WO2013101724A2 - Inhibition of clostridium difficile toxins - Google Patents

Abstract

The present disclosure pertains to compositions and methods related to intracellular inhibition of the virulence factors Toxin A and Toxin B produced by the pathogenic bacterium Clostridium difficile.

Description

INHIBITION OF CLOSTRIDIUM DIFFICILE TOXINS

FIELD OF THE DISCLOSURE

[0001]The subject matter disclosed herein relates to compositions and methods for inhibiting Clostridium difficile toxins and for treating disease related to Clostridium difficile infection.

BACKGROUND OF THE DISCLOSURE

[0002] The bacterium Clostridium difficile (C. difficile) is a clinically important opportunistic pathogen, which causes severe diarrhea and other intestinal diseases when competing gut flora are eliminated, as through antibiotic treatment, and is the most serious cause of antibiotic-associated diarrhea (AAD). C. difficile most frequently affects those in hospitals and nursing homes. For example, while C. difficile is present in about 3% of the healthy adult U.S. population, it occurs in 20-40% of the hospitalized community. C. difficile is the cause of C. difficile associated diarrhea (CDAD), an antibiotic-associated diarrhea. See FIG. 1 (A). Infection with C. difficile is the leading cause of pseudomembranous colitis, which can lead to life-threatening toxic megacolon. CDAD is a growing concern among the nation's health care providers and in nursing homes, due to an increasing frequency and severity of outbreaks of CDAD. A recent statistical analysis of patient outcomes in Pennsylvania, for example, indicated that approximately eighteen patients die each day in that state due to CDAD and complications thereof. The Center for Disease Control has identified C. difficile as one of the most important targets to understand and control over the next ten to twenty years. These statistics combined with an aging population, increased stress on hospital and nursing home care, and the extensive use of broad-spectrum antibiotics, all point toward growth of C. difficile -related infections and associated CDAD disease.

[0003] Current standard therapies for CDAD include antibiotics regimens usually including metronidazole or vancomycin. These are reasonably effective but only if coupled with discontinuation of the primary antibiotic, and unfortunately there is a high relapse rate after completion of the regimen.

[0004] Two current approaches toward mitigating the effects of CDAD include the development of vaccines and novel antibiotics. Efforts to identify appropriate immunogens for a vaccine have been slow, however, and it is unclear when such work might yield an effective vaccine. The effort to identify novel antibiotics is also difficult and the useful lifetime of such therapies before resistant strains are observed is becoming shorter.

[0005] Most of the cellular damage resulting from CDAD is caused by two primary virulence factors, C. difficile Toxins A and B (TcdA and TcdB, respectively; or collectively and together, TcdA B, or simply "toxins"), and the infected individual's immune response to these toxins and their effects. FIG. 1 (B) shows the overall pathway towards cellular damage that follows from C. difficile intoxication. The toxins bind intestinal epithelial cell surface receptors and are translocated into the cells through endocytosis. Once inside the cell, the toxins glucosylate and inactivate the RhoA family of G-proteins. Inactivation of RhoA and its relatives has numerous physiological effects, ultimately resulting in an inflammatory response, diarrhea and cell death. Therefore, interrupting the inactivation of these G-proteins could be effective at combating the effects of C. difficile toxins and CDAD. To date, however, compounds that interrupt the toxins' ability to inactivate RhoA have not been effective in treatment for several reasons, including because the compounds are also toxic to cells, or because they are not transported with the toxins into the cell from the extracellular matrix.

SUMMARY OF THE DISCLOSURE

[0006] The present disclosure provides peptide inhibitors of C. difficile Toxins A and B that are non-toxic and remain associated with the toxin through entry into the cell. The maintained association allows the inhibitor to inhibit the toxin in the environment where it causes damage; that is, intracellularly. The present disclosure achieves this effect by creating peptides that inhibit the toxins and also remain associated with them through their transport into the cell. The present disclosure also relates to methods of identifying and optimizing these peptide inhibitors.

[0007] Some embodiments of the present disclosure relate to a composition comprising the peptide represented by HQSPXHH, using the standard single-letter nomenclature for identifying amino acids, and wherein X is selected from an acrylamide, an acrylate, an acyloxymethyl ketone (AOMK), an O-aryloxycarbonyl hydroxamate, a chloroacetamide, a chloroformate, a chloromethylketone, a diazo-methylketone, an epoxide, a fluoromethylketone, a halomethylketone, a hydroxamate, an isothiocyanate, a ketomethylene, a propynamide, a 2-pyridone, a 2-pyrrolidone, and a pyrrolopyrimidine derivatized amino acid. In particular embodiments, the composition comprises the peptide represented by HQSPGepoxyHH. Some embodiments relate to a composition comprising a peptide selected from the peptides represented by HESPGepoxyHH, HKSPGepoxyHH, HQAPGepoxyHH; HQNPGepoxyHH, HEAPGepoxyHH, HENPGepoxyHH, HKAPGepoxyHH, or HKNPGepoxyHH. In some embodiments, the amino acid represented by Gepoxy derives from allylglycine. In some embodiments, one or more amino acids are added to the C-terminus of the peptide of the composition. In some embodiments, the one or more amino acids comprise the amino acids represented by -GGGC (a -GGGC "tail").

[0008] Certain embodiments of the present disclosure further relate to a composition comprising pharmaceutically acceptable salts, tautomers, isomers and/or prodrugs of one or more of the compositions comprising one or more of the peptides described herein.

[0009] Certain embodiments relate to a therapeutic composition comprising a composition comprising one or more of the peptides described herein.

[0010] Some embodiments of the present disclosure relate to a method of inhibiting TcdA/B, comprising exposing the TcdA B to a composition comprising one or more of the peptides described herein. Some particular embodiments relate to a method of inhibiting TcdA/B in a vertebrate cell, comprising administering to the cell a composition comprising one or more of the peptides described herein.

[0011] Some embodiments relate to a method of identifying compounds that inhibit TcdA/B intracellularly, comprising: selecting one or more peptides for analysis; derivatizing the peptides in silico with an electrophilic group at various amino acid sites within each peptide to create a set of in silico derivatized peptides, and performing docking studies on each peptide thus derivatized to determine a docking score for each derivatized peptide; selecting one or more derivatized peptides based on their docking scores thus obtained; from the derivatized peptides that were selected based on docking scores, further selecting a final set of derivatized peptides for testing where the electrophilic group in the derivatized peptide is susceptible to reactivity with potential nucleophilic groups in the active site of the toxin; and, testing the final set of peptides for inhibition of toxin in cellulo.

[0012] Some embodiments relate to a method of treating infection by C. difficile in a subject, comprising administering to the subject a composition comprising one or more of the peptides described herein.

BRIEF DESCRIPTION OF THE FIGURES

[0013] Exemplary embodiments are illustrated in the referenced figures. It is intended that the embodiments and figures disclosed herein are to be considered illustrative rather than restrictive.

[0014] FIG. 1 (A) shows the pathogenesis and etiology of C. difficile infection. FIG. 1 (B) shows the pathway for TcdA B induced cellular pathogenesis including: 1 ) binding to cell surface receptors, 2) endocytosis, 3) endosomal escape, 4) proteolytic release of the catalytic domain into the cytosol and 5) glycosylation of target G-proteins that disrupts control of actin filament assembly/disassembly which induces cellular rounding, loss of adhesion, disruption of gap junctions and apoptosis.

[0015] FIG. 2 shows a cell viability assay indicating cell death as a function of the amount of toxin administered, with cells exposed to toxin for 24, 48, or 72 hours. The x- axis indicates molar concentration of full-length recombinant TcdA. The y-axis indicates cell viability, where 1 .00 = 100% viability and 0 = total cell death.

[0016] FIG. 3 shows images from a cell viability assay illustrating protection of Vero cells from TcdA by epoxy-derivatized peptides. (A) shows PBS (negative control), (B) shows 0.4 nM TcdA alone, and (C) shows 0.4 nM TcdA pre-incubated with 600 μΜ HQSPGepoxyHHGGGC.

[0017] FIG. 4 shows the results of a cell viability assay, indicating % cell protection from TcdA by derivatized peptides HQSPGepoxyHHGGGC and HQSPWHGepoxyGGGC, and native, underivatized peptide HQSPWHHGGGC.

[0018] FIG. 5 shows the results of a cell viability assay, indicating % cell protection from TcdA by substituted but underivatized peptide HQSPWHGallylGGGC, derivatized peptides HQSPWHGepoxyGGGC and HQSPGepoxyHHGGGC, and native, underivatized peptide HQSPWHHGGGC.

DETAILED DESCRIPTION OF THE DISCLOSURE

[0019]The subject matter disclosed herein relates to compositions and methods for inhibiting C. difficile toxins and for treating disease related to C. difficile infection.

[0020] Some embodiments of the present disclosure relate to peptides that are nontoxic and inhibit the toxins while remaining associated with them through transport into the cell, thus retaining their inhibitory effectiveness intracellularly.

[0021]As used herein, "in silico" refers to a process performed by computer or computer simulation, computer modeling or the like, and "in cellulo" refers to an assay or process relating to live cells. As used herein "non-toxic" means, as understood by one of ordinary skill, minimal adverse biological effects within clinically acceptable parameters.

[0022] A high-throughput screening approach was used to identify peptide compounds that bind toxin competitively as related to RhoA and inhibit toxin extracellularly, are suitable for toxin inhibition studies and analog development, and covalently bind toxin so as to be transported with the toxin across cell membranes and thus maintain their inhibitory effects intracellularly. Although the disclosure is not bound by a specific mechanism of action of the compounds in vivo, the compounds are selected for their inhibition of the glucosyltransferase activity of C. difficile Toxins A and B. Compounds and methods disclosed herein function to protect cells by inhibiting toxin activity in cell culture models of C. difficile intoxication.

[0023] The starting set of peptides for identifying intracellular toxin inhibitors can be obtained, for example, experimentally from a set of peptides shown to bind and inhibit TcdA/B extracellularly, or as another example could be derived from peptidomimetic studies as is known in the art. See, e.g., Walensky L.D. et al. 2004, Science 305(5689):1466-1470; and, Li L. et al. 2004, Science 305(5689):1471-1474, both of which are incorporated herein by reference for their teachings regarding the same. In the embodiments described herein, phage display was used to screen for peptides which bind to the active site of the toxins and behave competitively with respect to the biologically relevant RhoA, the target of toxin glucosylation. See Abdeen S., Feig A. et al. 2010, ACS Chem. Biol. 5:1097-1 103 ("Abdeen and Feig"), for methods, which is incorporated herein by reference for its teachings regarding the same. Phage display identified 17 peptides with the tightest toxin binding affinity. From these 17, two peptides were selected for further analysis, EGWHAHT and HQSPWHH, the former based on its observed binding affinity for TcdA, and the latter because it showed the highest affinity in computational docking studies. In vitro inhibition studies were then performed. Both peptides inhibited TcdA glucosyltransferase activity on RhoA in phage-based inhibition studies; both peptides inhibited glucosyltransferase activity of both TcdA and TcdB in peptide-based inhibition studies; and, both peptides inhibited glucosylhydrolase activity of TcdA in peptide-based inhibition studies.

[0024] One of ordinary skill in the art would understand that TcdA results can be extrapolated to TcdB and vice versa because, inter alia, each parent peptide described herein bound and inhibited both toxins in vitro, and it is known that both catalyze glucosyltransferase and glucosylhydrolase activity, the catalytic domains of the two exhibit 74% sequence homology, they have identical substrate specificities, and both target Rho proteins, Rac1 , and Cdc42.

[0025] Cell viability studies were then performed to determine whether the peptides protected cells from the effects of the toxin. Neither of the parent peptides protected cells from the toxin in cell viability studies. Without being bound by theory, it is believed this lack of protection was due to the loss of the peptides from the toxin during transport into the cell. Accordingly HQSPWHH, already demonstrated to bind the toxin active site and inhibit toxin in vitro, was selected for derivatization. Other factors favoring this peptide for selection included computer modeling docking studies and molecular dynamics simulations, as described below, indicated its amino acids surrounding the toxin active site should tolerate modification without affecting toxin binding and inhibition, and would contribute favorably to the binding energy for the toxin-peptide interaction. Docking studies were performed using the crystal structure of the TcdB toxin, as TcdA to date has not been crystallographically resolved.

[0026] By derivatization it is meant that an amino acid residue within the parent peptide is replaced with a non-natural amino acid, such that the newly formed derivatized peptide still binds the toxin active site and inhibits toxin activity, but now also possesses the appropriate chemistry to form a covalent bond with the toxin, thus permitting its transport across the cell membrane with the toxin into the cell. Possible amino acid residues within the peptide for derivatization can be initially selected based on results of computational analyses such as docking studies. In certain embodiments, the peptide is derivatized to create an electrophile, Michael acceptor, or similar mechanism which can facilitate and undergo a nucleophilic "attack" by catalytic residues in the toxin active site, to create a covalent bond between peptide and toxin. In some embodiments, this derivative is an epoxide. Epoxy derivatization was selected for convenience in the Examples that follow, but a variety of types of amino acid derivatives can be used which possess the appropriate chemistry to produce the desired effects and functionality described herein. Examples of some of these derivatives include, without limitation, an acrylamide, an acrylate, an acyloxymethyl ketone (AOMK), an O-aryloxycarbonyl hydroxamate, a chloroacetamide, a chloroformate, a chloromethylketone, a diazo- methylketone, an epoxide (or oxirane), a fluoromethyl ketone, a halomethylketone, a hydroxamate, an isothiocyanate, a ketomethylene, a propynamide, a 2-pyridone, a 2- pyrrolidone, and a pyrrolopyrimidine derivative. See, e.g., Cohen, M.S. et al. 2005, Science 308, 1318-1321 ; Dragovich, P.S. et al. 2002, J. Med. Chem. 45, 1607-1623; Dragovich, P.S. et al. 1999, J. Med. Chem. 42, 1203-1212; Dragovich, P.S. et al. 2002, Bioorg. Med. Chem. Lett. 12, 733-738; Dragovich, P.S. et al. 2003, J. Med. Chem. 46, 4572-4585; Estacio, S.G. et al. 201 1 , J. Chem. Inf. Model. 51 , 1690-1702; Henise, J.C. et al. 201 1 , J. Med. Chem. 54, 4133-4146; Johnson, T.O. et al. 2002, J. Med. Chem. 45, 2016-2023; Kona, J. 2008, Org. Biomol. Chem. 6, 359-365; Matthews, D.A. et al. 1999, Proc. Natl. Acad. Sci. U. S. A. 96, 1 1000-1 1007; Ouertatani-Sakouhi, H. et al. 2009, Biochemistry 48, 9858-9870; Rose, R.B. et al. 1993, Biochemistry 32, 12498-12507; Singh, J. et al. 1997, J. Med. Chem. 40, 1 130-1 135; Tarnowska, M. et al. 1992, Eur. Biophys. J. 21 , 217-222; Totir, M.A. et al. 2007, Biochemistry 46, 8980-8987; Wong, T.W. et al. 201 1 , AAPS PharmSciTech 12, 201 -214; Wouters, J. et al. 2003, Proteins: Struct., Funct., Bioinf. 54, 216-221 ; and, Wyrembak, P.N. et al. 2007, J. Am. Chem. Soc. 129, 9548-9549; Yang, H. et al. 2005, PLoS Biol. 3, 1742-1752, which are incorporated herein by reference for their teachings regarding the same. [0027] In the example of epoxide denvatization, the following process can be applied. First an amino acid can be substituted with an allylglycine (Gallyl) amino acid during synthesis of the peptide, then the allylglycine can be converted to the epoxide (Gepoxy), for example by treatment with p-chloroperbenzoic acid.

Gallyl Gepoxy

[0028] When the epoxy-derivatized peptide is then mixed with the toxin under appropriate conditions, a nucleophilic attack on the epoxide electrophile occurs by residues in the active site of the toxin. The following is a schematic of this chemical reaction, in which Nu can be ROH, R-NH₂ or RCOO^".

[0029] This derivatization procedure is not stereospecific and generates a pair of diastereomers. In the Examples herein these diastereomers were not separated. Some embodiments further comprise stereoselective incorporation of derivatized peptides, using stereoselective methods as are currently available.

[0030] In a docking study, a peptide is placed in the active site of the toxins in a computer model to create a docking score which serves as an estimation of the tightness of bonding (bonding affinity) that could be expected between the peptide and toxin. The docking score can be thought of as akin to a measurement of free energy, but it is a relative not absolute term. See Example 4 for more details. Such docking scores were utilized herein to govern selection of peptides and also to determine which amino acids within the peptides would be the best targets for derivatization. After docking studies were performed, contributions of amino acids to the binding interaction were further assessed by computational alanine scanning (CAS). In this process, a single amino acid in the peptide is replaced with an alanine and the binding energy recalculated. The difference between the original binding energy of the peptide to the toxin and the new alanine-containing binding energy provides the contribution to binding based on that alanine-containing side chain. From these calculations one can select amino acid sites in the peptide for derivatization based on the lowest contribution to binding.

[0031] Docking studies were performed of the underivatized parent peptide HQSPWHH, and the parent peptide derivatized with Gepoxy at various sites. A docking score of greater magnitude than the parent's was considered an advantageous modification, while a docking score lesser than the parent's was considered disadvantageous. See Table 1 , below. Two peptides derivatized at different residues were selected for cell viability assays, both with docking scores of greater magnitude than the parent peptide (parent docking score, -35.58): HQSPGepoxyHH and HQSPWHGepoxy. One reason for selecting the first was because of its exceptional binding affinity as evidenced by the magnitude of its docking score, -70.03. One reason for selecting the second, with a docking score of -41 .29, was because of the number of nearby potentially nucleophilic amino acids in the toxin active site that would be capable of attacking the epoxide. Other reasons for selecting particular derivatives for further analysis include, for example but without limitation, ease of their synthesis, rapidity of trapping or detection chemistry when the peptide is bound to the toxin, peptide stability when not bound to toxin, and whether any toxic byproducts are formed when the peptide degrades or reacts non-specifically with another entity.

[0032] In vitro inhibition properties of the derivatized peptides were tested with assays for glucosyltransferase and glucosylhydrolase activity. The epoxide-derivatized peptides bound to toxin and provided in vitro inhibition comparable to the unmodified peptides, the first generation inhibitors. (Data not shown; for methods see Abdeen and Feig, incorporated herein by reference for its teachings regarding the same.)

[0033] Cell viability assays were then performed using the selected derivatized peptides in conjunction with TcdA/B to establish whether the peptides, when mixed with toxin extracellularly, inhibited toxin activity within cells. As stated above, the first generation peptides without derivatization, although good toxin inhibitors in vitro, failed to provide protection to cells. The derivatized peptide HQSPWHGepoxy did not show any protection of cells against TcdA (nor did its Gallyl-substituted (non-epoxy-derivatized) parent HQSPWHGallyl; FIG. 5). Without being bound by theory, one possible reason for this lack of effect could be that there was no suitable nucleophile in the toxin nearby the derivatized residue to react rapidly enough with and covalently bind the peptide. Additionally, the derivatized amino acid must be properly positioned within the active site of the toxin for covalent binding. The peptide HQSPGpeoxyHH, however, showed increased levels of cell protection in a concentration-dependent manner, with 95% cell protection at 600 μΜ concentration peptide. See FIGS. 5 and 6.

[0034] Note that in synthesizing the peptides a -GGGC tail was added at the C- terminus, and a C-terminal amide modification. This tail was added to facilitate lab handling of the peptides, such as for selection of the peptides in phage display and/or so the C-terminal cysteine could be tagged with, e.g., biotin, fluorophores, etc. Adding the tail did not adversely affect the peptides' ability to bind or inhibit the toxins. See Abdeen and Feig.

[0035] Following is the structure of the parent, underivatized peptide HQSPWHHGGGC:

_{H s} [0036] Following is the structure of the Gallyl-substituted peptide HQSPGallylHHGGGC:

[0037] For the epoxy-derivatized peptide HQSPGepoxyHHGGGC it was necessary to protect the sulfur group of the C-terminal cysteine so it would not react with the epoxide. Accordingly, the following is the structure of the epoxy-derivatized peptide HQSPGepoxyHHGGGC, with the C-terminal cysteine modified:

[0038] The results herein demonstrate that the derivatized peptide HQSPGepoxyHH bound the toxin while still extracellular and was successfully transported with the toxin into cells, where it was able to protect the cells from the toxin. As demonstrated by the Examples herein and stated above, approximately 95% cell survival was achieved by simultaneously administering to cells the toxin plus the peptide HQSPGepoxyHH. The results also indicate that the position of the derivatized amino acid in the peptide inhibitor is crucial for toxin inhibition. Accordingly, one embodiment of the present disclosure relates to a compound represented by HQSPGepoxyHH. Analogs and derivatives of HQSPGepoxyHH are also considered to be within the scope of the present description, such as for example HQSPGepoxyHH containing one or more conservative amino acid substitutions, and/or one or more amino acids add at the C- terminus of the peptide. By a conservative amino acid substitution it is meant an amino acid residue in the peptide is replaced by another amino acid residue which has similar chemical properties. A determination of which amino acids can be more easily replaced by conservative substitutions can be estimated by, for example, the amino acids' Blosum (block substitution matrix) scores. Based on their Blosum62 scores, conservative amino acid substitutions of Q include E and K, and conservative amino acid substitutions of S include A and N. See Lehninger Principles of Biochemistry 4^th ed. 2005 (WH Freeman and Co., New York, NY), which is incorporated herein by reference for its teachings regarding the same. Accordingly, the following peptides containing conservative amino acid substitutions of HQSPGepoxyHH are also considered to be within the scope of the present description: HESPGepoxyHH, HKSPGepoxyHH, HQAPGepoxyHH; HQNPGepoxyHH, HEAPGepoxyHH, HENPGepoxyHH, HKAPGepoxyHH, and HKNPGepoxyHH. The results described herein also indicate that there is flexibility at the C-terminus to make alterations or additions to the peptide. For example, adding a -GGGC tail to peptides that bound and inhibited toxin did not adversely affect their toxin binding or decrease their inhibitory activity. As another example, in the phage display assays the peptide connected to the rest of the bacteriophage coat protein via its C-terminal domain; hence, the C-terminus must be accessible to solvent. As another example, in the structural models from docking studies one can see that the C-terminus of the peptides are exposed to the solvent, where they can pull away from the toxin easily.

[0039] Pharmaceutically acceptable salts, tautomers, isomers and prodrugs of the compounds above are also considered to be within the scope of the present description. [0040] The compositions and methods disclosed herein also find use in treating disease caused by C. difficile. "Treating," as used herein, refers to ameliorating, reducing the effects of, and/or preventing disease. "Treatment," "therapy," "therapeutics," "therapeutic agents" and the like may be used interchangeably herein. The disclosure provides for a therapeutic agent comprising the compounds, alone or in combination with other compounds, adjuvants, etc., for the purpose of treating CDAD caused by C. difficile in an animal including vertebrate animal. The disclosure also provides for the use of the compounds as adjuvants.

[0041]The compounds and methods disclosed herein can be additive or synergistic with other therapies currently in development or use. For example, the antibiotics metronidazole and/or vancomycin currently provide the treatment for C. difficile infection. Their efficacy in combination with the compounds as described herein can exceed the efficacy of either or both antibiotic drug products when used alone. Accordingly, the compositions of the disclosure can be administered alone or in combination or conjunction with metronidazole, vancomycin, and/or a variety of other antibiotics or therapeutic agents as are in use or being developed against bacterial targets. The compositions of the present disclosure can also be administered in combination or conjunction with non-antibacterial therapeutic agents that are administered to treat C. difficile infection or the symptoms thereof but which do not specifically target the bacteria, such as anti-diarrheal medications, analgesics, antiinflammatories, etc.

[0042] A pharmaceutical composition comprising a compound of the present disclosure can be formulated in a variety of forms; e.g., as a liquid, gel, lyophilized, or as a compressed solid. The preferred form will depend upon the particular indication being treated and will be apparent to one of ordinary skill in the art. In one embodiment, the disclosed pharmaceutical composition comprises inhibitor peptide and formulations for oral delivery that can be small-molecule drugs that employ straightforward medicinal chemistry processes.

[0043] The administration of the formulations of the present disclosure can be performed in a variety of ways, including without limitation orally, subcutaneously, intravenously, intracerebrally, intranasally, transdermally, intraperitoneally, intramuscularly, intrapulmonary, intrathecally, vaginally, rectally, intraocularly, or in any other acceptable manner. The formulations can be administered continuously by infusion, although bolus injection is acceptable, using techniques well known in the art, such as pumps (e.g., subcutaneous osmotic pumps) or implantation. In some instances the formulations can be directly applied as a solution or spray.

[0044] Although in many cases pharmaceutical formulations are provided in liquid form, appropriate for immediate use, formulations can also be provided in frozen or in lyophilized form. In the former case, the composition must be thawed prior to use. The latter form is often used to enhance the stability of the active compound contained in the composition under a wider variety of storage conditions, as it is recognized by those of ordinary skill in the art that lyophilized preparations are generally more stable than their liquid counterparts. Such lyophilized preparations are reconstituted prior to use by the addition of one or more suitable pharmaceutically acceptable diluents such as, without limitation, sterile physiological saline solution.

[0045] Pharmaceutical formulations can be prepared for storage as lyophilized formulations or aqueous solutions by mixing, as appropriate, the compound having the desired degree of purity with one or more pharmaceutically acceptable carriers, excipients or stabilizers typically employed in the art (all of which are termed "excipients"), for example buffering agents, stabilizing agents, preservatives, isotonifiers, non-ionic detergents, antioxidants and/or other miscellaneous additives.

[0046] Buffering agents help to maintain the pH in the range which approximates physiological conditions. They are typically present at a concentration ranging from about 2 mM to about 50 mM. Suitable buffering agents for use with the present disclosure include both organic and inorganic acids and salts thereof such as citrate buffers {e.g., monosodium citrate-disodium citrate mixture, citric acid-trisodium citrate mixture, citric acid-monosodium citrate mixture, etc.), succinate buffers {e.g., succinic acid-monosodium succinate mixture, succinic acid-sodium hydroxide mixture, succinic acid-disodium succinate mixture, etc.), tartrate buffers {e.g., tartaric acid-sodium tartrate mixture, tartaric acid-potassium tartrate mixture, tartaric acid-sodium hydroxide mixture, etc.), fumarate buffers {e.g., fumaric acid-monosodium fumarate mixture, fumaric acid- disodium fumarate mixture, monosodium fumarate-disodium fumarate mixture, etc.), gluconate buffers {e.g., gluconic acid-sodium glyconate mixture, gluconic acid-sodium hydroxide mixture, gluconic acid-potassium glyuconate mixture, etc.), oxalate buffer {e.g., oxalic acid-sodium oxalate mixture, oxalic acid-sodium hydroxide mixture, oxalic acid-potassium oxalate mixture, etc.), lactate buffers {e.g., lactic acid-sodium lactate mixture, lactic acid-sodium hydroxide mixture, lactic acid-potassium lactate mixture, etc.) and acetate buffers {e.g., acetic acid-sodium acetate mixture, acetic acid-sodium hydroxide mixture, etc.). Additional possibilities are phosphate buffers, histidine buffers and trimethylamine salts such as Tris.

[0047] Preservatives can be added to the formulations to retard microbial growth, and are typically added in amounts of about 0.2%-1 % (w/v). Suitable preservatives for use with the present disclosure include, without limitation, phenol, benzyl alcohol, meta- cresol, methyl paraben, propyl paraben, octadecyldimethylbenzyl ammonium chloride, benzalkonium halides {e.g., benzalkonium chloride, bromide or iodide), hexamethonium chloride, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol and 3-pentanol.

[0048] Isotonifiers can be added to formulations to ensure isotonicity of liquid compositions and include, without limitation, polyhydric sugar alcohols, preferably trihydric or higher sugar alcohols, such as glycerin, erythritol, arabitol, xylitol, sorbitol and mannitol . Polyhydhc alcohols can be present in an amount between 0.1 % and 25% by weight, typically 1 % to 5%, taking into account the relative amounts of the other ingredients.

[0049] Stabilizers refer to a broad category of excipients which can range in function from a bulking agent to an additive which solubilizes the therapeutic agent or helps to prevent denaturation or adherence to the container wall. Typical stabilizers can be polyhydric sugar alcohols (examples enumerated above); amino acids such as arginine, lysine, glycine, glutamine, asparagine, histidine, alanine, ornithine, L-leucine, 2- phenylalanine, glutamic acid, threonine, etc., organic sugars or sugar alcohols, such as lactose, trehalose, stachyose, mannitol, sorbitol, xylitol, ribitol, myoinisitol, galactitol, glycerol and the like, including cyclitols such as inositol; polyethylene glycol; amino acid polymers; sulfur-containing reducing agents, such as urea, glutathione, thioctic acid, sodium thioglycolate, thioglycerol, alpha-monothioglycerol and sodium thiosulfate; low molecular weight polypeptides (i.e., of fewer than ten amino acid residues); proteins such as human serum albumin, bovine serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; monosaccharides such as xylose, mannose, fructose and glucose; disaccharides such as lactose, maltose and sucrose; trisaccharides such as raffinose, and polysaccharides such as dextran. Stabilizers are typically present in the range of from 0.1 to 10,000 parts by weight based on the active compound weight.

[0050] Additional miscellaneous excipients include bulking agents or fillers (e.g., starch), chelating agents (e.g., EDTA), antioxidants (e.g. , ascorbic acid, methionine, vitamin E) and cosolvents.

[0051] The active ingredient of the formulations described herein can also be entrapped in microcapsules prepared, for example, by coascervation techniques or by interfacial polymerization, for example hydroxymethylcellulose, gelatin or poly-(methylmethacylate) microcapsules, in colloidal drug delivery systems (for example liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences (Mack Pub. Co.).

[0052] Formulations to be used for in vivo administration generally are sterile. This is readily accomplished, for example, by filtration through sterile filtration membranes.

[0053] Suitable examples of sustained-release preparations include semi-permeable matrices of solid hydrophobic polymers containing the compound or composition, the matrices having a suitable form such as a film or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (for example, poly(2- hydroxyethyl-methacrylate) or poly(vinylalcohol)), polylactides, copolymers of L-glutamic acid and ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as the PROLEASE^® technology or LUPRON DEPOT^® (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(-)-3-hydroxybutyric acid. While polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid enable release of molecules for long periods such as up to or over 100 days, certain hydrogels release compounds for shorter time periods.

[0054] Oral administration of the compounds and compositions is one intended practice of the disclosure. For oral administration, the pharmaceutical composition can be in solid or liquid form; e.g., in the form of a capsule, tablet, powder, granule, suspension, emulsion or solution. The pharmaceutical composition is preferably made in the form of a dosage unit containing a given amount of the active ingredient. A suitable daily dose for a human or other vertebrate can vary widely depending on the condition of the patient and other factors, but can be determined by persons of ordinary skill in the art using routine methods. Administration of the compounds and compositions to the rectum and/or colon via the anus is also an intended practice of the disclosure, such as for example by enema, suppository, etc. [0055] In solid dosage forms, the active compound can be admixed with at least one inert diluent such as sucrose, lactose, or starch. Such dosage forms can also comprise, as is normal practice, additional substances; e.g., lubricating agents such as magnesium stearate. In the case of capsules, tablets and pills, the dosage forms can also comprise buffering agents. Tablets and pills can additionally be prepared with enteric coatings.

[0056] The compounds or compositions can be admixed with adjuvants such as lactose, sucrose, starch powder, cellulose esters of alkanoic acids, stearic acid, talc, magnesium stearate, magnesium oxide, sodium and calcium salts of phosphoric and sulphuric acids, acacia, gelatin, sodium alginate, polyvinyl-pyrrolidine, and/or polyvinyl alcohol, and tableted or encapsulated for conventional administration. Alternatively, they can be dissolved in saline, water, polyethylene glycol, propylene glycol, ethanol, oils (such as corn oil, peanut oil, cottonseed oil or sesame oil), tragacanth gum, and/or various buffers. Other adjuvants and modes of administration are well known in the pharmaceutical art. The carrier or diluent can include time delay material, such as glyceryl monostearate or glyceryl distearate alone or with a wax, or other materials well known in the art.

[0057] Although the invention has been described with reference to particular embodiments and examples thereof, the scope of the present invention is not limited only to those described embodiments. As will be apparent to persons of ordinary skill in the art, modification and adaptations to the present disclosure can be made without departing from the spirit and scope of the invention, which is defined and circumscribed by the appended claims.

EXAMPLES

[0058] Example 1 - Identification of first generation toxin inhibitors

[0059] Phage display assays were initially performed to identify a set of first generation peptides which bound TcdA competitively with RhoA, and inhibited the toxin of TcdA. For methods and results generally see Abdeen and Feig, which is incorporated by reference herein for the methods and results described in this example. For in vitro (non-cellular) studies herein such as inhibition of glucosylation, the catalytic domain of the toxin was used, rTcdA⁵⁴⁰, and for cellular studies herein such as cell viability assays, recombinant full-length toxin was used, rTcdA²⁷¹⁰. Id.

[0060] DNA sequencing of phage which bound TcdA in phage display assays was followed by an ELISA-based screen to measure the peptides' affinity for TcdA, which revealed 17 peptides with the tightest binding to the catalytic domain of Toxin A (rTcdA⁵⁴⁰). These initial 17 peptides bound the catalytic domain of TcdA with varying affinities, from low nanomolar to low micromolar, with 88% of peptides exhibiting a K_d below 500 nM.

[0061]Two peptides were selected for further study: EGWHAHT due to its tight binding affinity (K_d ~= 100 nM) and HQSPWHH, due to its high predicted toxin affinity based on computational modeling docking studies. The two peptides were analyzed in vitro for glucosyltransferase (GT) inhibition of both TcdA and TcdB, and both peptides inhibited TcdA and TcdB in vitro (see Abdeen and Feig), thus making them appropriate candidates for peptide inhibitors.

[0062] Example 2 - Cell viability assays

[0063] The native, underivatized first generation peptides obtained above (HQSPWHH and EGWHAHT) were analyzed for their ability to protect cells against Toxin A, as were the various derivatives of HQSPWHH.

[0064] Initially, cell viability assays were performed on Vera cells in the presence of toxin alone (rTcdA) to determine cell viability over time in the presence of varying amounts of toxin, in order to select the appropriate timeframe and amount of toxin for subsequent studies of cell viability in the presence of toxin + inhibitor. (The amount of time and toxin for inhibition studies should be sufficient to result in near total cell death in the absence of inhibitor.) FIG. 2 shows the results of this assay. Cell viability methods were performed as described below. Based on these results, a toxin concentration of 0.4 nM (4E-10) and a time of 48 hours were selected for cell viability studies in the presence of inhibitor.

[0065] Vero cells were plated in 96-well plates (10000 cells/well) in Essential Minimal Eagle's Media (EMEM, ATCC) with 10 % fetal bovine serum (FBS, USA Scientific) and 1 x antibiotic-antimycotic (from 100x stock, Invitrogen) and then incubated 24 hours at 370°C, 5% CO2. The next day serum was removed and exchanged to 200 μΙ serum-free EMEM and briefly incubated at 370°C and 5% CO2 while preparing the samples. Different peptide inhibitors at varying concentrations were titrated into 0.4 nM rTcdA (i.e., full-length TcdA). Serum-free EMEM was removed from each well and 0.4 nM rTcdA or 0.4 nM rTcdA + inhibitor was added, to a final volume in each well of 50 μΙ in serum-free EMEM. Cells were incubated 48 hours at 370°C and 5% CO₂. Cell viability was measured using the CELLTITER-GLO^® luminescent assay (Promega, Madison, Wisconsin). This is an ATP-based assay, which monitors cell viability by direct correlation with intracellular ATP. CellTiter-Glo reagent was thawed to room temperature from -20°C. Cells were exchanged into 50 μΙ fresh serum-free EMEM at room temperature. Plates were maintained at room temperature for 45 minutes, after which 50 μΙ of the CellTiter-Glo reagent was added to each well. Plates were shaken on an orbital shaker for two minutes at moderate speed and maintained at room temperature for an additional ten minutes before luminescence measurements were obtained using the TECAN^® GENIOS™ plus (Tecan Group, Switzerland) microplate reader.

[0066] In cell viability studies, neither of the first generation, underivatized peptides EGWHAHT or HQSPWHH protected cells from TcdA (data not shown). HQSPWHH was selected for derivatization and further study based on computational modeling and docking studies (see Example 4), which predicted a high toxin binding affinity. See Abdeen and Feig. The peptide HQSPWHH was epoxy-derivatized (see Example 3) at various amino acid residues, and the derivatives analyzed by computational modeling (see Example 4). Two derivatives were selected for cell viability/protection assays, the first based on its computed binding affinity (HQSPGepoxyHH), and the second based on the number of nearby polar amino acids in the toxin capable of attacking the epoxide (HQSPWHGepoxy).

[0067] FIG. 3 shows images from a cell viability assay comparing the effects of administering to Vera cells (A) PBS (negative control), (B) 0.4 nM TcdA alone, and (C) 0.4 nM TcdA pre-incubated with 600 μΜ HQSPGepoxyHH, thus illustrating the protective effects of this derivatized peptide.

[0068] FIG. 4 shows results of a cell viability assay comparing inhibitory effects of the two derivatives HQSPGepoxyHH and HQSPWHGepoxy. The results show that HQSPGepoxyHH provided 95% cell protection from TcdA, whereas HQSPWHGepoxy provided virtually no protection. FIG. 5 also demonstrates that derivative HQSPGepoxyHH provided 95% cell protection, whereas the other derivative HQSPWHGepoxy provided virtually no protection, nor did the underivatized peptide or the Gallyl-substituted peptide. Clearly, accurate choice of derivative position along the peptide chain is critical for the peptide's inhibitory activity.

[0069] Example 3 - Derivatization of peptides

[0070] HQSPWHH derivatized with allyglycine at various positions were subjected to epoxidation by meta-chloroperoxybenzoic acid (mCPBA). Purified allylglycine-modified peptides were obtained from American Peptide Company (Sunnyvale, California). Modified peptides were dissolved in 0.1 M (NH )₂CO3 buffer (pH 8.0), and the reaction vial was flushed with N₂. An equimolar (1 :1 ) ratio of freshly prepared DTT was added and incubated at 540°C for 30 minutes. To the reaction mix a 1 :10 molar excess of iodoacetamide solid (Sigma) was added, kept under N₂ and incubated in the dark at room temperature with continuous stirring for two hours, lodoacetamide-labeled peptides were purified by reverse-phase HPLC over a C18 column (Beckman Coulter) using a gradient of 0 to 100% acetonitrile containing 0.1 % trifluoroacetic acid and monitored by UV absorption. After purification, the peptide was lyophilized and redissolved. The identity of each product was confirmed by electrospray mass spectrometry.

[0071]To a stirred solution of CH₂CI₂:0.01 M Na₂HPO₄/NaH₂PO₄ buffer (pH 8.0) (ratio of organic to aqueous phase, 2:1 ), peptide and p-chloroperbenzoic acid (PCBA) were added (molar ratio of peptide to PCBA, 1 :10). The reaction was kept under N₂ and incubated at room temperature with stirring for five hours. Reaction progress was monitored by TLC: a 34% propanol:water mix was made, then using that as a solvent a solution was made of 70% NH4OH:30% of the propanol:water. After completion of the reaction, the epoxy derivative was precipitated by ether and the precipitate was washed with several-fold of ether. Peptide was lyophilized and redissolved in water, desalted and purified by reverse-phase HPLC over a C18 column, using a gradient of 0 to 100% acetonitrile containing 0.1 % trifluoroacetic acid and monitored by UV absorption. After purification, the peptide was lyophilized and stored at -200°C until used. The identity of the final product was confirmed by liquid chromatography electrospray mass spectrometry (LC-MS).

[0072] Example 4 - Modeling of toxin catalytic domain and docking studies

[0073] Computational models of the toxin, and especially its active site where peptide inhibitors would bind, were built based on X-ray crystallographic analysis of the toxins. These models were particularly useful for docking studies (i.e., modeling of toxin binding to substrate).

[0074] Molecular models of the peptides with toxin were built using the Spartan '02 molecular modeling program (Wavefunction, Irvine, California), minimizing at the AM1 (Austin Model 1 ) level of theory. Models thus obtained were saved in Sybyl Mol2 file format and catenated into a library for docking studies. The crystal structure of TcdB (2BVL) was retrieved from the Research Collaboratory for Structural Bioinformatics (RCSB) Protein Data Bank (PDB) for use as the docking receptor for the peptide. Structures were flexibly docked to the crystal structure of TcdB. Flexible docking was performed using the FlexX 3.1 .0 ligand docking program. See Schellhammer, I. and Rarey, M. 2004, Proteins 57, 504-517. Crystallographic phasing markers and counter ions were removed from the model, retaining crystallographic water molecules. The active site for docking was defined by 20A spheres around each atom of the crystallographically observed UDP. The crystallographic catalytic manganese was replaced with a magnesium ion for ease of calculation. A docking pharmacophore with two optional constraints was constructed utilizing the two octahedral coordination sites of the magnesium occupied by the crystallographic UDP molecule. Water molecules within the active site were included in the docking, designated as fully rotatable and displaceable. Docking scores were obtained from the FlexX output and using the FlexX internal scoring protocol. See Gohlke, H. et al. 2000, J. Mol. Biol. 295, 337-356. Results were viewed and all images were generated using the UCSF Chimera visualization program versionl .4.1 . The docked structures were then simulated for 10 nanoseconds to determine the stability of the docked conformation. The complete toxin/peptide complex was solvated and ionized to 0.5mm NaCI and simulated using the CHARMM27 force field and the NAMD molecular dynamics simulation package, on the Wayne State University grid supercomputer. A time step of one femtosecond was used, periodic boundary conditions were applied, and Langevin dynamics utilized to maintain constant temperature at 300K. A scaled cutoff was employed in the calculation of the long range electrostatics.

[0075] The first generation peptide of HQSPWHH was selected for further study due to its high predicted toxin affinity, based on this computational modeling docking study.

[0076] Each position in the peptide was replaced with an epoxide structure and its docking score determined. Dockings were evaluated using a standard scoring algorithm and compensating for differences in peptide sizes. Docking data provided information on the side chain residues in the peptides that could be modified. Table 1 demonstrates the docking scores from initial computational investigation for parent and variously epoxy-derivatized peptide HQSPWHH.

Table 1

[0077] The two derivatized peptides HQSPGepoxyHH and HQSPWHGepoxy were selected for further study, the former because of its exceptional binding affinity (docking score of -70.03), and the latter because of the number of nearby polar amino acids in the toxin active site capable of attacking the epoxide.

[0078] The present disclosure identifies and describes the development and optimization of inhibitors of C. difficile toxins, as well as methods to improve their efficacy and further develop their utility in vivo.

[0079] Unless otherwise indicated, all numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term "about." Accordingly, unless indicated to the contrary, the numerical parameters set forth in the specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements.

[0080] The terms "a," "an," "the" and similar referents used in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. Recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language {e.g., "such as") provided herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.

[0081] Groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations. Each group member may be referred to and claimed individually or in any combination with other members of the group or other elements found herein. It is anticipated that one or more members of a group may be included in, or deleted from, a group for reasons of convenience and/or patentability. When any such inclusion or deletion occurs, the specification is deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims. [0082] Certain embodiments of this invention are described herein. Of course, variations on these described embodiments will become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventor expects skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

[0083] Specific embodiments disclosed herein may be further limited in the claims using consisting of or consisting essentially of language. When used in the claims, whether as filed or added per amendment, the transition term "consisting of excludes any element, step, or ingredient not specified in the claims. The transition term "consisting essentially of limits the scope of a claim to the specified materials or steps and those that do not materially affect the basic and novel characteristic(s). Embodiments of the invention so claimed are inherently or expressly described and enabled herein.

[0084] Furthermore, wherever reference has been made to patents and/or printed publications throughout this specification, each of the above-cited references and/or printed publications are individually incorporated herein by reference in their entirety.

[0085] In closing, it is to be understood that the embodiments of the invention disclosed herein are illustrative of the principles of the present invention. Other modifications that may be employed are within the scope of the invention. Thus, by way of example, but not of limitation, alternative configurations of the present invention may be utilized in accordance with the teachings herein. Accordingly, the present invention is not limited to that precisely as shown and described.

Claims

1 . A composition comprising the peptide represented by HQSPXHH, wherein X is selected from an acrylamide, an acrylate, an acyloxymethyl ketone (AOMK), an O- aryloxycarbonyl hydroxamate, a chloroacetamide, a chloroformate, a chloromethylketone, a diazo-methylketone, an epoxide, a fluoromethyl ketone, a halomethylketone, a hydroxamate, an isothiocyanate, a ketomethylene, a propynamide, a 2-pyridone, a 2-pyrrolidone, and a pyrrolopyrimidine derivatized amino acid.

2. A composition comprising the peptide represented by HQSPGepoxyHH.

3. A composition comprising a peptide selected from the peptides represented by HESPGepoxyHH, HKSPGepoxyHH, HQAPGepoxyHH; HQNPGepoxyHH, HEAPGepoxyHH, HENPGepoxyHH, HKAPGepoxyHH, or HKNPGepoxyHH.

4. A composition of any of claims 1 -3, wherein the amino acid represented by Gepoxy derives from allylglycine.

5. A composition of any of claims 1 -4, wherein one or more amino acids are added to the C-terminus of the peptide.

6. A composition of claim 5, wherein the one or more amino acids added to the C-terminus of the peptide comprise the amino acids represented by -GGGC.

7. A composition of any of claims 1 -6 comprising pharmaceutically acceptable salts, tautomers, isomers and prodrugs of the composition.

8. A therapeutic composition comprising a composition according to any of claims 1 -7.

9. A method of inhibiting TcdA/B, comprising exposing the at least one of TcdA and TcdB to a composition according to any of claims 1 -8.

10. A method of inhibiting TcdA B in a vertebrate cell, comprising administering to the cell a composition according to any of claims 1 -8.

1 1 . A method of identifying compounds that inhibit TcdA B intracellular^ comprising: selecting one or more peptides for analysis; derivatizing the peptides in silico with an electrophilic group at various amino acid sites within each peptide to create a set of in silico derivatized peptides; performing docking studies on each derivatized peptide to determine a docking score for each derivatized peptide; selecting one or more derivatized peptides based on their docking scores; from the derivatized peptides that were selected based on docking scores, further selecting a final set of derivatized peptides for testing where the electrophilic group in the derivatized peptide is susceptible to reactivity with potential nucleophilic groups in the active site of the toxin; and, testing the final set of peptides for inhibition of toxin in cellulo.

12. A method of treating infection by Clostridium difficile in a subject, comprising administering to the subject a composition according to any of claims 1 -8.