WO2014102570A1

WO2014102570A1 - Use of the compound pm181104, for the treatment of clostridium difficile associated infections

Info

Publication number: WO2014102570A1
Application number: PCT/IB2012/057785
Authority: WO
Inventors: Girish Badrinath Mahajan
Original assignee: Piramal Enterprises Limited; Council Of Scientific And Industrial Research
Priority date: 2012-12-28
Filing date: 2012-12-28
Publication date: 2014-07-03

Abstract

The present invention relates to use of the compound PM181104, or a stereoisomer or a tautomer thereof, or a pharmaceutically acceptable salt thereof, for the treatment of Clostridium difficile associated infections. The invention further relates to a pharmaceutical composition comprising the compound PM181104 and at least one pharmaceutically acceptable carrier, for use in the treatment of Clostridium difficile associated infections. The invention also relates to a method for the treatment of Clostridium difficile associated infections, comprising administering to a subject in need thereof, a therapeutically effective amount of the compound PM181104.

Description

USE OF THE COMPOUND PM181 104, FOR THE TREATMENT OF

CLOSTRIDIUM DIFFICILE ASSOCIATED INFECTIONS

FIELD OF THE INVENTION

The present invention relates to a method of treating Clostridium difficile (C. difficile) associated infections or diseases by administering a therapeutically effective amount of the compound PM181 104, or a stereoisomer or a tautomer thereof, or a pharmaceutically acceptable salt thereof, to a subject in need thereof. BACKGROUND OF THE INVENTION

Clostridium difficile (C. difficile), is a gram-positive, spore-forming, anaerobic, toxin-producing Bacillus. It was first described in 1935 as a component of the intestinal flora in healthy newborn infants. The role of C. difficile in human disease was found in 1980s, when it was identified as the causative agent of pseudomembranous colitis. C. difficile accounts for approximately 20% of cases of antibacterial-associated diarrhea and the majority of cases of antibacterial- associated colitis. C. difficile associated disease (CDAD) is a serious condition with mortality up to 25 per cent in frail elderly people. C. difficile is now recognized as the primary cause of hospital acquired colitis in patients who receive antibiotics, chemotherapeutics or other drugs that alter their normal flora. Although virtually all antimicrobial agents have been implicated in the development of CDAD, some recent studies of CDAD caused by the current NAP1/BI/027 strain, have found that fluoroquinolones were the antimicrobials most closely associated with the disease. Antibacterial disturbance of normal anaerobic gut flora allows the over growth of toxigenic strains of C. difficile and the production of toxins A and B by the strain. Reactions to these toxins can result in lesions and severe damage to the epithelial lining of the colon [Clinical Infectious Diseases, 2007, 45, 222-227].

Clostridium difficile Infection (CDI) is the most common cause of infectious diarrhea in healthcare settings. Although there is no mandatory reporting of CDI in the United States, it is estimated that there are at least 500000 cases in the US hospitals and nursing homes per year. Data from Canadian surveillance studies estimate their incidence to be approximately 4.6 cases per 10000 patient admissions. A recently completed pan-European hospital based survey shows a similar European incidence of 4.1 cases per 10 000 patient days [Current Opinion in Gastroenterology, 201 1 , 27(1 ), 38-47].

In addition to disease in healthcare settings, C. difficile can cause disease in healthy individuals. Reports of community associated CDAD [CA-CDAD] from the USA suggested that CA-CDAD is very uncommon. However, studies performed in Sweden few years ago suggested that as many as 1 in 5 cases of CDAD may be CA-CDAD.

Present remedial options for Clostridium difficile Infection (CDI) include the off label use of metronidazole and oral vancomycin, the only FDA approved treatment. However, approximately 20% to 30% of CDI patients who initially respond to these treatments experience a clinical recurrence following cessation of antibiotic administration, which is of more concern. Primary risk factors for CDI include broad- spectrum antibiotic (such as cephalosporins and fluoroquinolones) usage, advanced age (over 65 years) and exposure to emerging hyper virulent strains of C. difficile, such as NAP1/BI/027. Increasing incidence, higher treatment failures and recurrence with current therapies have resulted in greater awareness and concern for CDI among medical professionals and public health officials. At present, few agents are being developed as new therapies for CDAD. Tolevamer, a C. difficile toxin binding agent, is in the approval stage of development, and is being specifically developed for CDAD. Other agents such as rifaximin and nitazoxanide are already approved for other gastrointestinal infections and are currently being investigated as potential therapies for CDAD. Novel agents in early phases of development include Fidaxomicin / OPT-80 (completed Phase III), CB-183,315 (in phase II), MK-3415A (in phase II), Ramoplanin (in phase II), Oritavancin (in phase II) and monoclonal antibodies. It is to be noted that the current antibiotics under development are either very narrow spectrum or successive generations of earlier drugs, which make them more liable to rapid resistance development. Thus, there is demand for more novel skeletons and wider spectrum antibiotics for the treatment of Clostridium difficile associated infections.

PCT Application Publication WO20071 19201 , which is incorporated herewith in its entirety as reference, describes the compound PM181 104 as a novel anti bacterial compound, produced by the fermentation of the microorganism belonging to Kocuria species (ZMA B-1 / MTCC 5269), active against the bacterial infections caused by bacteria belonging to Staphylococcus, Streptococcus, Enterococcus and aerobic Bacillus species.

Inventors of the present invention have found that the compound PM181 104 is active against Clostridium difficile, which is an obligate anaerobic, spore forming, gram positive Bacillus species, and hence, useful for the treatment of Clostridium difficile associated infections or diseases.

SUMMARY OF THE INVENTION

According to one aspect of the present invention, there is provided a method for the treatment of Clostridium difficile (C. difficile) associated infections or diseases, comprising administering to a subject in need thereof, a therapeutically effective amount of the compound PM181 104, or a stereoisomer or a tautomer thereof, or a pharmaceutically acceptable salt thereof.

According to another aspect of the present invention, there is provided a method of inhibiting growth of Clostridium difficile (C. difficile) organism, comprising contacting the C. difficile organism with the compound PM181 104, or a stereoisomer or a tautomer thereof, or a pharmaceutically acceptable salt thereof in an amount sufficient to inhibit growth of C. difficile.

According to another aspect of the present invention, there is provided a method for providing prophylaxis of Clostridium difficile (C. difficile) associated infections or diseases in a subject, comprising administering to the subject in need thereof the compound PM181 104 or a stereoisomer or a tautomer thereof, or a pharmaceutically acceptable salt thereof in an amount sufficient to achieve prophylaxis of Clostridium difficile (C. difficile) associated infections or diseases.

According to one aspect of the present invention, there is provided use of the compound PM181 104 (structure as indicated herein below), or a stereoisomer or a tautomer thereof, or a pharmaceutically acceptable salt thereof, for the treatment of Clostridium difficile associated infections or diseases.

According to a further aspect of the present invention, there is provided a pharmaceutical composition comprising the compound PM181 104, or a stereoisomer or a tautomer thereof, or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable carrier, for use in the treatment of Clostridium difficile associated infections or diseases. The present invention in a further aspect concerns use of the compound PM181 104, or a stereoisomer or a tautomer thereof, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for the treatment of Clostridium difficile associated infections or diseases.

DETAILED DESCRIPTION OF THE INVENTION

In an embodiment, the present invention provides the compound PM181 104:

PM181 104

or a stereoisomer or a tautomer thereof, or a pharmaceutically acceptable salt thereof, for use in the treatment of Clostridium difficile associated infections or diseases.

Definitions:

For the purpose of the disclosure, listed below are definitions of various terms used to describe the present invention. These definitions apply to the terms as they are used throughout the specification and the appended claims (unless they are otherwise limited in specific instances) either individually or as part of a larger group. They should not be interpreted in the literal sense. They are not general definitions and are relevant only for this application.

In the specification where the term "compound PM181 104" is used alone, wherever appropriate, it is deemed to include a stereoisomer, or a tautomer or a pharmaceutically acceptable salt of the compound PM181 104.

The term "pharmaceutically acceptable" means that the carrier or salt must be compatible with the other ingredients of the formulation, and not deleterious to the recipient thereof.

The term "pharmaceutically acceptable carrier" as used herein means a diluent, excipient, encapsulating material or formulation auxiliary, which is non-toxic, and inert, which is compatible with a subject, preferably a mammal, more preferably a human, and is suitable for delivering an active agent to the target site without terminating the activity of the agent.

The term, "therapeutically effective amount" as used herein means an amount of the compound PM181 104 or a composition comprising said compound, sufficient to significantly induce a positive modification in the condition to be treated, but low enough to avoid side effects if any (at a reasonable benefit/risk ratio), within the scope of sound medical judgment. The requirement of therapeutically effective amount of the compound PM181 104 or the composition containing said compound, for the treatment will vary with the particular Clostridium difficile associated infection or disease being treated, the age and physical condition of the subject (patient), the severity of the condition being treated, the duration of the treatment, the nature of concurrent therapy, the particular pharmaceutically acceptable carrier utilized, and like factors.

The terms "sufficient amount" and "in an amount sufficient" are used interchangeably and means in relation to inhibiting the growth of Clostridium difficile (C. difficile) organism, means an amount of the compound PM181 104 or a composition comprising said compound, sufficient to significantly induce inhibition of the growth of Clostridium difficile (C. difficile) organism.

The term 'treating", "treat" or "treatment" as used herein have their ordinary or customary meanings, and include alleviating the infection or disease, slowing the progression of, attenuation or cure of the existing disease (e.g., antibacterial associated diarrhea or antibacterial associated colitis).

The term 'prophylaxis" as used herein has its ordinary or customary meaning, and includes inhibiting the development of a productive or progressive infection by C. difficile in a subject, where the prophylaxis lasts at least from about 1 to about 50 or more days after administration of the compound PM181 104 or a composition containing the compound PM181 104 of the present invention. Inhibition against development of a productive or progressive infection by C. difficile means that the severity of a C. difficile infection in a subject is reduced by about 20% to about 100% versus a subject to which the compound PM181 104 or the composition containing the compound of the present invention has not been administered.

The term "contacting" is meant to broadly refer to bringing Clostridium difficile (C. difficile) organism(s) and the compound PM 181 104 of the present invention into sufficient proximity that the compound can exert an effect on the bacterial cell. The compound may be transported to the location of the C. difficile organism, or the compound may be situated in a location to which the C. difficile organism travels or is brought into contact. The skilled artisan will understand that the term "contacting" includes physical interaction between the compound and C. difficile, as well as interactions that do not require physical interaction.

The term "subject" as used herein refers to an animal, preferably a mammal, and most preferably a human.

The term "mammal" used herein refers to warm-blooded vertebrate animals of the class Mammalia, including humans, characterized by a covering of hair on the skin and, in the female, milk-producing mammary glands for nourishing the young. The term mammal includes without limitations animals such as cat, dog, horse, rabbit, bear, fox, wolf, monkey, deer, mouse, pig as well as human.

The term "Clostridium difficile associated infection" or "Clostridium difficile Infection (CDI)" or "Clostridium difficile associated disease (CDAD)" as used herein, refers to an infection or a disease, such as antibacterial associated diarrhea; antibacterial associated colitis; diarrhea; or colitis; caused by Clostridium difficile (C. difficile), which is a gram-positive, spore-forming, anaerobic, toxin producing bacillus.

The term "antibacterial associated diarrhea" or "antibacterial associated colitis" as used herein refers to an infection or disease caused after receiving treatment with antibiotics, chemotherapeutics or other drugs that alter their normal intestinal flora.

The term "Bacillus species" refers to a large number of diverse, rod-shaped Gram positive bacteria, which can be obligate aerobes or facultative anaerobes, and test positive for the enzyme catalase. It is also a member of the division Firmicutes. Members of this genus are capable of producing endospores, that can stay dormant, and hence they can resist unfavourable environmental conditions. Clostridium difficile (C. difficile) belongs to anaerobic Bacillus species. The term "pharmaceutically acceptable salt(s)" is meant to include salt(s) of the compound PM181 104, which are prepared with acids or bases. Examples of pharmaceutically acceptable base addition salts include sodium, potassium, calcium, magnesium, ammonium or organic base salt. Examples of pharmaceutically acceptable organic base addition salts include those derived from organic bases such as lysine, arginine, guanidine, and the like. Examples of pharmaceutically acceptable acid addition salts include those derived from inorganic acids such as hydrochloric, nitric, phosphoric, sulfuric and the like, as well as the salts derived from organic acids such as acetic, propionic, oxalic, maleic, benzoic, succinic, fumaric, phthalic, benzenesulfonic, p-tolylsulfonic, citric, tartaric, methanesulfonic acids and the like.

The compound PM181 104, was obtained according to the method disclosed in PCT publication WO20071 19201 , which is incorporated herein by reference.

The compound PM181 104, was isolated from the fermented broth of the microorganism belonging to Kocuria species (ZMA B-1/ MTCC 5269) and was further purified. The microorganism, a strain of Kocuria species (ZMA B-1/ MTCC 5269), which was used for the production of the compound PM181 104, (herein after referred to as culture no. ZMA B-1 ) was isolated from a marine sample collected in Palk Bay, Tamil Nadu coast, India.

The compound PM181 104 has the molecular formula

(molecular weight 1514) and was characterised by any one or more of its physico- chemical and spectral properties, such as high performance liquid chromatography (HPLC), mass spectrum (MS), ultra violet (UV), infra red (IR) and nuclear magnetic resonance (NMR) spectroscopic data as discussed herein below.

The structure of the compound PM181 104 was elucidated and its complete characterization was done by HPLC, MS, UV, IR and NMR spectroscopic data. The structure was confirmed by the three-dimensional (3D) NMR study of bioactive ¹⁵N and ¹³C- labeled PM181 104.

The process for the production of the compound PM181 104 from culture no. ZMA B-1 , comprised the steps of: growing the culture no. ZMA B-1 under submerged aerobic conditions in a nutrient medium containing one or more sources of carbon and one or more sources of nitrogen and optionally nutrient inorganic salts and/or trace elements; isolating the compound PM181 104 from the culture broth; and purifying the compound PM181 104 using purification procedures generally used in the related art.

Preliminary identification of culture no. ZMA B-1 , which is the producer of PM181 104 was performed by examination of its colony morphology, wet mount observations and Gram stain reaction. Culture no. ZMA B-1 has been deposited with Microbial Type Culture Collection (MTCC), Institute of Microbial Technology, Sector

39- A, Chandigarh -160 036, India, a World Intellectual Property Organization (WIPO) recognized International Depository Authority (IDA) and has been given the accession number MTCC 5269.

The production of the compound PM181 104 was carried out by cultivating culture no. ZMA B-1 by fermentation at a temperature ranging from 26°C to 36°C and a pH of about 6.5 to 8.5, for 24-96 hours at 60-140 rpm and 100-200 Ipm aeration. Typically, culture no. ZMA B-1 is cultivated at 30°C-32°C and pH 7.4-7.8 for

40- 72 hours at 90-1 10 rpm and 140-160 Ipm aeration.

The production of the compound PM181 104 was carried out by cultivating culture no. ZMA B-1 in a suitable nutrient broth under conditions described herein, preferably under submerged aerobic conditions, for example in shake flasks, as well as in laboratory fermenters. The progress of fermentation and production of the compound PM181 104 was monitored by high performance liquid chromatography (HPLC) and by measuring the bioactivity of the culture broth against Staphylococcus aureus 3066 (by the procedure described in PCT publication WO20071 19201 ). In the resulting culture broth, the compound PM181 104 was present in the culture filtrate as well as in cell mass and was isolated using known separation techniques such as solvent extraction and column chromatography. The compound PM181 104 was extracted with ethyl acetate from the whole broth. Concentration of the extracts gave the active crude material.

The compound PM181 104 of the present invention was recovered from the crude material by fractionation using chromatography over reverse phase silica gel (RP-18).

Those skilled in the art will recognize that stereocentres exist in the compound

PM181 104. Accordingly, the present invention includes all possible stereoisomers and tautomers of the compound PM181 104. The compound PM181 104, may be converted into their pharmaceutically acceptable salts. The salts can be prepared by standard procedures known to one skilled in the art, for example, salts like sodium and potassium salts, can be prepared by treating the compound PM181 104, with a suitable sodium or potassium base, for example sodium hydroxide, potassium hydroxide.

In one embodiment of the invention, the compound PM181 104, or a stereoisomer or a tautomer thereof, or a pharmaceutically acceptable salt thereof, is active against Clostridium difficile, and hence find use in the treatment of Clostridium difficile associated infections or diseases.

In another embodiment, the present invention as used herein, Clostridium difficile associated infections or diseases include: antibacterial associated diarrhea; antibacterial associated colitis; diarrhea; or colitis.

In another embodiment, the present invention provides a method for the treatment or prophylaxis of Clostridium difficile associated infection or disease, comprising administering to a subject in need thereof, a therapeutically effective amount of the compound PM181 104, or a stereoisomer or a tautomer thereof, or a pharmaceutically acceptable salt thereof.

The present invention further relates to pharmaceutical compositions which contain an therapeutically effective amount of the compound PM181 104, or a stereoisomer or a tautomer thereof, or a pharmaceutically acceptable salt thereof, together with a pharmaceutically acceptable carrier, wherein the said pharmaceutical composition is adapted for use in the treatment of Clostridium difficile associated infections or diseases. The therapeutically effective amount of the compound PM181 104, or a stereoisomer or a tautomer thereof, or a pharmaceutically acceptable salt thereof, as the active ingredient in the pharmaceutical preparations normally is between about 1 mg to about 1000 mg, preferably from 5 to 750 mg, more preferably from 10 to 500 mg.

Furthermore, in addition to the compound PM181 104, as active ingredient, the pharmaceutical compositions may also contain one or more other therapeutically active ingredient/s such as antimicrobials selected from metronidazole or vancomycin.

In an embodiment, the present invention relates to a method of inhibiting growth of Clostridium difficile (C. difficile) organism, comprising contacting the C. difficile organism with the compound PM181 104, or a stereoisomer or a tautomer thereof, or a pharmaceutically acceptable salt thereof in an amount sufficient to inhibit growth of C. difficile.

In an embodiment, the present invention relates to a method of inhibiting growth of Clostridium difficile (C. difficile) organism, comprising contacting the C. difficile organism with a pharmaceutical composition comprising the compound PM181 104, or a stereoisomer or a tautomer thereof, or a pharmaceutically acceptable salt thereof in an amount sufficient to inhibit growth of C. difficile.

The method of inhibiting the growth of the C. difficile organism, may be in vitro, in vivo or both, comprising contacting organism with the compound PM181 104, or a stereoisomer or a tautomer thereof, or a pharmaceutically acceptable salt thereof or a pharmaceutical composition in an amount sufficient to inhibit the growth of the bacteria C. difficile.

In an embodiment, the present invention relates to a method of inhibiting growth of Clostridium difficile (C. difficile) organism, comprising contacting the C. difficile organism in vitro with the compound PM181 104, or a stereoisomer or a tautomer thereof, or a pharmaceutically acceptable salt thereof in an amount sufficient to inhibit growth of C. difficile.

In an embodiment, the present invention relates to a method of inhibiting growth of Clostridium difficile (C. difficile) organism, comprising contacting the C. difficile organism in vivo with the compound PM181 104, or a stereoisomer or a tautomer thereof, or a pharmaceutically acceptable salt thereof in an amount sufficient to inhibit growth of C. difficile.

The present invention also relates to the use of the compound PM181 104, or a stereoisomer or a tautomer thereof, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for the treatment of infection or disease caused by Clostridium difficile.

The compound of the present invention can be administered orally, nasally, topically, subcutaneously, intramuscularly, intravenously, or by other modes of administration.

Pharmaceutical compositions which contain the compound PM181 104, or a stereoisomer or a pharmaceutically acceptable salt thereof, can be prepared by mixing the active compounds with one or more pharmacologically tolerated auxiliaries and/or excipients such as wetting agents; solubilisers such as surfactants; vehicles; tonicity agents; fillers; colorants; masking flavors; lubricants; disintegrants; diluents; binders; plasticizers; emulsifiers; ointment bases; emollients; thickening agents; polymers; lipids; oils; cosolvents; or complexation agents; and converting the mixture into a suitable pharmaceutical form such as, for example, tablets, coated tablets, capsules, granules, powders, creams, ointments, gels, syrup, emulsions, suspensions, or solutions suitable for parenteral administration.

Examples of auxiliaries and/or excipients that may be used are cremophor, poloxamer, benzalkonium chloride, sodium lauryl sulfate, dextrose, glycerin, magnesium stearate, polyethylene glycol, starch, dextrin, lactose, cellulose, carboxymethylcellulose sodium, gelatin, talc, agar-agar, mineral oil, animal oil, vegtetable oil, organic and mineral waxes, paraffin, gels, propylene glycol, benzyl alcohol, dimethylacetamide, ethanol, polyglycols, tween 80, solutol HS 15, and water.

It is also possible to administer the active substances as such, without vehicles or diluents, in a suitable form, for example, in capsules.

Formulations for parenteral administration can be in the form of aqueous or non-aqueous isotonic sterile injection solutions, suspensions or fat emulsions. The parenteral form used for injection must be fluid to the extent that easy syringability exists. These solutions or suspensions can be prepared from sterile concentrated liquids, powders or granules.

Excipients used in parenteral preparations also include, without limitation, stabilizing agents (e.g. carbohydrates, amino acids and polysorbates, such as 5% dextrose), solubilizing agents (e.g. cetrimide, sodium docusate, glyceryl monooleate, polyvinylpyrolidone (PVP) and polyethylene glycol (PEG)), surfactants (e.g. polysorbates, tocopherol PEG succinate, poloxamer and CremophorTM), buffers (e.g. acetates, citrates, phosphates, tartrates, lactates, succinates, amino acids and the like), antioxidants and preservatives (e.g. BHA, BHT, gentisic acids, vitamin E, ascorbic acid, sodium ascorbate and sulfur containing agents such as sulfites, bisulfites, metabisulfites, thioglycerols, thioglycolates and the like), tonicity agents (for adjusting physiological compatibility), suspending or viscosity agents, antibacterials (e.g. thimersol, benzethonium chloride, benzalkonium chloride, phenol, cresol and chlorobutanol), chelating agents, and administration aids (e.g. local anesthetics, anti-inflammatory agents, anti-clotting agents, vaso-constrictors for prolongation and agents that increase tissue permeability), and combinations thereof. Parenteral formulations using hydrophobic carriers include, for example, fat emulsions and formulations containing lipids, lipospheres, vesicles, particles and liposomes. Fat emulsions include in addition to the above-mentioned excipients, a lipid and an aqueous phase, and additives such as emulsifiers (e.g. phospholipids, poloxamers, polysorbates, and polyoxyethylene castor oil), and osmotic agents (e.g. sodium chloride, glycerol, sorbitol, xylitol and glucose). Liposomes include natural or derived phospholipids and optionally stabilizing agents such as cholesterol.

In an embodiment, the parenteral unit dosage form of the compound

PM181 104 can be a ready-to-use solution of the compound PM181 104 in a suitable carrier in sterile, hermetically sealed ampoules or in sterile pre-loaded syringes. The suitable carrier optionally comprises any of the above-mentioned excipients.

Alternatively, the unit dosage of the compound PM181 104 of the present invention can be in a concentrated liquid, powder or granular form for ex tempore reconstitution in the appropriate pharmaceutically acceptable carrier, such as sterile water, at the time of delivery. In addition to the above-mentioned excipients, powder forms optionally include bulking agents (e.g. mannitol, glycine, lactose, sucrose, trehalose, dextran, hydroxyethyl starch, ficoll and gelatin), and cryo or lyoprotectants.

In intravenous (IV) use, a sterile formulation of the pharmaceutical compositions of the present invention and optionally one or more additives, including solubilizers or surfactants, can be dissolved or suspended in any of the commonly used intravenous fluids and administered by infusion. Intravenous fluids include, without limitation, physiological saline, phosphate buffered saline, 5% dextrose in water or Ringer'sTM solution.

In intramuscular preparations, a sterile formulation of the pharmaceutical compositions of the present invention can be dissolved and administered in a pharmaceutical diluent such as Water-f or- Injection (WFI), physiological saline or 5% dextrose in water. A suitable insoluble form of the pharmaceutical compositions may be prepared and administered as a suspension in an aqueous base or a pharmaceutically acceptable oil base, e.g. an ester of a long chain fatty acid such as ethyl oleate. The method of administration which is suitable in a specific case depend on the species of Clostridium difficile to be treated and on the state of the respective infection or disease caused by Clostridium difficile. Further, this can be optimized using methods known in the art.

The daily dose of the compound PM181 104 may vary depending upon the physical characteristics of the subject, the severity of the subject's symptoms, the formulation and the means used to administer the active ingredient or compound, and the method being practiced. The specific dose for a given subject is usually set by the judgment of the attending physician. However, a therapeutically effective and/or sufficient amount of the compound PM181 104 of the present invention is typically between about 0.5 mg/kg body weight to about 100 mg/kg body weight, preferably from 1 to 50 mg/kg, more preferably from 5 to 30 mg/kg, regardless of the formulation. In equally preferred embodiments, a therapeutically effective amount used for single dosing or infrequent dosing is about 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34 or 35 mg/kg body weight, regardless of the formulation. In some situations, a dose less than 0.5 mg/kg body weight or greater than 100 mg/kg body weight may be effective.

Suitable frequencies of administration may vary based on whether administration is for the purposes of treatment or prophylaxis of the infections or diseases associated with C. difficile. Administration frequencies of doses for the treatment of a subject having a C. difficile infection, prophylaxis or prevention of C. difficile infection include 4, 3, 2 or once daily, every other day, every third day, every fourth day, every fifth day, every sixth day, once weekly, every eight days, every nine days, every ten days, bi-weekly, monthly and bi-monthly. In certain methods and embodiments of the present invention a single dose or infrequent dose (e.g., 2, 3, 4, 5 or six doses) can be sufficient to achieve the desired results. In other embodiments, the course of treatment may require the administration of many doses over many days, such as administration of a dose 4, 3, 2 or once daily over 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15 or more days.

Depending on the means of administration, the dosage may be administered all at once, such as with an oral formulation in a capsule, or slowly over a period of time, such as with an intravenous administration. For slower means of administration, the administering period can be a matter of minutes, such as about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 1 10, 1 15, 120 or more minutes, or a period of hours, such as about 0.5, 1 , 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5 or more hours.

A pharmaceutical composition, containing the compound PM181 104, can also be administered to a subject, in particular a human, with another pharmaceutically active ingredient or a pharmaceutical composition containing the active ingredient. The pharmaceutically active ingredient that may be used in conjunction with the compound PM181 104 may be an antimicrobial agent such as metronidazole or vancomycin, known to be useful in treating infections or diseases caused by Clostridium difficile.

The invention is explained in detail in the examples given below and should not be construed to limit the scope of the invention. The compound PM181 104 was obtained according to the method disclosed in PCT publication WO20071 19201 , which is incorporated herein in its entirety by reference.

Example 1

Isolation of culture no. ZMA B-1 from marine source

a) Composition of the isolation medium:

Zobell Marine Broth 2216 (agarified by 1.5% agar agar)

Peptic digest of animal tissue 5.0 g, yeast extract 1 .0 g, ferric citrate 0.1 g, sodium chloride 19.45 g, magnesium chloride 8.8 g, sodium sulphate 3.24 g, calcium chloride 1 .8 g, potassium chloride 0.55 g, sodium bicarbonate 0.16 g, potassium bromide 80.0 mg, strontium chloride 34.0 mg, boric acid 22.0 mg, sodium silicate 4.0 mg, sodium fluorate 2.4 mg, ammonium nitrate 1 .6 mg, disodium phosphate 8.0 mg, agar powder 15.0 g, double distilled water 1 .0 L, final pH (at 25°C) 7.4-7.8.

b) Procedure

The sponge sample, Spirastrella inconstans var. digitata (Dendy) was collected from Palk Bay, Tamil Nadu coast, India, by SCUBA diving, from a depth of three meters. The sponge sample was rinsed in sterile seawater and immediately transferred into sterile polyethene containers. The containers were stored at -20°C and transported by maintaining the temperature below 0°C, to the laboratory for further studies. On reaching the lab, the sponge samples were stored at less than 0°C and later thawed to room temperature (25+2 °C) just before isolation of the culture. The sponge sample was cut aseptically into 2 x 2 cm pieces and suspended in 5 ml. of sterile seawater in a 25 ml. sterilized test tube. The test tube was vortexed for 30 seconds; the seawater was drained out and fresh seawater was added. The same process was repeated three times. Finally, the seawater was drained out and the sponge piece was placed on petri plates containing above mentioned isolation medium [Zobell Marine Broth 2216 (agarified by 1 .5% agar agar); HiMedia]. The petri plate was incubated at room temperature (25+2°C) till growth was observed in the plates. The colonies grown on the plates were isolated on the basis of colony characteristics and streaked on petri plates containing above mentioned isolation medium [Zobell Marine Broth 2216 (agarified by 1 .5% agar agar); HiMedia]. The isolates were repeatedly subcultured till pure culture no. ZMA B-1 was obtained. The culture no. ZMA B-1 was thus isolated from amongst the growing microorganisms as a single isolate.

Example 2

Purification of culture no. ZMA B-1

a) Composition of the isolation medium:

Zobell Marine Broth 2216 (agarified by 1.5% agar agar)

Peptone 5.0 g, yeast extract 1 .0 g, ferric citrate 0.1 g, sodium chloride 19.45 g, magnesium chloride 8.8 g, sodium sulfate 3.24 g, calcium chloride 1 .8 g, potassium chloride 0.55 g, sodium bicarbonate 0.16 g, potassium bromide 0.08 g, strontium chloride 34.0 mg, boric acid 22.0 mg, sodium silicate 4.0 mg, sodium fluorate 2.4 mg, ammonium nitrate 1 .6 mg, disodium phosphate 8.0 mg, agar 15.0 g, demineralised water 1 .0 L, pH 7.4-7.8.

b) Procedure:

The culture was available on Zobell Marine Broth 2216 (agarified by 1 .5% agar agar) in 15 mm diameter petriplate. The growth on the petriplate was streaked on Zobell Marine Broth 2216 (agarified by 1 .5% agar agar) slant. The slant was incubated for 2 days at 25°C. One of the single colonies from the upper portion of the slant bed was transferred to fresh slants. The slants were incubated for 2 days at 25°C. These were then used for shake flask fermentation for the purpose of primary anti-infective screening.

Example 3

Maintenance of producer strain - culture no. ZMA B-1

a) Composition of the medium (Zobell Marine Broth 2216):

b) After dissolving the ingredients thoroughly by heating, the resultant solution was distributed in test tubes and sterilized at 121 °C for 30 min. The test tubes were cooled and allowed to solidify in a slanting position. The agar slants were streaked with the growth of culture no. ZMA B-1 by a wire loop and incubated at 27-29°C until a good growth was observed. The well-grown cultures were stored in the refrigerator at 4-8°C. Example 4

Fermentation of the culture no. ZMA B-1 in shake flasks

a) Composition of seed medium (Zobell Marine Broth 2216):

Peptone 5.0 g, yeast extract 1 .0 g, ferric citrate 0.1 g, sodium chloride 19.45 g, magnesium chloride 8.8 g, sodium sulfate 3.24 g, calcium chloride 1 .8 g, potassium chloride 0.55 g, sodium bicarbonate 0.16 g, potassium bromide 0.08 g, strontium chloride 34.0 mg, boric acid 22.0 mg, sodium silicate 4.0 mg, sodium fluorate 2.4 mg, ammonium nitrate 1.6 mg, disodium phosphate 8.0 mg, demineralised water 1 .0 L, pH 7.4-7.8.

b) The above medium was distributed in 40 ml amounts in 500 ml Erlenmeyer flasks and autoclaved at 121 °C for 30 mins. The flasks were cooled to room temperature and each flask was inoculated with a loopful of the well-grown producing strain (culture no. ZMA B-1 ) on the slant and shaken on a rotary shaker for 24-48 hours at 230-250 rpm at 30°C (±1 °C) to give seed culture. c) Composition of the production medium:

Peptone 5.0 g, yeast extract 1.0 g, ferric citrate 0.1 g, sodium chloride 19.45 g, magnesium chloride 8.8 g, sodium sulfate 3.24 g, calcium chloride 1 .8 g, potassium chloride 0.55 g, sodium bicarbonate 0.16 g, potassium bromide 0.08 g, strontium chloride 34.0 mg, boric acid 22.0 mg, sodium silicate 4.0 mg, sodium fluorate 2.4 mg, ammonium nitrate 1 .6 mg, disodium phosphate 8.0 mg, demineralised water 1 .0 L, pH 7.4-7.8

d) 40 ml of the production medium in 500 ml capacity Erlenmeyer flasks was autoclaved at 121 °C for 30 mins, cooled to 29-30°C and seeded with 2 ml of the seed culture mentioned in example 4b.

e) Fermentation parameters

Temperature 29-30°C; agitation 230-250 rpm; harvest time 48-72 hours.

The production of the compound PM181 104 in the fermentation broth was determined by testing the bioactivity S.aureus 3066 MRSA strain using the agar well diffusion method (by the procedure as described in PCT publication WO20071 19201 ). The harvest pH of the culture broth was 7.0-8.0. The culture broth was harvested and the whole broth was used for bioactivity testing, which is indicative of presence of the compound PM181 104 in the fermented broth. Example 5

Preparation of seed culture in shake flasks for fermentation

a) Composition of the medium:

Peptone 5.0 g, yeast extract 1 .0 g, ferric citrate 0.1 g, sodium chloride 19.45 g, magnesium chloride 8.8 g, sodium sulfate 3.24 g, calcium chloride 1 .8 g, potassium chloride 0.55 g, sodium bicarbonate 0.16 g, potassium bromide 0.08 g, strontium chloride 34.0 mg, boric acid 22.0 mg, sodium silicate 4.0 mg, sodium fluorate 2.4 mg, ammonium nitrate 1 .6 mg, disodium phosphate 8.0 mg, demineralised water 1 .0 L, pH 7.4-7.8.

b) The above medium was distributed in 200 ml amounts in 1 L Erlenmeyer flasks and autoclaved at 121 °C for 30 mins. The flasks were cooled to room temperature and each flask was inoculated with a loopful of the well-grown producing strain (culture no. ZMA B-1 ) on the slant and shaken on a rotary shaker for 24-48 hours at 230-250 rpm at 29-31 °C to give a seed culture. Example 6

Cultivation of the culture no. ZMA B-1 in fermenter

a) Composition of the production medium:

Glucose 50.0 g, yeast extract 1 1 .0 g, peptone 4.0 g, beef extract 4.0 g , calcium carbonate 5 g , sodium chloride 2.5 g, demineralized water 1 L, pH 7.6 (before sterilization).

b) 250 L of the production medium in 300 L fermenter along with 80 ml of desmophen as an antifoaming agent was sterilized in situ for 30 mins. at 121 °C, cooled to 29-31 °C and seeded with 6 L of the seed culture mentioned in example 5b. c) Fermentation parameters:

Temperature 30-32°C; agitation 100 rpm; aeration 150 Ipm; harvest time 44-66 hours.

The production of the compound PM181 104 in the fermentation broth was determined by testing the bioactivity against S. aureus 3066 (MRSA strain) using the agar well diffusion method (by the procedure as described in PCT publication WO20071 19201 ). The harvest pH of the culture broth was 7.0-8.0. The culture broth was harvested and the whole broth was used for isolation and purification of the compound PM181 104. Example 7

Isolation and purification of the compound PM181 104

The whole broth (240 L) of Example 6 was harvested and extracted using ethyl acetate (240 L) by stirring in a glass vessel. The organic layer was separated using disc stack separator (Alfa-laval, model No. LAPX404) and concentrated to obtain the crude extract (296 g). The crude material obtained was stirred and sonicated for 30 min using petroleum ether (3 X 1 L) and filtered to obtain insoluble residue (38 g), which was chromatographed by vacuum liquid chromatography using following method.

The insoluble residue (35.5 g) was dissolved in a mixture of methanol and acetonitrile (3:1 , 400 ml) and preadsorbed on to LiChroprep RP-18 [25-40 μ, 40 g] and applied to a fritted filter funnel (G-4 grade; 10 cm x 10.5 cm) packed with LiChroprep RP-18 (25-40 10g) adsorbent. Elution using house vacuum (100-120 mm) was done initially with water (4 L), followed by water:methanol (1 :1 , 5 L), methanol (3 L), methanohacetonitrile (2.5 L) and acetonitrile. The monitoring of the purification was done by bioassay against S. aureus 3066 (by the procedure as described in PCT publication WO20071 19201 ) and/or analytical HPLC. The compound PM181 104 was detected in methanol and methanokacetonitrile & acetonitrile fractions. Like fractions were pooled and concentrated to obtain the semi pure material (1 .826 g).

The final purification was done by repeated preparative HPLC using the following conditions:

Column Eurospher RP-18 (10μ, 32x250 mm)

Eluent acetonitrile:water (56:44)

Flow rate 50 ml/min

Detection (UV) 220 nm

Retention time 12-14 min

Purity of fractions was checked by bioassay against S. aureus 3066 (by the procedure as described in PCT publication WO20071 19201 ) and/or analytical HPLC. The eluates containing the compound PM181 104 were pooled and concentrated under reduced pressure to remove the solvent to obtain 600 mg of pure compound.

The physico-chemical and spectral properties of the compound PM181 104

Appearance White amorphous solid

Melting point >300°C (decomposes)

Solubility Methanol, DMSO

HPLC Rt 5.61 mins

Column Kromasil C18 (5 μ;150x4.6 mm I.D.)

(column temperature 40°C)

Mobile phase acetonitrile:water (1 :1 )

Injection vol. 10 μΙ (0.1 mg/ml concentration in mobile ph

Flow rate 1 ml/min

Detection 220 nm

HR-ESI(+)MS m/z: 1515.3733 (M+H)

Mol. Formula C₆₉H₆₆ 1₈O1₃S5

Mol. Weight 1514

UV (MeOH) 205.2, 220.6 and 306.2 nm IR(KBr) 3368, 2981,1654, 1516, 1429, 1314, 1199, 1269, 1074,

1034, 805, 580 cm^"1

1H NMR : refer to table 1.

¹³C NMR : refer to table 2.

Table 1 : ¹H NMR of the compound PM181104 in DMSO-d₆

Peak δ Peak δ Peak δ

1 1.33-1.34 (d, 3H) 21 5.33 (m, 1H) 41 8.57-8.58

(d, 1H)

2 1.94 (m, 4H) 22 5.52 (s, 1H) 42 8.65 (s, 1H)

3 2.05-2.06 23 5.63 (s, 1H) 43 8.81 (t, 2H)

(brm, 1H)

4 2.12 (brm, 1H) 24 5.83 (s, 1H) 44 9.07 (s, 1H)

5 2.25-2.28 25 5.92 (s, 1H) 45 9.16 (s, 1H)

(brd, 1H)

6 2.40-2.42 26 6.07 (s, 1H) 46 9.48 (s, 1H)

(brm, 1H)

7 2.68 (s, 3H) 27 6.50 (s, 1H) 47 10.03 (s, 1H)

8 2.76-2.82 (m, 2H) 28 6.59-6.61 (d, 2H)

9 2.97-2.99 (d, 1H) 29 6.86 (s, 1H)

10 3.17- 3.21 (m, 1H) 30 7.04-7.06 (d, 2H)

11 3.24-3.26 (m, 1H) 31 7.17-7.18 (m, 1H)

12 3.62 (t, 2H) 32 7.26 (d, 4H)

13 3.69 (m, 1H) 33 7.30 (s, 1H)

14 3.77 (s, 2H) 34 7.33-7.35 (d, 1H)

15 4.49-4.50 (d, 1H) 35 7.53 (s, 1H)

16 4.69-4.71 (t, 1H) 36 7.69 (s, 1H)

17 4.80 (m, 1H) 37 7.90 (s, 1H)

18 4.89 (m, 1H) 38 7.95 (s, 1H)

19 4.93-4.97 (t, 1H) 39 8.27-8.29 (d, 2H)

20 5.23 (t, 1H) 40 8.49-8.50 (d, 1H) Table 2: ^1JC NMR of the compound PM181 104 in DMSO-d₆

^* two carbons BIOLOGICAL EVALUATION OF THE COMPOUND PM181 104

In vitro assay

Example 8

The in vitro potency of the compound PM181 104 against multiple clinical isolates of Clostridium difficile was established by minimum inhibitory concentration (MIC) determinations. Due to concern over the possible deleterious effects of the blood component in the standard reference medium (Supplemented Brucella Agar), the compound was also tested in Wilkens-Chalgren Agar medium. This medium supports the growth of most anaerobic bacteria but is not supplemented with blood or blood products.

Materials and Methods

The compound PM181 104 was stored at 4°C until assayed. DMSO was used as solvent for the preparation of the stock solution of the compound PM181 104 [concentration was 1280 μg ml].

Information regarding the standards used is as follows:

DM water: Deminaralised water

Organisms

The test organisms for the assays were clinical isolates or reference strains acquired from the American Type Culture Collection (ATCC), USA. The quality control organisms included in the assay were: Bacteroides fragilis 0123 (ATCC 25285) & Clostridium difficile 4381 (ATCC 700057). The growth and test media were those recommended by the Clinical and Laboratory Standards Institute [Methods for Antimicrobial Susceptibility Testing of Anaerobic Bacteria; Approved Standard— Seventh Edition. CLSI document M1 1 -A7 [ISBN 1 -56238-626-3]; Clinical and Laboratory Standards Institute, 940 West Valley Road, Suite 1400, Wayne, Pennsylvania, 19087-1898, USA, 2007], for growth and susceptibility testing of anaerobes. The test organisms were maintained frozen at -80 °C. The isolates were subcultured on Supplemented Brucella Agar (SBA) plates (Cat. No. B0155; Teknova, Hollister, CA) in a Bactron II anaerobic chamber and incubated for 48 hours at 35- 36 °C in the Bactron II anaerobe chamber. Media

The reference medium employed for the agar dilution MIC assay of anaerobic bacteria was Brucella Agar (Becton Dickinson, Sparks, MD # 21 1086, Lot # 7166673) supplemented with hemin (Sigma, Lot #027K2055), Vitamin K1 (Sigma, Lot #106K1523), and 5% lysed sheep blood (Cleveland Scientific, Lot S03582). In addition, the compound was assayed using Wilkins-Chalgren Agar (Becton- Dickinson #218051 , Lot #91 10652).

Minimal Inhibitory Concentration (MIC) Assay Procedures

Anaerobic bacteria were assayed using a reference agar dilution method [Methods for Antimicrobial Susceptibility Testing of Anaerobic Bacteria; Approved Standard— Seventh Edition. CLSI document M1 1 -A7 [ISBN 1 -56238-626-3]; Clinical and Laboratory Standards Institute, 940 West Valley Road, Suite 1400, Wayne, Pennsylvania, 19087-1898, USA, 2007]. Dilutions of the compound PM181 104 or the standards were prepared manually. The compound PM181 104 supplemented agar plates were prepared manually. Similarly, standards supplemented agar plates were prepared manually. Organisms were grown in a Bactron II Anaerobic Chamber (Sheldon Manufacturing Inc., Cornelius, OR) for 48 hours prior to assay. Following inoculation, the compound PM181 104 supplemented agar plates or standards supplemented agar plates were incubated at 35 °C for 48 hours in the anaerobic environment (5% hydrogen, 5% carbon dioxide, 90% nitrogen) of the Bactron II. The MIC was read as per CLSI guidelines [Methods for Antimicrobial Susceptibility Testing of Anaerobic Bacteria; Approved Standard— Seventh Edition. CLSI document M1 1 -A7 [ISBN 1 -56238-626-3]; Clinical and Laboratory Standards Institute, 940 West Valley Road, Suite 1400, Wayne, Pennsylvania, 19087-1898, USA, 2007]. Results

The MIC results are summarized in Table 3 and Table 4. The MIC values for the quality control strains and standards, when tested in the reference SBA medium all fell within the published quality control ranges, thereby validating the assay procedure. The compound PM181 104 demonstrated potent activity against C. difficile, inhibiting all strains at 0.5 μg/ml or less. The activity of the compound was similar to that of metronidazole and greater than that of vancomycin and clindamycin. For the compound PM181 104, the MIC for 90% of the test isolates (MIC₉₀) was 0.5 μg/ml. The compound PM181 104 was inactive against the Gram-negative quality control organism B. fragilis. When tested in Wilkens-Chalgren Agar (in the absence of blood), the MIC values for the compound PM181 104 were two fold lower than those in SBA for 13 of 16 strains, and four fold lower than those in SBA for 3 of 16 strains. A difference in MIC value of two fold is not considered significant since it is within the standard error of the assay. Minimal differences in MICs in the two test media were also seen for the standards, though where they occurred, the result was always lower in Wilkens-Chalgren Agar than SBA.

Conclusion

The compound PM181 104 is a potent inhibitor of C. difficile, with activity similar to that of metronidazole.

Table 3: MIC ^g/ml) of PM181 104 and standards against anaerobic bacteria using

Supplemented Brucella Agar medium

¹ : Range of MIC values;

2: Concentration required to inhibit 50% of the test strains;

3: Concentration required to inhibit 90% of the test strains;

4: American Type Culture Collection;

⁵ ( ): Clinical and Laboratory Standards Institute Acceptable Limits for Quality Control Strains Table 4: MIC ^g/ml) of PM181 104 and standards against anaerobic bacteria using

Wilkens-Chalgren Agar medium

Range of MIC values;

Concentration required to inhibit 50% of the test strains;

Concentration required to inhibit 90% of the test strains;

American Type Culture Collection. It should be noted that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to a composition containing "a compound" includes a mixture of two or more compounds. It should also be noted that the term "or" is generally employed in its sense including "and/or" unless the content clearly dictates otherwise.

The invention has been described with reference to various specific and preferred embodiments and techniques. However, it should be understood that many variations and modifications may be made while remaining within the spirit and scope of the invention.

Claims

We claim:

1 . A compound PM181 104,

PM181 104

or a stereoisomer or a tautomer thereof, or a pharmaceutically acceptable salt thereof, for use in the treatment of Clostridium difficile associated infection or disease.

2. The compound PM181 104 for use according to claim 1 , wherein the Clostridium difficile associated infection or disease include: antibacterial associated diarrhea; antibacterial associated colitis; diarrhea; or colitis.

3. A method for the treatment or prophylaxis of Clostridium difficile associated infection or disease, comprising administering to a subject in need thereof, a therapeutically effective amount of the compound PM181 104, as defined in claim 1 , or a stereoisomer or a tautomer thereof, or a pharmaceutically acceptable salt thereof.

4. The method according to claim 3, wherein the Clostridium difficile associated infection or disease include: antibacterial associated diarrhea; antibacterial associated colitis; diarrhea; or colitis.

5. A method of inhibiting growth of Clostridium difficile (C. difficile) organism, comprising contacting C. difficile organism with the compound PM181 104 as defined in claim 1 or a stereoisomer or a tautomer thereof, or a pharmaceutically acceptable salt thereof in an amount sufficient to inhibit growth of C. difficile organism, thereby inhibiting growth of C. difficile.

6. The method according to claim 5, wherein C. difficile is contacted in vitro or in vivo.

7. The method according to claim 6, wherein C. difficile is contacted in vitro.

8. The method according to claim 6, wherein C. difficile is contacted in vivo.

9. A pharmaceutical composition comprising a therapeutically effective amount of the compound PM181 104, as defined in claim 1 , or a stereoisomer or a tautomer thereof, or a pharmaceutically acceptable salt thereof together with a pharmaceutically acceptable carrier, wherein the said pharmaceutical composition is adapted for use in the treatment of Clostridium difficile associated infection or disease.

10. The pharmaceutical composition according to claim 9, wherein the Clostridium difficile associated infection or disease include: antibacterial associated diarrhea; antibacterial associated colitis; diarrhea; or colitis.

1 1 . The pharmaceutical composition as claimed in claim 9, wherein the pharmaceutical composition is in the form of a tablet, coated tablet, capsule, granule, powder, cream, ointment, gel, syrup, emulsion, suspension, or solution for injection.