapeutics for the treatment of a neuropathy. Alternatively, virtually any number of unknown chemical extracts or compounds can be screened using the methods described herein. Examples of such extracts or compounds include, but are not limited to, plant-, fungal-, prokaryotic- or animal-based extracts, fermentation broths, and synthetic compounds, as well as the modification of existing polypeptides.

Libraries of natural polypeptides in the form of bacterial, fungal, plant, and animal extracts are commercially available from a number of sources, including Biotics (Sussex, UK), Xenova (Slough, UK), Harbor Branch Oceangraphics Institute (Ft. Pierce, Fla.), and PharmaMar, U.S.A. (Cambridge, Mass.). Such polypeptides can be modified to include a protein transduction domain using methods known in the art and described herein. In addition, natural and synthetically produced libraries are produced, if desired, according to methods known in the art, e.g., by standard extraction and fractionation methods. Examples of methods for the synthesis of molecular libraries can be found in the art, for example in: DeWitt et al., Proc. Natl. Acad. Sci. U.S.A. 90:6909, 1993; Erb et al., Proc. Natl. Acad. Sci. USA 91 : 11422, 1994; Zuckermann et al, J. Med. Chem. 37:2678, 1994; Cho et al, Science 261 : 1303, 1993; Carrell et al., Angew. Chem. Int. Ed. Engl. 33 :2059, 1994; Carell et al, Angew. Chem. Int. Ed. Engl. 33 :2061, 1994; and Gallop et al, J. Med. Chem. 37: 1233, 1994. Furthermore, if desired, any library or compound is readily modified using standard chemical, physical, or biochemical methods.

Numerous methods are also available for generating random or directed synthesis (e.g., semi-synthesis or total synthesis) of any number of polypeptides, chemical compounds, including, but not limited to, saccharide-, lipid-, peptide-, and nucleic acid-based compounds. Synthetic compound libraries are commercially available from Brandon Associates

(Merrimack, N.H.) and Aldrich Chemical (Milwaukee, Wis.). Alternatively, chemical compounds to be used as candidate compounds can be synthesized from readily available starting materials using standard synthetic techniques and methodologies known to those of ordinary skill in the art. Synthetic chemistry transformations and protecting group

methodologies (protection and deprotection) useful in synthesizing the compounds identified by the methods described herein are known in the art and include, for example, those such as described in R. Larock, Comprehensive Organic Transformations, VCH Publishers (1989); T. W. Greene and P. G. M. Wuts, Protective Groups in Organic Synthesis, 2nd ed., John Wiley and Sons (1991); L. Fieser and M. Fieser, Fieser and Fieser's Reagents for Organic Synthesis, John Wiley and Sons (1994); and L. Paquette, ed., Encyclopedia of Reagents for Organic Synthesis, John Wiley and Sons (1995), and subsequent editions thereof.

Libraries of compounds may be presented in solution (e.g., Houghten, Biotechniques 13 :412-421, 1992), or on beads (Lam, Nature 354:82-84, 1991), chips (Fodor, Nature 364:555-556, 1993), bacteria (Ladner, U.S. Patent No. 5,223,409), spores (Ladner U.S. Patent No. 5,223,409), plasmids (Cull et al, Proc Natl Acad Sci USA 89: 1865-1869, 1992) or on phage (Scott and Smith, Science 249:386-390, 1990; Devlin, Science 249:404-406, 1990; Cwirla et al. Proc. Natl. Acad. Sci. 87:6378-6382, 1990; Felici, J. Mol. Biol. 222:301-310, 1991; Ladner supra. ) . In addition, those of ordinary skill in the art of drug discovery and development readily understand that methods for dereplication (e.g., taxonomic dereplication, biological dereplication, and chemical dereplication, or any combination thereof) or the elimination of replicates or repeats of materials already known for their activity should be employed whenever possible.

When a crude extract is found to have MMP-13 inhibitory activity, further fractionation of the positive lead extract is necessary to isolate molecular constituents responsible for the observed effect. Thus, the goal of the extraction, fractionation, and purification process is the careful characterization and identification of a chemical entity within the crude extract that reduces neoplastic cell proliferation or viability. Methods of fractionation and purification of such heterogeneous extracts are known in the art. If desired, compounds shown to be useful as therapeutics are chemically modified according to methods known in the art. Pharmaceutical Therapeutics

In other embodiments, agents discovered to have medicinal value using the methods described herein are useful as a drug or as information for structural modification of existing compounds, e.g., by rational drug design. Such methods are useful for screening agents having an effect on a neuropathy.

For therapeutic uses, the compositions or agents identified using the methods disclosed herein may be administered systemically, for example, formulated in a

pharmaceutically-acceptable buffer such as physiological saline. Preferable routes of administration include, for example, subcutaneous, intravenous, intraperitoneally, intramuscular, or intradermal injections that provide continuous, sustained levels of the drug in the patient. Treatment of human patients or other animals will be carried out using a therapeutically effective amount of a therapeutic identified herein in a physiologically- acceptable carrier. Suitable carriers and their formulation are described, for example, in Remington's Pharmaceutical Sciences by E. W. Martin. The amount of the therapeutic agent to be administered varies depending upon the manner of administration, the age and body weight of the patient, and with the clinical symptoms of the neuropathy. Generally, amounts will be in the range of those used for other agents used in the treatment of other diseases associated with neuropathy, although in certain instances lower amounts will be needed because of the increased specificity of the compound. A compound is administered at a dosage that inhibits the toxic effects of a chemotherapeutic on a neuronal or epithelial cell or that reduces MMP-13 expression or biological activity as determined by a method known to one of ordinary skill in the art , or using any that assay that measures the expression or the biological activity of a MMP-13 polypeptide.

Formulation of Pharmaceutical Compositions

The administration of a compound for the treatment of a neuropathy may be by any suitable means that results in a concentration of the therapeutic that, combined with other components, is effective in ameliorating, reducing, or stabilizing a neuropathy. The compound may be contained in any appropriate amount in any suitable carrier substance, and is generally present in an amount of 1-95% by weight of the total weight of the composition. The composition may be provided in a dosage form that is suitable for parenteral (e.g., subcutaneously, intravenously, intramuscularly, or intraperitoneally) or oral administration route. The pharmaceutical compositions may be formulated according to conventional pharmaceutical practice (see, e.g., Remington: The Science and Practice of Pharmacy (20th ed.), ed. A. R. Gennaro, Lippincott Williams & Wilkins, 2000 and Encyclopedia of

Pharmaceutical Technology, eds. J. Swarbrick and J. C. Boylan, 1988-1999, Marcel Dekker, New York).

Human dosage amounts can initially be determined by extrapolating from the amount of compound used in mice, as an artisan of ordinary skill recognizes it is routine in the art to modify the dosage for humans compared to animal models. In certain embodiments it is envisioned that the dosage may vary from between about 1 μg compound/Kg body weight to about 5000 mg compound/Kg body weight; or from about 5 mg/Kg body weight to about 4000 mg/Kg body weight or from about 10 mg/Kg body weight to about 3000 mg/Kg body weight; or from about 50 mg/Kg body weight to about 2000 mg/Kg body weight; or from about 100 mg/Kg body weight to about 1000 mg/Kg body weight; or from about 150 mg/Kg body weight to about 500 mg/Kg body weight. In other embodiments this dose may be about 1, 5, 10, 25, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1600, 1700, 1800, 1900, 2000, 2500, 3000, 3500, 4000, 4500, or 5000 mg/Kg body weight. In other embodiments, it is envisaged that doses may be in the range of about 5 mg compound/Kg body to about 20 mg compound/Kg body. In other embodiments the doses may be about 8, 10, 12, 14, 16 or 18 mg/Kg body weight. Of course, this dosage amount may be adjusted upward or downward, as is routinely done in such treatment protocols, depending on the results of the initial clinical trials and the needs of a particular patient.

Pharmaceutical compositions according to the invention may be formulated to release the active compound substantially immediately upon administration or at any predetermined time or time period after administration. The latter types of compositions are generally known as controlled release formulations, which include (i) formulations that create a substantially constant concentration of the drug within the body over an extended period of time; (ii) formulations that after a predetermined lag time create a substantially constant concentration of the drug within the body over an extended period of time; (iii) formulations that sustain action during a predetermined time period by maintaining a relatively, constant, effective level in the body with concomitant minimization of undesirable side effects associated with fluctuations in the plasma level of the active substance (sawtooth kinetic pattern); (iv) formulations that localize action by, e.g., spatial placement of a controlled release composition adjacent to or in contact with the thymus; (v) formulations that allow for convenient dosing, such that doses are administered, for example, once every one or two weeks; and (vi) formulations that target a neoplasia by using carriers or chemical derivatives to deliver the therapeutic agent to a particular cell type (e.g., neuronal or epithelial cell). For some applications, controlled release formulations obviate the need for frequent dosing during the day to sustain the plasma level at a therapeutic level.

Any of a number of strategies can be pursued in order to obtain controlled release in which the rate of release outweighs the rate of metabolism of the compound in question. In one example, controlled release is obtained by appropriate selection of various formulation parameters and ingredients, including, e.g., various types of controlled release compositions and coatings. Thus, the therapeutic is formulated with appropriate excipients into a pharmaceutical composition that, upon administration, releases the therapeutic in a controlled manner. Examples include single or multiple unit tablet or capsule compositions, oil solutions, suspensions, emulsions, microcapsules, microspheres, molecular complexes, nanoparticles, patches, and liposomes.

Parenteral Compositions

The pharmaceutical composition may be administered parenterally by injection, infusion or implantation (subcutaneous, intravenous, intramuscular, intraperitoneal, or the like) in dosage forms, formulations, or via suitable delivery devices or implants containing conventional, non-toxic pharmaceutically acceptable carriers and adjuvants. The formulation and preparation of such compositions are well known to those of ordinary skill in the art of pharmaceutical formulation. Formulations can be found in Remington: The Science and Practice of Pharmacy, supra.

Compositions for parenteral use may be provided in unit dosage forms (e.g., in single- dose ampoules), or in vials containing several doses and in which a suitable preservative may be added (see below). The composition may be in the form of a solution, a suspension, an emulsion, an infusion device, or a delivery device for implantation, or it may be presented as a dry powder to be reconstituted with water or another suitable vehicle before use. Apart from the active agent that reduces or ameliorates a neuropathy, the composition may include suitable parenterally acceptable carriers and/or excipients. The active therapeutic agent(s) may be incorporated into microspheres, microcapsules, nanoparticles, liposomes, or the like for controlled release. Furthermore, the composition may include suspending, solubilizing, stabilizing, pH-adjusting agents, tonicity adjusting agents, and/or dispersing, agents.

As indicated above, the pharmaceutical compositions according to the invention may be in the form suitable for sterile injection. To prepare such a composition, the suitable active antineoplastic therapeutic(s) are dissolved or suspended in a parenterally acceptable liquid vehicle. Among acceptable vehicles and solvents that may be employed are water, water adjusted to a suitable pH by addition of an appropriate amount of hydrochloric acid, sodium hydroxide or a suitable buffer, 1,3-butanediol, Ringer's solution, and isotonic sodium chloride solution and dextrose solution. The aqueous formulation may also contain one or more preservatives (e.g., methyl, ethyl or n-propyl p-hydroxybenzoate). In cases where one of the compounds is only sparingly or slightly soluble in water, a dissolution enhancing or solubilizing agent can be added, or the solvent may include 10-60% w/w of propylene glycol or the like. In one embodiment, paclitaxel or another MMP-13 inhibitor is delivered in cremophor EL.

Controlled Release Parenteral Compositions

Controlled release parenteral compositions may be in form of aqueous suspensions, microspheres, microcapsules, magnetic microspheres, oil solutions, oil suspensions, or emulsions. Alternatively, the active drug may be incorporated in biocompatible carriers, liposomes, nanoparticles, implants, or infusion devices. Materials for use in the preparation of microspheres and/or microcapsules are, e.g., biodegradable/bioerodible polymers such as polygalactia poly-(isobutyl cyanoacrylate), poly(2-hydroxyethyl-L-glutam- nine) and, poly(lactic acid). Biocompatible carriers that may be used when formulating a controlled release parenteral formulation are carbohydrates (e.g., dextrans), proteins (e.g., albumin), lipoproteins, or antibodies. Materials for use in implants can be non-biodegradable (e.g., polydimethyl siloxane) or biodegradable (e.g.,

poly(caprolactone), poly(lactic acid), poly(glycolic acid) or poly(ortho esters) or

combinations thereof).

Formulations for oral use include tablets containing the active ingredient(s) in a mixture with non-toxic pharmaceutically acceptable excipients. Such formulations are known to an artisan of ordinary skill. Excipients may be, for example, inert diluents or fillers (e.g., sucrose, sorbitol, sugar, mannitol, microcrystalline cellulose, starches including potato starch, calcium carbonate, sodium chloride, lactose, calcium phosphate, calcium sulfate, or sodium phosphate); granulating and disintegrating agents (e.g., cellulose derivatives including microcrystalline cellulose, starches including potato starch, croscarmellose sodium, alginates, or alginic acid); binding agents (e.g., sucrose, glucose, sorbitol, acacia, alginic acid, sodium alginate, gelatin, starch, pregelatinized starch, microcrystalline cellulose, magnesium aluminum silicate, carboxymethylcellulose sodium, methylcellulose, hydroxypropyl methylcellulose, ethylcellulose, polyvinylpyrrolidone, or polyethylene glycol); and lubricating agents, glidants, and antiadhesives (e.g., magnesium stearate, zinc stearate, stearic acid, silicas, hydrogenated vegetable oils, or talc). Other pharmaceutically acceptable excipients can be colorants, flavoring agents, plasticizers, humectants, buffering agents, and the like.

The tablets may be uncoated or they may be coated by known techniques, optionally to delay disintegration and absorption in the gastrointestinal tract and thereby providing a sustained action over a longer period. The coating may be adapted to release the active drug in a predetermined pattern (e.g., in order to achieve a controlled release formulation) or it may be adapted not to release the active drug until after passage of the stomach (enteric coating). The coating may be a sugar coating, a film coating (e.g., based on hydroxypropyl methylcellulose, methylcellulose, methyl hydroxyethylcellulose, hydroxypropylcellulose, carboxymethylcellulose, acrylate copolymers, polyethylene glycols and/or

polyvinylpyrrolidone), or an enteric coating (e.g., based on methacrylic acid copolymer, cellulose acetate phthalate, hydroxypropyl methylcellulose phthalate, hydroxypropyl methylcellulose acetate succinate, polyvinyl acetate phthalate, shellac, and/or ethylcellulose). Furthermore, a time delay material, such as, e.g., glyceryl monostearate or glyceryl distearate may be employed.

The solid tablet compositions may include a coating adapted to protect the

composition from unwanted chemical changes, (e.g., chemical degradation prior to the release of the active anti- neuropathy therapeutic substance). The coating may be applied on the solid dosage form in a similar manner as that described in Encyclopedia of

Pharmaceutical Technology, supra.

At least two anti- neuropathy therapeutics may be mixed together in the tablet, or may be partitioned. In one example, the first active anti- neuropathy therapeutic is contained on the inside of the tablet, and the second active anti- neuropathy therapeutic is on the outside, such that a substantial portion of the second anti- neuropathy therapeutic is released prior to the release of the first anti- neuropathy therapeutic.

Formulations for oral use may also be presented as chewable tablets, or as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent (e.g., potato starch, lactose, microcrystalline cellulose, calcium carbonate, calcium phosphate or kaolin), or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium, for example, peanut oil, liquid paraffin, or olive oil. Powders and granulates may be prepared using the ingredients mentioned above under tablets and capsules in a conventional manner using, e.g., a mixer, a fluid bed apparatus or a spray drying equipment.

Controlled Release Oral Dosage Forms

Controlled release compositions for oral use may, e.g., be constructed to release the active anti- neuropathy therapeutic by controlling the dissolution and/or the diffusion of the active substance. Dissolution or diffusion controlled release can be achieved by appropriate coating of a tablet, capsule, pellet, or granulate formulation of compounds, or by

incorporating the compound into an appropriate matrix. A controlled release coating may include one or more of the coating substances mentioned above and/or, e.g., shellac, beeswax, glycowax, castor wax, carnauba wax, stearyl alcohol, glyceryl monostearate, glyceryl distearate, glycerol palmitostearate, ethylcellulose, acrylic resins, dl-polylactic acid, cellulose acetate butyrate, polyvinyl chloride, polyvinyl acetate, vinyl pyrrolidone, polyethylene, polymethacrylate, methylmethacrylate, 2-hydroxymethacrylate, methacrylate hydrogels, 1,3 butylene glycol, ethylene glycol methacrylate, and/or polyethylene glycols. In a controlled release matrix formulation, the matrix material may also include, e.g., hydrated metylcellulose, carnauba wax and stearyl alcohol, carbopol 934, silicone, glyceryl tristearate, methyl acrylate-methyl methacrylate, polyvinyl chloride, polyethylene, and/or halogenated fluorocarbon.

A controlled release composition containing one or more therapeutic compounds may also be in the form of a buoyant tablet or capsule (i.e., a tablet or capsule that, upon oral administration, floats on top of the gastric content for a certain period of time). A buoyant tablet formulation of the compound(s) can be prepared by granulating a mixture of the compound(s) with excipients and 20-75% w/w of hydrocolloids, such as

hydroxyethylcellulose, hydroxypropylcellulose, or hydroxypropylmethylcellulose. The obtained granules can then be compressed into tablets. On contact with the gastric juice, the tablet forms a substantially water-impermeable gel barrier around its surface. This gel barrier takes part in maintaining a density of less than one, thereby allowing the tablet to remain buoyant in the gastric juice.

Combination Therapies

In one embodiment, a having MMP-13 inhibitory activity, such as CL-82198 and DB04760, may be administered in combination with any conventional chemotherapeutic agent, such as paclitaxel for use in treating or preventing peripheral neuropathy(e.g., paclitaxel-induced peripheral neuropathy, diabetic neuropathy) and wound healing; such methods are known to the an artisan of ordinary skill and described in Remington's

Pharmaceutical Sciences by E. W. Martin. If desired, agents of the invention are

administered in combination with other conventional anti-neoplastic therapy, including but not limited to, surgery, radiation therapy, or chemotherapy. Conventional chemotherapeutic agents include, but are not limited to, alemtuzumab, altretamine, aminoglutethimide, amsacrine, anastrozole, azacitidine, bleomycin, bicalutamide, busulfan, capecitabine, carboplatin, carmustine, celecoxib, chlorambucil, 2-chlorodeoxyadenosine, cisplatin, colchicine, cyclophosphamide, cytarabine, Cytoxan, dacarbazine, dactinomycin,

daunorubicin, docetaxel, doxorubicin, epirubicin, estramustine phosphate, etodolac, etoposide, exemestane, floxuridine, fludarabine, 5-fluorouracil, flutamide, formestane, gemcitabine, gentuzumab, goserelin, hexamethylmelamine, hydroxyurea, hypericin, ifosfamide, imatinib, interferon, irinotecan, letrozole, leuporelin, lomustine,

mechlorethamine, melphalen, mercaptopurine, 6-mercaptopurine, methotrexate, mitomycin, mitotane, mitoxantrone, nilutamide, nocodazole, paclitaxel, pentostatin, procarbazine, raltitrexed, rituximab, rofecoxib, streptozocin, tamoxifen, temozolomide, teniposide, 6- thioguanine, topotecan, toremofine, trastuzumab, vinblastine, vincristine, vindesine, and vinorelbine. In one embodiment, the therapeutic agent is paclitaxel. In one embodiment, an MMP-13 inhibitor is administered before, during, or after administration of paclitaxel.

Kits or Pharmaceutical Systems

The present compositions may be assembled into kits or pharmaceutical systems for use in ameliorating a neuropathy. Kits or pharmaceutical systems according to this aspect of the invention comprise a carrier means, such as a box, carton, tube or the like, having in close confinement therein one or more container means, such as vials, tubes, ampoules, bottles and the like. The kits or pharmaceutical systems of the invention may also comprise associated instructions for using the agents of the invention. Kits of the invention include an MMP-13 inhibitor. If desired, the kit also includes a chemotherapeutic that has neurotoxic activity (e.g., paclitaxel. Optionally, the kit includes instructions for administering the MMP-13 inhibitor and paclitaxel, thereby ameliorating the neurotoxic activity of the chemotherapeutic agent administered alone.

The practice of the present invention employs, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are well within the purview of the an artisan of ordinary skill. Such techniques are explained fully in the literature, such as, "Molecular Cloning: A Laboratory Manual", second edition (Sambrook, 1989);

"Oligonucleotide Synthesis" (Gait, 1984); "Animal Cell Culture" (Freshney, 1987);

"Methods in Enzymology" "Handbook of Experimental Immunology" (Weir, 1996); "Gene Transfer Vectors for Mammalian Cells" (Miller and Calos, 1987); "Current Protocols in Molecular Biology" (Ausubel, 1987); "PCR: The Polymerase Chain Reaction", (Mullis, 1994); "Current Protocols in Immunology" (Coligan, 1991). These techniques are applicable to the production of the polynucleotides and polypeptides of the invention, and, as such, may be considered in making and practicing the invention. Particularly useful techniques for particular embodiments will be discussed in the sections that follow.

The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the assay, screening, and therapeutic methods of the invention, and are not intended to limit the scope of what the inventors regard as their invention.

EXAMPLES

Example 1: Paclitaxel induces neurotoxicity in the zebrafish caudal fin

To induce peripheral neuropathy in adult zebrafish, ΙΟμΜ paclitaxel in DMSO was administered by intraperitoneal injections on four consecutive days. This concentration is based on studies in 1) various mammalian species showing a Cmax of ΙΟμΜ after paclitaxel infusion, and 2) humans in which up to 6μΜ paclitaxel bound to plasma proteins. Since in mammals, paclitaxel preferentially affects the distal extremities, the equivalent distal caudal fin in zebrafish was analyzed. Immunofluorescence staining using anti-acetylated-tubulin (FIGs. IB, 1C) and Neurofilament 160 antibodies revealed a selective loss of fine cutaneous axons and those projecting along the bony rays within the distal but not proximal fin regions when analyzed one day after the last injection (Day 5). The distal fin is primarily innervated by axons of dorsal root ganglion (DRG) neurons, whereas motor axons innervate the muscle within the proximal fin. Distal nerves projecting along the bony rays harbor lateral line besides DRG axons, which innervate the neuromasts located along bony rays. Since primarily fine cutaneous axons were lost and axon bundle diameters at the bony rays were reduced but not absent in regions in which the skin was denervated (FIG. IB), in conclusion, the data indicated paclitaxel treatment primarily affects DRG axons.

Paclitaxel neurotoxicity manifests as numbness, paresthesia, and hyperalgesia. Since no established protocols are available to readily assess pain in zebrafish, further the touch response was examined as indicator of neuropathy to corroborate the findings (FIG. 1 A). Daily touch response assays prior to injections showed that the number of stimuli given to the distal caudal fin until a response was evoked was significantly reduced in paclitaxel-treated animals compared with controls. Some, but not all, animals responded similar to controls after a 10-day recovery period (Day 14) (FIG. ID). Next the effects of paclitaxel on locomotor activity were determined since prolonged treatment and high dosage has been found to induce motor deficits. By utilizing an automated tracking device, the swimming distances of zebrafish were measured over a 1 hour period daily. This did not show significant differences between groups, neither during nor after paclitaxel treatment (FIG. IE). Collectively, these findings indicate that adult zebrafish develop signs of paclitaxel neurotoxicity that specifically affects DRG sensory neurons in the distal caudal fin. To obtain a higher temporal resolution of axonal changes in the presence of paclitaxel, axon degeneration in zebrafish larvae was investigated using in vivo imaging. In larval fish (2- 6 days post fertilization, dpf), the skin consists of two layers: the superficial periderm of ectodermal origin and the epidermal basal cell layer. The epidermis is separated by a basement membrane from the underlying rudimentary dermis. DRG neurons are not yet functional, and the epidermis is innervated by unmyelinated Rohon-beard (RB) neurons, which are molecularly and functionally similar to trigeminal sensory neurons and DRGs. To induce neuropathy in RB neurons, two approaches were used, either incubated larvae starting at 2 days post fertilization (dpf) up to 96 hrs in 22uM paclitaxel, which was determined to be most effective (FIG. 2A), or alternatively microinjection of larval fish with ΙΟμΜ paclitaxel into the cardinal vein once daily on three consecutive days (FIG. 2H). The axon branch density was assessed in transgenic Tg(/s72£:GFP) larvae with fluorescently labeled RB neurons (FIG. 2B). Incubated larvae had a slightly decreased caudal fin diameter, but apoptosis was not increased, suggesting some degree of developmental growth restriction due to paclitaxel treatment. Similar to adults, paclitaxel incubation induced axon degeneration, which was most significant following 96hrs of treatment (FIGs. 2C-2E). Also the touch response (but not locomotor activity, FIG. 2G) was significantly reduced (FIG. 2F).

Microinjections similarly induced axon degeneration (FIG. 21), and loss of touch sensitivity (FIG. 2J). Both were prominent after the last injection and rapidly recovered thereafter. At 1 ldpf, both control and paclitaxel-injected larvae harbored fewer axon branches, likely due to the onset of programmed RB neuron death during which DRG neurons become functional. Collectively, these findings demonstrate that paclitaxel induces axon degeneration and loss of touch sensation in the caudal fin of larval zebrafish without affecting motor activity. Example 2: Paclitaxel damages the fin epithelium prior to onset of axon

degeneration

The data indicated that the caudal fin-fold of paclitaxel-injected larvae displayed altered morphologies visible as early as lhr after injection (FIGs. 3A-3C). Caudal fins had a disheveled appearance and were prone to injury under mechanical stress conditions imposed by gentle pipetting (FIG. 3D). When analyzed with scanning electron microscopy, microtearing of the skin in the distal caudal fin was frequently observed following 3hrs of paclitaxel treatment ( FIGs. 3E, 3F), suggesting that the skin is fragile. The skin phenotype worsened in larvae treated with paclitaxel for 96hrs. Keratinocytes spanning both layers delaminated from the fin, thereby exposing collagen-rich actinotrichia present within the mesenchyme beneath the skin (FIG. 6H, panel FT). Paclitaxel dependent morphological changes were also evident in the adult fin, where the skin appeared disorganized and cells were more rounded compared with an elongated shape in vehicle controls. These findings indicate that paclitaxel damages the zebrafish skin epithelium, which is exacerbated under mechanical stress conditions. To further investigate the role of mechanical stress in epithelial damage, the formation of reactive oxygen species (ROS) was assessed with the H₂O₂- selective sensor pentafluorobenzenesulfonyl-fluorescein. Three-hour paclitaxel treatment in combination with mechanical stress led to more widespread ROS/ H₂0₂ formation compared with control animals (FIG. 3G). Intriguingly, adjacent wounds remained devoid of ROS/ H₂O₂, suggesting that stress-related ROS formation may be regulated by different

mechanisms than ROS induced by injury. Next,the effects of paclitaxel on the F-κΒ stress response pathway was examined in a transgenic Tg(NF-KB:EGFP) reporter strain, in which F-KB is activated in keratinocytes. The data indicated that increased F-κΒ activity was present under both unstressed and mechanically stressed conditions in paclitaxel-treated larvae but not in the controls (FIGs. 3H-3I). Since F-κΒ is known to be regulated by H₂O₂, the relationship between F-κΒ activity and ROS/ H₂0₂formation was also assessed (Figure 31). Both the superoxide scavenger diphenyleneiodonium (DPI) and apocynin, a bona fide NOX inhibitor, attenuated F-κΒ activity, suggesting that NF-κΒ activation in keratinocytes is in part mediated by paclitaxel-induced oxidative stress. The rapid phenotypic changes in the fin-fold epithelium observed upon brief paclitaxel treatment suggested that the epithelium might either be susceptible to paclitaxel-induced damage or accumulate higher doses. To test this, paclitaxel accumulation in the fin epithelium and axons was tracked over time using tubulin tracker injections. Tubulin tracker is fluorescently activated after cleavage by intracellular esterases. This probe selectively binds to microtubules with high affinity

(Kd 10-7). The fluorescence increase was monitored in both the caudal fin epithelium and fluorescently labeled RB neurons of transgenic Tg(CREST3 :tdTomato) larvae using 12-hour time-lapse confocal imaging (FIG 3 J-3L). Quantification of normalized fluorescence intensity showed a transient increase of tubulin tracker fluorescence in the caudal fin within 3hrs (FIG. 3 J, 3K). Paclitaxel accumulation in some RB neurons, in contrast, peaked between 5-8hrs (FIG. 3L). To further determine the cell type of tubulin tracker accumulation in the fin, colocalization studies were performed in transiently injected animals in which RB neurons and axons innervating the caudal fin mosaically express tdTomato, and in a Tg(?p<53:dsRed) transgenic strain that was generated in which basal keratinocytes are fluorescently labeled. Axonal tubulin tracker was not detected up to 12hrs following injections (FIG. 3M), but co- localization with basal keratinocytes was present within lhr (FIG. 3N). Interestingly, suprabasal cells of the enveloping layer remained devoid of fluorescence and only epidermal basal cells showed tubulin tracker accumulation. Thus these findings indicate that basal keratinocytes are highly susceptible to paclitaxel accumulation.

Example 3: Paclitaxel impairs cutaneous axon regeneration

It was previously demonstrated that epithelial keratinocytes stimulate injury-induced cutaneous axon regeneration through release of H₂0₂ into the wound environment. Since paclitaxel rapidly damages the skin epithelium, it was hypothesized that it might also impair axon regeneration by perturbing keratinocyte function. Single-labeled RB axons were tracked following caudal fin amputation in 12hr time-lapse movies following 3hr pre- treatment with either vehicle or paclitaxel and continuous incubation (FIGs. 4A-4C).

Paclitaxel significantly impaired axon regeneration compared with control larvae. The growth cone core domain of regenerating axons is rich in microtubules that undergo a process of dynamic instability, which due to continuous polymerization and depolymerization permits shrinkage and growth of nerve endings. This growth and retraction behavior is also characteristic for cutaneous axons of RB neurons. To assess the effects of paclitaxel on microtubule dynamics, the average growth and retraction distance was measured over the course of 12hrs, showing a reduction in paclitaxel incubated animals (FIG. 4D). These findings demonstrate that growth cone dynamics are altered by paclitaxel treatment but not abolished. Example 4: Paclitaxel delays axon debris clearance

Zebrafish cutaneous axons that are severed by laser axotomy degenerate by Wallerian degeneration. The process of Wallerian degeneration is defined by a lag phase during which the severed axons remain intact, a fragmentation phase during which severed branches fragment, and a clearance phase during which axon debris is phagocytosed. Keratinocytes in both Drosophila and zebrafish have been shown to act as non-professional phagocytes and are the sole source for clearing axon debris. To assess the effects of paclitaxel on keratinocyte function, their ability to clear axon debris was examined following 3hr incubations. The duration between fragmentation onset and clearance of the last fragment was measured for individual axon branches, which showed that the mean time of clearance in control animals was 50 minutes (FIGs. 4A, 4E). Paclitaxel-treated larvae needed significantly longer and were moreover inefficient in clearing the debris (FIGs. 4B, 4E). These finding demonstrate that paclitaxel impairs axon debris clearance, but not the onset of Wallerian degeneration, pointing to dysfunctional keratinocytes.

Example 5: Axon regeneration is rescued upon MMP-13 inhibition

The aqueous environment in which zebrafish live provides an opportunity for pharmacological screening. This feature was exploited to screen for chemical compounds that can restore impaired axon regeneration and debris clearance in paclitaxel-treated larval zebrafish. Compounds were pre-selected for targeting genes that were differentially regulated in H₂O₂ treated larval zebrafish in which a whole transcriptome sequencing analysis was performed (FIG. 4F). Each compound was co-administered with paclitaxel 3hrs prior to amputation and larvae remained in the drugs for 12hrs during time-lapse imaging. This identified one compound, CL-82198 (10[xM)(FIG 4G) for restoring axon regeneration (FIG. 4C). CL-82198 is a MMP-13 inhibitor and displays no activity against MMP-1 or MMP-9. To confirm MMP-13 as a target, another selective, non-zinc-chelating MMP-13 inhibitor, DB04760 was further tested (FIG. 4G), which also significantly rescued axon regeneration (FIG. 4C). MMP-13 inhibition with either CL-82198 or DB04760 in paclitaxel-treated larvae also restored axon debris clearance to control levels (FIGs. 4E, 4H). These findings demonstrate that MMP-13 inhibition rescues paclitaxel-induced neurotoxicity in larval zebrafish. Next MMP-13 inhibition was assessed to determine whether MMP-13 prevents or delays axon degeneration by analyzing axon branch density and touch response following a 96hr incubation period. Intriguingly, both inhibitors when co-administered with paclitaxel prevented axon degeneration in the caudal fin (Figure 41). Each inhibitor also restored the touch response, with DB04760 being more efficient than CL-82198 (FIG. 4J). Prolonged CL- 82198 coadministration showed adverse effects since the touch response in the head region was diminished compared with the other groups (FIG. 4J). CL-82198 or DB04760 administered alone however did not cause any adverse effects. It is noteworthy that only -30% of animals treated with paclitaxel+CL-82198 were unresponsive, as compared to -50% with paclitaxel alone (FIG. 4K), suggesting that a subset of animals still benefited from this compound. Also adult fish benefited from CL-82198, which rescued axon branch number and touch response (FIGs. 1A-1D). Long-term MMP-13 inhibition with DB04760 similarly showed complete rescue (FIGs. 8C, 8D). Thus MMP-13 inhibition prevents neurotoxicity associated with paclitaxel treatment in larval and adult zebrafish.

Example 6: Paclitaxel induces ectopic MMP-13 expression

MMP-13 is expressed at relatively low levels in the uninjured skin epithelium but is upregulated in response to acute tissue injury, where it is important for proper wound repair. On the contrary, increased MMP-13 activity in uninjured tissues can promote injury and cancer metastasis, suggesting that precisely controlled levels are essential for tissue homeostasis. It was hypothesized that paclitaxel induces ectopic MMP-13 expression within the skin, which would be consistent with the beneficial effects when inhibited. To test this, the mRNA expression levels of the zebrafish MMP-13 homolog, mmpl3a, were determined with quantitative PCR following 3hr paclitaxel incubation. This showed elevated transcript levels in uninjured paclitaxel-treated larvae as compared with uninjured vehicle controls, which were enhanced upon amputation (FIG. 5 A). MMP-13 activity was further examined in western blots. MMP-13 exists as uncleaved (pro-enzyme) and cleaved active forms. Various isoforms of MMP-13 were reported, including 35, 48, and 54kDa for the active enzyme, and 60 and 80kDa for the pro-enzyme. This variability could relate to species, age or tissue type. Western analysis using 2dpf zebrafish extracts following 3hr treatment revealed expected 48 and 54kDa bands for the cleaved isoforms and an 80kDa band for the pro-enzyme (FIG. 5B). Quantifications further revealed that the intermediate 54kDa but not 48kDa cleaved MMP-13 isoform was more abundant in uninjured and injured paclitaxel-treated larvae as compared with the respective vehicle control group (FIGs. 5C, 5D). Given the preferential accumulation of tubulin tracker in basal keratinocytes, it was hypothesized that MMP-13 is upregulated in this cell type to modulate axon function. To test this, a whole-mount immunofluorescence staining was utilized to determine the precise MMP-13 expression domains. In mice, MMP- 13 has been detected in dermal fibroblasts of skin wounds and in the leading edge of migratory epithelial cells following corneal injury . In zebrafish embryos, MMP-13 was detected after caudal fin amputation , consistent with the studies showing MMP-13 immunofluorescence at the wound edge in amputated 3dpf zebrafish larvae (FIG. 5E, panel E"). In contrast, 3hr paclitaxel incubation induced ubiquitous MMP-13 expression within the epidermis (FIG. 5E, panels F-F", G, H), but not in cutaneous axons (FIG. 5E, panelsl, J-K). Intriguingly, similar to tubulin tracker accumulation in basal keratinocytes, also MMP-13 expression was localized to the deep, basal keratinocyte layer (FIG.5E, panels L-L"). MMP- 13 expression was also examined in adult fish, which was confined to regions in the skin adjacent to, but not within, cutaneous sensory axons (FIG. 8E). Collectively, these findings suggest that paclitaxel stimulates MMP-13 expression in epidermal keratinocytes but not axons. MMP-13 impairs epithelial barrier function in paclitaxeltreated larvae Increased MMP-13 activity has been linked to defects in epithelial barrier function, such as in the gut epithelium where it destabilizes intercellular tight junctions (TJs), thereby promoting pathogenic inflammation after LPS challenge. Experiements were conducted to determine whether MMP-13 upregulation is also associated with decreased skin barrier function. For this, transgenic Tg(krt4:HyPer) zebrafish larvae expressing the ratiometric genetic H₂0₂ sensor HyPer were examined in keratinocytes. The submicromolar affinity of HyPer for hydrogen peroxide and its insensitivity to other ROS permits the detection of fast changes in H₂0₂ concentrations under various physiological and pathological conditions. It was expected that diffusion of low-level exogenous H₂0₂ (0.01%) into intact skin, which has been previously shown to stimulate cutaneous axon regeneration , is minimal whereas a defective barrier increases H₂0₂ influx and thus Hy- Per oxidation ratios

(oxidized(488nm):unoxidized(405nm)). As expected, the mean HyPer ratio was 1.3-fold upon addition of low-level H₂0₂ to vehicle control larvae (FIGs. 6A, 6D), whereas larvae pre-treated with paclitaxel for 3hrs displayed significantly increased ratios of 1.6-fold (FIGs. 6B, 6D). To further assess whether ectopic MMP-13 activity might underlie these barrier defects CL-82198 was co-administered, which decreased HyPer oxidation ratios to levels below those observed with DMSO vehicle (FIGs. 6C, 6D). Interestingly, CL-82198 administration alone further decreased HyPer oxidation, indicating that either DMSO induces some degree of MMP-13 activity or that low-level MMP- 13 activity is necessary under basal conditions to maintain optimal skin barrier function.

To assess MMP-13 functions in skin homeostasis, mmpl3a was cloned and injected mPvNA into 1-cell stage embryos. In addition, a non-functional, truncated control variant (mmpl3aA373) was generated in which the zinc-binding domain (active site) and hemopexin- like repeats are deleted (FIG. 9A). Applying mechanical stress to mmpl3a overexpressing larvae at developmental stage 2dpf promoted rupturing of the yolk and fins, whereas larvae expressing the deletion variant were largely unaffected (FIG. 6E). To further assess, if besides rescuing neurotoxicity MMP-13 inhibition also rescues paclitaxel-induced skin phenotypes, scanning electron microscopy was performed on larvae co-treated with paclitaxel and either CL82918 or DB04760 (FIGs. 6G-6H", FIG. 9B). Both inhibitors improved the skin defects observed with paclitaxel alone. Finally, the skin resistance to mechanical stress was analyzed in larvae co-treated with these inhibitors, which also showed some

improvement (FIG. 6F). Taken together, these findings implicate MMP-13 in paclitaxel - induced skin damage.

Example 7: Increased MMP-13 activity impairs wound repair

Given the role of paclitaxel and MMP-13 in keratinocyte damage, their role in wound repair was examined, since basal keratinocytes have a critical role in this process. Moreover, evidence shows that implantation of paclitaxel-eluting stents following coronary lesion leads to dose-dependent incomplete arterial healing (48). Twelve hour time-lapse movies were recorded of puncture-wounded caudal fins in transgenic Tg(?p<53:CAAXGFP) zebrafish larvae in which the plasma membrane of basal keratinocytes was fluorescently labeled.

Punctured vehicle control animals showed a rapid healing response, marked by a slight increase in wound diameter within the first 2hrs, followed by wound closure around 5hrs post-wounding (FIGs. 7A, 7C). Despite a similar initial wound size in paclitaxel-treated larvae, the wound diameter continuously increased and wounds failed to close (FIGs. 7B, 7C). Co-administration of CL-82198 prevented the increase in wound size seen in larvae treated with paclitaxel alone (FIG. 7C), whereas DB04760 nearly restored wound healing to vehicle controls (FIG. 7C). These findings show that increased MMP-13 activity induced by paclitaxel underlies wound healing defects, which can be rescued upon MMP-13 inhibition. To further examine cell type-specific effects, an established in vitro scratch-assay was utilized using the human keratinocyte line, HEK01. This line retains normal epidermotropic responses during the first 24hrs after plating (not shown). Given the injury-specific production of H₂O₂, which is absent in paclitaxel treated larvae, H₂0₂production in scratched keratinocytes was assessed. In controls, the formation of H₂O₂ along the scratch margin was observed within 20min (FIGs. 7D, 7G), and ROS/ H₂0₂ remained present until scratch wound closure was completed (FIGs. 7D, 7E). In contrast, paclitaxel-treated keratinocytes showed a dose-dependent reduction in ROS/ H₂0₂levels at the scratch margin (FIGs. 7D', 7D", 7G), which recovered after 2hrs (not shown). At 12hrs, control gaps were nearly closed and few cells produced ROS/ H₂0₂, while gaps in paclitaxel-treated wells remained large despite that many cells produced ROS/ H₂O₂ (FIGs. 7E, 7E', 7H). By 24hrs, gaps were no longer visible in the control wells whereas paclitaxel-treated gaps had closed by 40% (FIG. 7F, panels F and F', FIG. 7H), suggesting that paclitaxel delays H₂0₂/ROS formation and impairs healing in a keratinocyte-specific manner.

To determine the role of MMP-13 in scratch wound repair, HEKOl cells were treated with paclitaxel and either CL-82198 or DB04670 for 30 minutes prior to scratching. This showed a dose-dependent partial improvement in the migration toward gap closure (FIGs. 71, 7J), indicating that impaired scratch healing is in part mediated by keratinocyte-specific MMP-13 activity. Interestingly, inhibition of MMP-13 in wildtype keratinocytes significantly enhanced scratch repair compared with vehicle controls. To analyze whether gap closure defects in paclitaxel -treated HEK01 cells are mediated by cytoskeletal defects, scratch margin cells were monitored over time (FIG. 7K). While migratory control cells formed lamellipodia at the leading edges, indicating migration, lamellipodia were absent in paclitaxel-treated HEKOl cells. Coadministration of DB04760 (or CL-82198, not shown) in contrast restored lamellipodia formation and migration, as did DB04760 treatment alone. These findings indicate that increased MMP- 13 activity induced by paclitaxel impairs keratinocyte migration however, without affecting cytoskeletal functions.

Without wishing to be bound by theory, these findings demonstrate a role for keratinocytes in paclitaxel-induced peripheral neuropathy in zebrafish. The data indicatesthat keratinocytes are highly susceptible to paclitaxel-induced damage prior to visible axon defects. This finding is intriguing since axon degeneration in rodent models is initially evident in the epidermis. The role of the epidermis in paclitaxel neurotoxicity is further supported by the specific upregulation of MMP-13 in the skin, but not in axons, which upon pharmacological inhibition prevents neurotoxicity (FIG. 7L). Despite this evidence it is possible that paclitaxel also directly damages axons, given that RB neurons accumulated tubulin tracker and because of the decreased growth cone dynamics after paclitaxel incubations. The latter could however also be attributed to repulsive actions of paclitaxel- damaged keratinocytes that may induce growth cone retraction. Intra-axonal damage may instead depend on prolonged treatment or higher doses. For example, studies in rats demonstrated that paclitaxel, when administered over four cumulative doses at 2 mg/kg, induced terminal arbor degeneration in only the intra-epidermal DRG axons, whereas higher doses (>8 mg/kg) induced distinct phenotypes, such as peripheral nerve-specific degeneration and neuronal death.

The finding that perturbations of skin homeostasis induce neurotoxicity is intriguing given that human patients undergoing chemotherapy with paclitaxel also develop various skin phenotypes and wound-healing deficits. However, data that correlate paclitaxel-induced skin defects in humans with the incidence of neuropathy is not available. The data described herein established a direct link between paclitaxel-induced skin damage in zebrafish and neurotoxicity through MMP- 13 upregulation. MMP- 13 is a collagenase that belongs to the MMP family of zinc-dependent neutral endopeptidases, which are important matrix degrading enzymes. While MMP- 13 has been primarily implicated in collagen degradation, it has also been associated with a role in cellular signaling. In the intestinal epithelium during sepsis and in inflammatory bowel disease, MMP-13 promotes LPS-induced goblet cell depletion, endoplasmic reticulum stress, permeability and tight junction destabilization through its role as TNF sheddase by cleaving pro-TNF into its bioactive form. Thus, MMP- 13 might directly regulate junction disassembly in keratinocytes, consistent with the reduced skin resistance to mechanical stress in paclitaxel-treated larval zebrafish. Alternatively, excessive collagen degradation could alter the mechanical properties of the skin, given the collagen-rich network within the ECM, which is essential to maintain tissue integrity. Since the glabrous skin is frequently exposed to biomechanical stresses, axons in this region may be more susceptible to mechanical stress-induced damage compared with other body regions. Nociceptors and small diameter mechanoreceptors have been shown to be modulated in hairy skin by mechanical stress through binding of collagen to integrins alpha 2 and beta 1. Parallel mechanisms in glabrous skin may exist, and disruptions pertaining to excessive MMP-13 mediated collagen degradation may promote axon degeneration. Without wishing to be bound by theory, it is possible that MMP-13 accumulates within the ECM due to reduced protein turnover, potentially due to altered microtubule associated protein transport within keratinocytes. It is further possible that microtubule stabilization alters signaling cascades within keratinocytes, thereby leading to increased mmpl3a expression. The link between mechanical stress and MMP-13 upregulation could be provided by ROS. First, increased ROS/ H₂0₂ formation in paclitaxel-treated, mechanically stressed zebrafish larvae was observed. Second, mechanical stress in cardiomyocytes and skeletal myofibers triggers Nox-2 dependent "X-ROS" formation and signaling in a microtubule-dependent manner. Moreover, X-ROS production is exacerbated in skeletal muscle of mice with Duchenne Muscular Atrophy due to enhanced microtubule stiffness . Third, the RNAseq data (GEO Accession number GSE75728, submitted) shows that H₂0₂ stimulates MMP- 13 expression, which is also evident after injury upon which H₂0₂ is produced (FIG. 5E, panel F'). While injury stimulates some degree of MMP-13 expression, the data suggest that paclitaxel further increases MMP-13 levels, leading to excessive tissue damage and wound healing defects. Delayed wound healing has also been reported in paclitaxel-treated patients. In the studies, impaired wound healing could have been caused by a number of factors, including paclitaxel- induced cytoskeletal stiffness in basal keratinocytes, which have been shown to contribute to wound repair. Alternatively, paclitaxel could induce changes in cellular signaling or ECM matrix composition. The latter is more likely given that MMP-13 inhibition rescued wound repair, implying that the cytoskeleton must be relatively intact. The short incubation time in paclitaxel or the concentrations used in the studies may have marginally stabilized cytoskeletal functions and thereby increased MMP-13 activity. Further studies are required to address this question.

MMP-13 serves as a new candidate target for the treatment of paclitaxel-induced peripheral neuropathy given that its inhibition prevented neurotoxicity, even after prolonged treatment. A number of MMP inhibitors are currently being developed as anticancer agents and the MMP-13 selective inhibitor, CL-82198, has also proven beneficial in decreasing cancer metastasis. Paradoxically, recent data showed that also paclitaxel promotes metastasis in TLR4-positive tumors. Therefore, inhibitors targeting MMP-13 may provide additional benefits when coadministered with paclitaxel. While MMP-13 inhibitors have not been directly tested in mammalian models for paclitaxel-induced peripheral neuropathy, general MMP inhibition using the potent MMP inhibitor, tetracycline-3, showed favorable effects in the treatment of paclitaxel-induced hyperalgesia in mice. A role for MMPs in paclitaxel- induced neuropathy is further supported by studies in rat models of neuropathic pain where paclitaxel induced MMP-3 expression in DRG neurons. MMP activity is determined by binding of a zinc ion to the active site. The first generation of MMP inhibitors was designed to chelate the zinc ion, thereby preventing enzymatic activity. Because of the low selectivity of these inhibitors due to sequence conservation within the active site, more selective MMP inhibitors were subsequently developed. CL-82198 belongs to the class of highly selective, non-zinc-chelating compounds and was shown to exhibit weak inhibition of MMP-13 (89% at lC^g/ml) without activity against MMP-1, 9 and TACE. This inhibitor binds to the large SI ' binding pocket without apparent interactions between the inhibitor and the catalytic zinc binding domain, justifying its micromolar potency. The weak binding may be favorable in the studies to reduce, but not abolish, MMP-13 expression to similar levels as seen in control animals ( FIGs. 5A, 5B, FIG. 5E, panel J). Also DB04760, a pyrimidine dicarboxamide inhibitor, belongs the class of non-zinc-chelating, SI ' pocket binding compounds and therefore may have exhibited similar effects as CL-82198. Collectively, the data obtained from the investigation points to MMP-13 dependent keratinocyte damage as the underlying cause of paclitaxel toxicity, which can be alleviated upon MMP-13 inhibition. Given the implications of MMP-13 in peripheral neuropathy, cancer and in a variety of other diseases, these compounds may provide benefits for the treatment of multiple conditions.

Example 8:Treatment with paclitaxel results in significant skin composition changes.

Transmission electron microscopy of zebrafish larvae treated for 4 consecutive days either with DMSO vehicle, paclitaxel, paclitaxel and DB04760 or paclitaxel and CL-82198 shows the presence of significant changes in skin composition in animals treated with paclitaxel. Unlike in DMSO vehicle-treated control animals, large gaps have formed between the outer periderm and inner basal cell layer (FIG. 10, panels B and B"). Mitochondrial cisternae are difficult to distinguish (FIG. 10, panel B', arrows) and the basement membrane is partially degraded evident by the lack of electron dense structures at the interface between the basal layer and mesenchyme (FIG. 10, panel B', white arrowheads). Also tight junctions are less strong (black arrowhead in FIG. 10, panel B") and other junctions such as

desmosomes connecting periderm and basal layer or largely absent or weak. While sensory axon degeneration is typical at this stage in paclitaxel-treated animals, those sensory axons that remained intact are enlarged and no longer tightly embedded into the skin. Instead they occupy large gaps between the two cell layers (black arrow in FIG. 10, panel B"). Treatment with either one of the MMP-13 inhibitors (DB04760 and CL-82198) ameliorates these changes and normalizes the skin structures.

FIGs 10-14 provide a series of electron micrographs of skin showing larval zebrafish skin following 4-day treatment with paclitaxel and MMP-13 inhibitors, vehicle-treated control skin (FIG. 10A), and skin treated with paclitaxel and the MMP-13 inhibitor,

DB04760. Organelle structures and basement membrane were significantly improved when DB04760 was co-administered (FIG. IOC). Similar results were observed when skin was treated with paclitaxel and the MMP-13 inhibitor, CL-82198. Organelle structures and basement membrane were also significantly improved when CL-82198 or DB04760 was co- administered.

The results described above were obtained using the following methods and materials. Chemical inhibitors Paclitaxel was kept as 5.8mM stock in DMSO and diluted either to ΙΟμΜ in PBS for microinjections or 22μΜ in Ringers solution for incubations. The MMP-13 inhibitors CL- 82198 hydrochloride (Tocris) and DB04760 (sc-205756, Santa Cruz Biotechnology) were kept as lOmM stock solutions in DMSO stored at -20°C and diluted to ΙΟμΜ prior to use. Control solutions were supplemented with equal volumes of DMSO (vehicle).

Zebrafish drug treatments and microinjections

Microinjections: Three-6nl ΙΟμΜ paclitaxel was injected into the cardinal vein of zebrafish larvae on three consecutive days (2, 3, 4dpf), using a pulled glass capillary.

Incubations: Larvae were incubated for either 3 or 96hrs starting at 2dpf to assess axon degeneration, touch response, MMP-13 expression and tubulin tracker co-localizations. Three dpf larvae were incubated in drugs for axon regeneration and wound healing studies.

Adult injections: Zebrafish 9-12 months of age were injected with 4μ1 injection solution once daily on four consecutive days using a 33 gauge Hamilton syringe.

Mechanical stress and wounding assay: Larvae pre-examined for absence of skin phenotypes were pipetted up and down three times using a glass Pasteur pipette. Skin phenotypes were assessed under a stereomicroscope. Larvae were assessed for injuries, including ruptured tail fins and yolks. For puncture and amputation wounding, larvae were anesthetized and placed sideward onto an agarose-coated plate. Caudal fin amputations were performed with a 23 -gauge syringe needle. Puncture wounds were introduced using a pulled glass capillary needle that created 20-50μπι diameter wounds.

NF-KB studies: 3dpf F-κΒ reporter larvae were pre-incubated in either vehicle, diphenyleneiodonium (DPI, 50μΜ), or apocynin (ΙΟΟμπι) for 2hrs prior to imaging and maintained in the drug during time-lapse recordings. Mechanical stress assays were performed immediately prior to mounting larvae for imaging. ROS detection: ROS were detected in 3dpf larvae using 4μπι pentafluorobenzenesulfonyl-fluorescein. Following incubation for lhr, animals were stressed (sometimes leading to injury), washed three times and immediately imaged on an Olympus FV1000 confocal microscope.

Tubulin tracker injections: ΙΟμΜ Oregon Green 488 bis-acetate (Life Technologies) was injected into the cardinal vein of 2dpf Tg(¾p<53:dsRed) larval fish, or into transiently- injected or transgenic CKES 3:Gal4 5xUAS-tdTomato fish, followed by immediate time- lapse imaging. MMP-13 stress assays: Stress assays were performed at 2dpf either on larvae injected at the one-cell stage with 160pg mmpl3a mRNA, or on wildtype larvae treated for 2hrs with 22μΜ paclitaxel and ΙΟμΜ of each MMP-13 inhibitor.

Membrane staining: Fixed caudal fins of injected adult animals were incubated in ΙμΜ Bodipy®FL C5-Ceramide (Molecular Probes) overnight at 4°C in the dark. Fins were subsequently washed three times in PBS prior to imaging.

Zebrafish imaging Zebrafish larvae were mounted as described in (71). For time-lapse imaging, 10-20 larvae per session were imaged either on a F VI 000 (Olympus) or Zeiss LSM510 confocal microscope with motorized stage for up to 12hrs per session (20x objective, 0.75NA). Stacks were projected into single images and processed in Imaris (Bitplane) or Image J. Movies were assembled in QuickTime Pro 7.

Other Embodiments

From the foregoing description, it will be apparent that variations and modifications may be made to the invention described herein to adopt it to various usages and conditions. Such embodiments are also within the scope of the following claims.

The recitation of a listing of elements in any definition of a variable herein includes definitions of that variable as any single element or combination (or subcombination) of listed elements. The recitation of an embodiment herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.

All patents and publications mentioned in this specification are herein incorporated by reference to the same extent as if each independent patent and publication was specifically and individually indicated to be incorporated by reference.

Claims

What is claimed is:

1. A method of inhibiting toxicity in a cell, the method comprising contacting a cell with an MMP-13 inhibitor in the presence of a toxin, thereby inhibiting toxicity in the cell.

2. The method of claim 1, wherein the cell is a neuron or an epithelial cell.

3. The method of claim 2, wherein the neuron is a sensory neuron, motor neuron, autonomous neuron, and the epithelial cell is a keratinocyte, fibroblast, Langerhans cell, or leukocyte.

4. The method of claim 1, wherein the toxin is associated with cell damage and/or a loss of cell function.

5. A method of inhibiting axonal degeneration in a neuron, the method comprising contacting a cell with an MMP-13 inhibitor in the presence of a chemotherapeutic selected from the group consisting of paclitaxel, cisplatin, carboplatin, and oxaliplatin, docetaxel, cabazitaxel, epothilones, ixabepilone, vinblastine, vincristine, vinorelbine, etoposide, thalidomide, lenalidomide, pomalidomide, bortezomib, carfilzomib, and eribulin, thereby inhibiting axonal degeneration in the neuron.

6. A method of restoring function to a neuron, the method comprising contacting the neuron with an MMP-13 inhibitor, wherein the neuron is in the presence of or was previously contacted with paclitaxel or another neurotoxic agent.

7. The method of any one of claims 1-6, wherein the neuron is in vitro or in vivo.

8. The method of claim 6, wherein the neuronal function is touch sensation, axon debris clearance, or axon regeneration.

9. A method of treating or preventing neuropathy or a symptom thereof in a subject, the method comprising administering to the subject an effective amount of an MMP-13 inhibitor before, during or after administration of paclitaxel or another chemotherapeutic agent having neurotoxic activity.

10. The method of claim 9, wherein the method reduces numbness, paresthesia, and/or hyperalgesia in the subject.

11. The method of claim 9, wherein the method increases sensation in the subject.

12. A method of promoting wound healing in a subject having a peripheral neuropathy, the method comprising administering to the subject an effective amount of an MMP-13 inhibitor.

13. A method of inhibiting MMP-13 activity in a subject being treated with paclitaxel or another chemotherapeutic agent, the method comprising administering to the subject an effective amount of an MMP-13 inhibitor before, during or after administration of paclitaxel or another chemotherapeutic agent having neurotoxic activity.

14. The method of any one of claims 1- 13, wherein the MMP-13 inhibitor is administered at least about 1, 3, 6, 12, 24, 48, or 72 hours prior to or following administration of paclitaxel or another chemotherapeutic agent.

15. The method of any one of claims 1-13, wherein the MMP-13 inhibitor is DB04760 or CL-82198.

16. A combination therapeutic composition comprising an MMP-13 inhibitor and paclitaxel or another chemotherapeutic agent, wherein the MMP-13 inhibitor and the paclitaxel or other chemotherapeutic agent are each present in a separate container or are co-formulated.