Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
  • A61K31/365—Lactones
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
  • A01N43/00—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds
  • A01N43/02—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms
  • A01N43/04—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms with one hetero atom
  • A01N43/22—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms with one hetero atom rings with more than six members
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
  • A61K31/4353—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems
  • A61K31/436—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems the heterocyclic ring system containing a six-membered ring having oxygen as a ring hetero atom, e.g. rapamycin
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
  • A61P1/12—Antidiarrhoeals
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/04—Antibacterial agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/10—Antimycotics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
  • C07H17/00—Compounds containing heterocyclic radicals directly attached to hetero atoms of saccharide radicals
  • C07H17/04—Heterocyclic radicals containing only oxygen as ring hetero atoms
  • C07H17/08—Hetero rings containing eight or more ring members, e.g. erythromycins

Definitions

• the present invention relates generally to the 18-membered macrocyclic antimicrobial agents called Tiacumicins, specifically, the R-Tiacumicin B or Tiacumicin B and its related compounds.
• substantially pure R-Tiacumicin B as a potent antibiotic agent for the treatment of bacterial infections, specifically GI infections caused by toxin producing strains of Clostridium difficile (C. difficile), Staphylococcus aureus (S. aureus) including methicillin-resistant Staphylococcus aureus (MRSA) and Clostridium perfringens (C. perfringens).
• C. difficile Clostridium difficile
• Staphylococcus aureus Staphylococcus aureus
• MRSA methicillin-resistant Staphylococcus aureus
• Clostridium perfringens C. perfringens
• Macrocycles are an important therapeutic class of antibiotics. These compounds are frequently produced as a family of closely related biogenetic congeners.
• the Tiacumicins are a series of 18-membered macrocyclic antibiotics in which the macrocyclic ring is glycosidically attached to one or two sugars. A seven-carbon sugar is esterf ⁇ ed at various positions with small fatty acids. The other sugar, when present, is esterified with an isomer of the fully substituted benzoic acid, everninic acid. (Journal of Liquid Chromatography, 1988, 11: 191-201).
• Tiacumicins are a family of related compounds that contain the 18-membered ring shown in Formula I below.
• Tiacumicin A-F Tiacumicin A-F
• substituents R 1 , R 2 , and R 3 US Patent No.4,918,174; J. Antibiotics, 1987, 40: 575-588, as shown in Table 1.
• Tiacumicins A-F have been characterized spectroscopically and by other physical methods.
• the chemical structures of Tiacumicins are based on spectroscopy: UV-vis, IR and 1 H and 13 C NMR, see for example J. Antibiotics, 1987, 40: 575-588. Inspection of Table 1 reveals that certain members of the family are structurally related isomers and/or differ by the presence or absence of certain moieties. Others differ in the nature of their ester groups.
• Tiacumicins are produced by bacteria, including Dactylosporangium aurantiacum subspecies hamdenensis, which may be obtained from the ARS Patent Collection of the Northern Regional Research Center, United States Department of Agriculture, 1815 North University Street, Peoria, IL 61604, accession number NRRL 18085. The characteristics of strain AB 718C-41 are given in J. Antibiotics, 1987, 40: 567-574 and US Patent No. 4,918,174.
• C. difficile-associated diarrhea is a disease characterized by severe and painful diarrhea. C. difficile is responsible for approximately 20% of the cases of antibiotic- associated diarrhea (AAD) and the majority of the cases of antibiotic-associated colitis (AAC). These diseases are typically caused by toxin producing strains of C. difficile, S. aureus including methicillin-resistant S. aureus (MRSA) and Clostridium perfringens (C. perfringens).
• AAD represents a major economic burden to the healthcare system that is conservatively estimated at $3-6 billion per year in excess hospital costs in the U.S. alone.
• VRE Vancomycin-resistant enterococci, for which intestinal colonization provides a constant reservoir for infection, has also emerged as a major nosocomial pathogen associated with increased health care cost and mortality.
• VRE can appear as coinfection in patients infected with C. difficile, or more commonly cause infection in certain high risk patients such as haematology and oncology patients, patients in intensive care units and patients receiving solid organ transplants.
• Methicillin-resistant Staphylococci such as MRSA
• Staphylococci are increasing in prevalence in both the hospital and community settings. Staphylococci are found on the skin and within the digestive and respiratory tracts but can infect open wounds and burns and can progress to serious systemic infection.
• the emergence of multi-drug resistant Staphylococci, especially, in the hospital where antibiotic use is frequent and selective pressure for drug-resistant organisms is high, has proven a challenge for treating these patients.
• the presence of MRSA on the skin of patients and health care workers promotes transmission of the multidrug resistant organisms. Similar diseases, including but not limited to clostridial enterocolitis, neonatal diarrhea, antibiotic-associated enterocolitis, sporadic enterocolitis, and nosocomial enterocolitis are also significant problems in some animal species.
• AAD is a significant problem in hospitals and long-term care facilities and in the community.
• C. difficile is the leading cause of AAD in the hospital setting, accounting for approximately 20% of cases of AAD and the majority of cases of antibiotic-associated colitis (AAC).
• AAC antibiotic-associated colitis
• CDAD Clostridium difficile-associated diarrhea
• PMC pseudomembranous colitis
• CDAD pseudomembranous colitis
• Diarrhea and colitis are caused by the elaboration of one or more C. difficile toxins.
• the organism proliferates in the colon in patients who have been given broad-spectrum antibiotics or, less commonly, cancer chemotherapy.
• CDAD is diagnosed in approximately 20% of hospitalized patients who develop diarrhea after treatment with such agents.
• VRE vancomycin-resistant Enterococcus
• VRSA vancomycin-resistant S. aureus
• Metronidazole is recommended as initial therapy out of concern for the promotion and selection of vancomycin resistant gut flora, especially enterococci. Despite reports that the frequency of C. difficile resistance may be >6% in some countries, metronidazole remains nearly as effective as vancomycin, is considerably less expensive, and can be used either orally or intravenously. Metronidazole is associated with significant adverse effects including nausea, neuropathy, leukopenia, seizures, and a toxic reaction to alcohol. Furthermore, it is not safe for use in children or pregnant women. Clinical recurrence occurs in up to 20% of cases after treatment with either vancomycin or metronidazole. Therapy with metronidazole has been reported to be an important risk factor for VRE colonization and infection.
• CDAD Clostridium difficile-associated diarrhea
• AAD Antibiotic-associated diarrhea
• AAC Antibiotic-associated colitis
• Tiacumicins specifically Tiacumicin B, show activity against a variety of bacterial pathogens and in particular against C. difficile, a Gram-positive bacterium (Antimicrob. Agents Chemother. 1991, 1108-1111).
• C. difficile is an anaerobic spore-forming bacterium that causes an infection of the bowel.
• C. difficile is a major causative agent of colitis (inflammation of the colon) and diarrhea that may occur following antibiotic intake. This bacterium is primarily acquired in hospitals and chronic care facilities. Because Tiacumicin B shows promising activity against C. difficile, it is expected to be useful in the treatment of bacterial infections, especially those of the gastrointestinal tract, in mammals. Examples of such treatments include but are not limited to treatment of colitis and treatment of irritable bowel syndrome. Tiacumicins may also find use for the treatment of gastrointestinal cancers.
• the present invention relates to new pharmaceutical compositions containing R- Tiacumicins, specifically the optically pure R-Tiacumicin B, and to the use of these new compositions in combination with existing drugs to treat infections caused by gram-positive anerobes.
• One embodiment of the present invention is directed towards the discovery that the chiral center at C- 19 of Tiacumicin B has great effect on biological activity. It has now been discovered that a substantially pure preparation of higher activity R-Tiacumicin B, which has an R-hydroxy group at C- 19 has surprisingly lower MIC values than the optically pure S- isomer of Tiacumicin B and other Tiacumicin B related compounds.
• the substantially pure R-Tiacumicin B has an unusually long post-antibiotic activity (PAE).
• PAE post-antibiotic activity
• This invention encompasses the composition of novel antibiotic agents, containing substantially pure R-Tiacumicins, by submerged aerobic fermentation of the microorganism Dactylosporangium aurantiacum subspecies hamdenensis.
• the production method is covered by WO 2004/014295 A2, which is hereby incorporated by reference.
• Figure 1 shows the Oak Ridge Thermal Ellipsoid Plot Program (ORTEP) chemical structure of R-Tiacumicin B.
• antibiotic-associated condition refers to a condition resulting when antibiotic therapy disturbs the balance of the microbial flora of the gut, allowing pathogenic organisms such as enterotoxin producing strains of C. difficile, S. aureus and C. perfringens to flourish. These organisms can cause diarrhea, pseudomembranous colitis, and colitis and are manifested by diarrhea, urgency, abdominal cramps, tenesmus, and fever among other symptoms. Diarrhea, when severe, causes dehydration and the medical complications associated with dehydration.
• asymmetrically substituted refers to a molecular structure in which an atom having four tetrahedral valences is attached to four different atoms or groups.
• the commonest cases involve the carbon atom.
• two optical isomers D- and L- enantiomers or R- and S- enantiomers
• R- and S- enantiomers two optical isomers per carbon atom
• Many compounds have more than one asymmetric carbon. This results in the possibility of many optical isomers, the number being determined by the formula 2 n , where n is the number of asymmetric carbons.
• broth refers to the fluid culture medium as obtained during or after fermentation. Broth comprises a mixture of water, the desired antibiotic(s), unused nutrients, living or dead organisms, metabolic products, and the adsorbent with or without adsorbed product.
• C- 19 Ketone refers to a Tiacumicin B related compound shown below in Formula II:
• enantiomer refers to a non-superimposable mirror image of itself.
• An enantiomer of an optically active isomer rotates plane polarized light in an equal but opposite direction of the original isomer.
• a solution of equal parts of an optically active isomer and its enantiomer is known as a racemic solution and has a net rotation of plane polarized light of zero.
• Enantiomers will have the opposite prefixes of each other: D- becomes L- or R- becomes S-. Often only one enantiomer is active in a biological system, because most biological reactions are enzymatic and the enzymes can only attach to one of the enantiomers.
• excipient refers to an inert substance added to a pharmacological composition to further facilitate administration of a compound.
• excipients include but are not limited to, calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols.
• halogen includes F, Cl, Br and I.
• isomeric mixture means a mixture of two or more configurationally distinct chemical species having the same chemical formula.
• An isomeric mixture is a genus comprising individual isomeric species.
• isomeric mixtures include stereoisomers (enantiomers and diastereomers), regioisomers, as might result for example from a pericyclic reaction.
• the compounds of the present invention comprise asymmetrically substituted carbon atoms. Such asymmetrically substituted carbon atoms can result in mixtures of stereoisomers at a particular asymmetrically substituted carbon atom or a single stereoisomer. As a result, racemic mixtures, mixtures of diastereomers, as well as single diastereomers of the compounds of the invention are included in the present invention.
• Lipiarmycin A4 refers to a Tiacumicin B related compound shown below in Formula III:
• lower alkyl refers to an optionally substituted straight-chain or optionally substituted branched-chain having from 1 to about 8 carbons (e.g., Ci, C 2 , C 3 , C 4 , C 5 , C 6 , C 7 , C 8 ,), more preferably 1 to 4 carbons (e.g., Ci, C 2 , C 3 , C 4 ,).
• alkyl radicals include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec- butyl, tert-butyl.
• a "lower alkyl” is generally a shorter alkyl, e.g., one containing from 1 to about 4 carbon atoms (e.g., Ci, C 2 , C 3 , C 4 ,).
• macrocycles refers to organic molecules with large ring structures usually containing over 10 atoms.
• 18-membered macrocycles refers to organic molecules with ring structures containing 18 atoms.
• membered ring can embrace any cyclic structure, including carbocycles and heterocycles as described above.
• membered is meant to denote the number of skeletal atoms that constitute the ring.
• pyridine, pyran and thiopyran are 6 membered rings and pyrrole, furan, and thiophene are 5 membered rings.
• MIC or "minimum inhibitory concentration” refers to the lowest concentration of an antibiotic that is needed to inhibit growth of a bacterial isolate in vitro.
• a common method for determining the MIC of an antibiotic is to prepare several tubes containing serial dilutions of the antibiotic, that are then inoculated with the bacterial isolate of interest. The MIC of an antibiotic can be determined from the tube with the lowest concentration that shows no turbidity (no growth).
• MIC 5 0 refers to the lowest concentration of antibiotic required to inhibit the growth of 50% of the bacterial strains tested within a given bacterial species.
• MIC 9 0 refers to the lowest concentration of antibiotic required to inhibit the growth of 90% of the bacterial strains tested within a given bacterial species.
• OPT-80 refers to a preparation containing approximately 70-100%, preferably, 90% (with respect to the whole antibiotic substance, by HPLC assay) of the optically pure R-Tiacumicin B (which has an R-hydroxy group at C-19, see Formula IV). The remaining portions consist essentially of small amounts of Tiacumicin B related compounds (including, but not limited to Lipiarmycin A4 and C-19 Ketone). Preparations of this type are described in detail in PCT application PCT/US03/21977, having an international publication number of WO 2004/014295 A2 and which preparations and are incorporated here by reference. However, for exclusive use in non-humans crude "OPT-80" that contains less than 70% of the optically pure R-Tiacumicin B (with respect to the whole antibiotic substance, by HPLC assay) may be used.
• ORTEP refers to the Oak Ridge Thermal Ellipsoid Plot computer program, written in Fortran, for drawing crystal structure illustrations. Ball-and-stick type illustrations of a quality suitable for publication are produced with either spheres or thermal- motion probability ellipsoids, derived from anisotropic temperature factor parameters, on the atomic sites. The program also produces stereoscopic pairs of illustrations which aid in the visualization of complex arrangements of atoms and their correlated thermal motion patterns.
• PAE or "post-antibiotic effect” refers to a well-established pharmacodynamic parameter that reflects the persistent suppression of bacterial growth following antibiotic exposure.
• patient refers to a human or animal in need of medical treatment.
• human patients are typically institutionalized in a primary medical care facility such as a hospital or nursing home.
• treatment of a disease associated with the use of antibiotics or cancer chemotherapies or antiviral therapies can occur on an outpatient basis, upon discharge from a primary care facility, or can be prescribed by a physician for home-care, not in association with a primary medical care facility.
• Animals in need of medical treatment are typically in the care of a veterinarian.
• pharmaceutically acceptable c arrier refers to a carrier or diluent that i s pharmaceutically acceptable.
• salts derived from appropriate bases include alkali metal (e.g., sodium or potasium), alkaline earth metal (e.g., magnesium), ammonium and N(Ci-C 4 alkyl) 4 + salts, and the like.
• alkali metal e.g., sodium or potasium
• alkaline earth metal e.g., magnesium
• ammonium and N(Ci-C 4 alkyl) 4 + salts and the like.
• Illustrative examples of some of these include sodium hydroxide, potassium hydroxide, choline hydroxide, sodium carbonate, and the like.
• composition refers to a mixture of one or more of the Tiacumicins described herein, or physiologically acceptable salts thereof, with other chemical components, such as physiologically acceptable carriers and/or excipients.
• the purpose of a pharmaceutical composition is to facilitate administration of a compound to an organism.
• physiologically acceptable carrier refers to a carrier or diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered compound.
• pseudomembranous colitis or "enteritis” refers to the formation of pseudomembranous material (i.e., material composed of fibrin, mucous, necrotic epithelial cells and leukocytes) due to inflammation of the mucous membrane of both the small and large intestine.
• R and S configuration, as used herein, are as defined by the IUPAC
• Chiral molecules can be named based on the atomic numbers of the atoms or groups of atoms, the ligands that are attached to the chiral center.
• the ligands are given a priority (the higher the atomic number the higher the priority) and if the priorities increase in a clockwise direction, they are said to be R-. Otherwise, if they are prioritized in a counterclockwise direction they are said to be S-.
• R-Tiacumicin B refers to the optically pure (R)-isomer of Tiacumicin B with an (R)-hydroxy group at C- 19, as shown below in Formula IV:
• S-Tiacumicin B refers to the optically pure (S)-isomer of Tiacumicin B with an (S)-hydroxy group at C-19, as shown below in Formula V:
• stereoisomers refers to compounds whose molecules have the same number and kind of atoms and the same atomic arrangement, but differ in their spatial arrangement.
• saccharide generally refers to mono-, di- or oligosaccharides.
• a saccharide may be substituted, for example, glucosamine, galactosamine, acetylglucose, acetylgalactose, N- acetylglucosamine, N-acetyl-galactosamine, galactosyl -N-acetylglucosamine, N- acetylneuraminic acid (sialic acid), etc., as well as sulfated and phosphorylated sugars.
• the saccharides are in their pyranose or furanose form.
• Tiacumicin refers to a family of compounds all of which comprise the 18-membered macrocycle shown below in Formula I:
• Tiacumicin B refers to the 18-membered macrocycle shown below in Formula VI:
• yield refers to an amount of crude Tiacumicin reconstituted in methanol to the same volume as the original fermentation broth. Yield is determined using standard HPLC techniques. Yield is reported in units of mg/L.
• This invention encompasses the composition of novel antibiotic agents, Tiacumicins, by submerged aerobic fermentation of the microorganism Dactylosporangiwn aurantiacum subspecies hamdenensis.
• the production method is covered by WO 2004/014295 A2.
• the present invention relates to new antibacterial compositions containing R- Tiacumicins, specifically the R-Tiacumicin B (which has an R-hydroxy at C- 19), and to the use of these new compositions in combination with existing drugs to treat infections caused by gram-positive anerobes.
• the present invention further relates to the novel OPT-80 preparation which contains 70-100% preferably, 90% (with respect to the whole antibiotic substance, by HPLC assay) of the R-Tiacumicin B.
• the remaining portions consist essentially of small amounts of Tiacumicin B related compounds (including, but not limited to Lipiarmycin A4 and C- 19 Ketone). Preparations of this type are described in detail in PCT application PCT/US03/21977, having an international publication number of WO 2004/014295 A2.
• PCT/US03/21977 having an international publication number of WO 2004/014295 A2.
• WO 2004/014295 A2 for exclusive use in non-humans crude OPT-80 that contains less than 70% of the R-Tiacumicin B (with respect to the whole antibiotic substance, by HPLC assay) may be used.
• Z is selected from H or lower alkyl, and wherein the term "lower alkyl” as used herein refers to branched or straight chain alkyl groups comprising one to five carbon atoms, including methyl, ethyl, propyl, isopropyl, n-butyl, t-butyl, and the like.
• the most preferred compounds of the invention are the compounds of Formula VII wherein X is ethyl, Y is OH R and Z is isopropyl.
• One embodiment of the present invention is directed towards the discovery that the chiral center at C- 19 of Tiacumicin B has great effect on biological activity. It has now been discovered that R-Tiacumicin B, which has an R-hydroxy group at C- 19 has significantly higher activity than the S-Tiacumicin B and other Tiacumicin B related compounds (Lipiarmycin A4 and C-19 Ketone). The higher activity is shown by much lowered MIC values, which can be seen below in Example 3, Tables 3 and 4 for several strains of C. difficile, S. aureus, E.faecalis, and E. faecium. This effect of the C-19 chiral center on biological activity is an unexpected and novel discovery.
• OPT-80 (which is composed almost entirely of the R-Tiacumicin B) has an unusually long post-antibiotic effect (PAE). This is discussed below in Example 4, where it is shown that OPT-80 has a PAE of greater than 24 hours. This PAE is unexpectedly longer than the usual antibiotic PAE of 1-5 hours.
• the present invention also relates to the disclosure of pharmaceutical compositions, which comprise a compound of the present invention in combination with a pharmaceutically acceptable carrier.
• Yet another aspect of the invention discloses a method of inhibiting or treating bacterial infections in humans, comprising administering to the patient a therapeutically effective amount of a compound of the invention alone or in combination with another antibacterial or antifungal agent.
• the 18-membered macrocycles and analogs thereof are produced by fermentation. Cultivation of Dactylosporangium aurantiacum subsp. hamdenensis AB 718C-41 NRRL 18085 for the production of the Tiacumicins is carried out in a medium containing carbon sources, inorganic salts and other organic ingredients with one or more absorbents under proper aeration conditions and mixing in a sterile environment.
• the microorganism to produce the active antibacterial agents was identified as belonging to the family Actinoplanaceae, genus Dactylosporangium (Journal of Antibiotics, 1987, 40: 567-574 and US patent 4,918, 174). It has been designated Dactylasporangium aurantiacum subspecies hamdenensis 718C-41.
• the subculture was obtained from the ARS Patent Collection of the Northern Regional Research Center, United States Department of Agriculture, 1815 North University Street, Peoria, IL. 61604, U.S.A., where it was assigned accession number NRRL 18085.
• the characteristics of strain AB 718C-41 are given in the Journal of Antibiotics, 1987, 40: 567-574 and US patent 4,918,174.
• This invention encompasses the composition of novel antibiotic agents, Tiacumicins, by submerged aerobic fermentation of the microorganism Dactylosporangium aurantiacum subspecies hamdenensis.
• the production method is covered by WO 2004/014295 A2, which is hereby incorporated by reference.
• compositions of the Tiacumicin compounds of the present invention may be formulated to release an antibiotic substantially immediately upon administration or at any predetermined time or time period after administration.
• compositions are generally known as modified release formulations, which include formulations that create a substantially constant concentration of the drug within the intestinal tract over an extended period of time, and formulations that have modified release characteristics based on temporal or environmental criteria as described in Modified-Release Drug Delivery Technology, ed. M. J. Rathbone, J. Hodgraft and M. S. Roberts. Marcel Dekker, Inc. New York.
• any oral biologically-acceptable dosage form, or combinations thereof, can be employed in the methods of the invention.
• dosage forms include, without limitation, chewable tablets, quick dissolve tablets, effervescent tablets, reconstitutable powders, elixirs, liquids, suppositories, creams, solutions, suspensions, emulsions, tablets, multi-layer tablets, bi-layer tablets, capsules, soft gelatin capsules, hard gelatin capsules, osmotic tablets, osmotic capsules, caplets, lozenges, chewable lozenges, beads, powders, granules, particles, microparticles, dispersible granules, ingestibles, infusions, health bars, confections, animal feeds, cereals, cereal coatings, foods, nutritive foods, functional foods and combinations thereof.
• the preparation of any of the above dosage forms is well known to persons of ordinary skill in the art.
• the pharmaceutical formulations may be designed to provide either immediate or controlled release of the antibiotic upon reaching the target site.
• the selection of immediate or controlled release compositions depends upon a variety of factors including the species and antibiotic susceptibility of Gram-positive bacteria being treated and the bacteriostatic/bactericidal characteristics of the therapeutics. Methods well known in the art for making formulations are found, for example, in Remington: The Science and Practice of Pharmacy (20th ed.), ed. A.R. Gennaro, 2000, Lippincott Williams & Wilkins, Philadelphia, or in Encyclopedia of Pharmaceutical Technology, eds. J. Swarbrick and J. C. Boylan, 1988-1999, Marcel Dekker, New York.
• Immediate release formulations for oral use include tablets or capsules containing the active ingredient(s) in a mixture with non-toxic pharmaceutically acceptable excipients.
• excipients may be, for example, inert diluents or fillers (e.g., sucrose, sorbitol, sugar, mannitol, microcrystalline cellulose, starches including potato starch, calcium carbonate, sodium chloride, lactose, calcium phosphate, calcium sulfate, or sodium phosphate); granulating and disintegrating agents (e.g., cellulose derivatives including microcrystalline cellulose, starches including potato starch, croscarmellose sodium, alginates, or alginic acid); binding agents (e.g., sucrose, glucose, mannitol, sorbitol, acacia, alginic acid, sodium alginate, gelatin, starch, pregelatmized starch, microcrystalline cellulose, magnesium aluminum silicate, carboxymethylcellulose sodium, methylcellulose,
• compositions can be colorants, flavoring agents, plasticizers, humectants, buffering agents, and the like as are found, for example, in The Handbook of Pharmaceutical Excipients, third edition, edited by Arthur H. Kibbe, American Pharmaceutical Association Washington DC.
• Dissolution or diffusion controlled release can be achieved by appropriate coating of a tablet, capsule, pellet, or granulate formulation of compounds, or by incorporating the compound into an appropriate matrix.
• a controlled release coating may include one or more of the coating substances mentioned above and/or, e.g., shellac, beeswax, glycowax, castor wax, carnauba wax, stearyl alcohol, glyceryl monostearate, glyceryl distearate, glycerol palmitostearate, ethylcellulose, acrylic resins, dl-polylactic acid, cellulose acetate butyrate, polyvinyl chloride, polyvinyl acetate, vinyl pyrrolidone, polyethylene, polymethacrylate, methylmethacrylate, 2-hydroxymethacrylate, methacrylate hydrogels, 1,3 butylene glycol, ethylene glycol methacrylate, and/or polyethylene glycols.
• the matrix material may also include, e.g., hydrated methylcellulose, carnauba wax and stearyl alcohol, carbopol 934, silicone, glyceryl tristearate, methyl acrylate-methyl methacrylate, polyvinyl chloride, polyethylene, and/or halogenated fluorocarbon.
• a controlled release composition may also be in the form of a buoyant tablet or capsule (i.e., a tablet or capsule that, upon oral administration, floats on top of the gastric content for a certain period of time).
• a buoyant tablet formulation of the compound(s) can be prepared by granulating a mixture of the antibiotic with excipients and 20-75% w/w of hydrocolloids, such as hydroxyethylcellulose, hydroxypropylcellulose, or hydroxypropylmethylcellulose. The obtained granules can then be compressed into tablets. On contact with the gastric juice, the tablet forms a substantially water-impermeable gel barrier around its surface. This gel barrier takes part in maintaining a density of less than one, thereby allowing the tablet to remain buoyant in the gastric juice.
• Other useful controlled release compositions are known in the art (see, for example, U.S. Patent Nos. 4,946,685 and 6,261,601).
• a modified release composition may be comprised of a compression-coated core whose geometric configuration controls the release profile of the encapsulated antibiotic. By varying the geometry of the core, the profile of the antibiotic release can be adjusted to follow zero order, first order or a combination of these orders.
• the system can also be designed to deliver more beneficial agents at the same time, each having a different release profile (see, for example U.S. Patent Nos. 4,111,202 and 3,279,995).
• Formulations that target the Tiacumicin compounds of the present invention, specifically OPT-80 (which is composed almost entirely of the R-Tiacumicin), that release to particular regions of the intestinal tract can also be prepared.
• the Tiacumicin compounds of the present invention, specifically OPT-80 can be encapsulated in an enteric coating that prevents release degradation and release from occurring in the stomach, but dissolves readily in the mildly acidic or neutral pH environment of the small intestine.
• a formulation targeted for release of antibiotic to the colon, utilizing technologies such as time-dependent, pH- dependent, or enzymatic erosion of polymer matrix or coating can also be used.
• the targeted delivery properties of the Tiacumicin compounds of the present invention, specifically OPT-80 (which is composed almost entirely of the R-Tiacumicin B), containing formulation may be modified by other means.
• the antibiotic may be complexed by inclusion, ionic association, hydrogen bonding, hydrophobic bonding, or covalent bonding.
• polymers or complexes susceptible to enzymatic or microbial lysis may also be used as a means to deliver drug.
• Microsphere encapsulation of the Tiacumicin compounds of the present invention, specifically OPT-80 (which is composed almost entirely of the R-Tiacumicin B), is another useful pharmaceutical formulation for targeted antibiotic release.
• the antibiotic-containing microspheres can be used alone for antibiotic delivery, or as one component of a two-stage release formulation. Suitable staged release formulations may consist of acid stable microspheres, encapsulating the compounds of the present invention, specifically OPT-80 (which is composed almost entirely of the R-Tiacumicin B), to be released later in the lower intestinal tract admixed with an immediate release formulation to deliver antibiotic to the stomach and upper duodenum.
• Microspheres can be made by any appropriate method, or from any pharmaceutically acceptable material. Particularly useful are proteinoid microspheres (see, for example, U.S. Patent Nos. 5,601,846, or 5,792,451) and PLGA-containing microspheres (see, for example, U.S. Patent Nos. 6,235,224 or 5,672,659). Other polymers commonly used in the formation of microspheres include, for example, poly- ⁇ -caprolactone, poly(e ⁇ caprolactone- Co-DL- lactic acid), poly(DL-lactic acid), poly(DL-lactic acid-Co-glycolic acid) and poly(s- caprolactone-Co-glycolic acid) (see, for example, Pitt et al, J. Pharm.
• Microspheres can be made by procedures well known in the art including spray drying, coacervation, and emulsification (see for example Davis et al. Microsphere and Drug Therapy, 1984, Elsevier; Benoit et al. Biodegradable Microspheres: Advances in Production Technologies, Chapter 3, ed. Benita, S, 1996, Dekker, New York; Microencapsulation and Related Drug Processes, Ed. Deasy, 1984, Dekker, New York; U.S. Patent No. 6,365,187).
• Powders, dispersible powders, or granules suitable for preparation of aqueous solutions or suspensions of the Tiacumicin compounds of the present invention, specifically OPT-80 (which is composed almost entirely of the R-Tiacumicin B), by addition of water are convenient dosage forms for oral administration.
• Formulation as a suspension provides the active ingredient in a mixture with a dispersing or wetting agent, suspending agent, and one or more preservatives.
• Suitable dispersing or wetting agents are, for example, naturally- occurring phosphatides (e.g., lecithin or condensation products of ethylene oxide with a fatty acid, a long chain aliphatic alcohol, or a partial ester derived from fatty acids) and a hexitol or a hexitol anhydride (e.g., polyoxyethylene stearate, polyoxyethylene sorbitol monooleate, polyoxyethylene sorbitan monooleate, and the like).
• Suitable suspending agents are, for example, sodium carboxymethylcellulose, methylcellulose, sodium alginate, and the like.
• the official chemical name is 3-[[[6-Deoxy-4- ⁇ 9-(3,5- dichloro-2-ethyl-4,6-dihydroxybenzoyl)-2-0-methyl- ⁇ -D-mannopyranosyl]oxy]-methyl]- 12(i?)-[[6-deoxy-5-C-methyl-4-O-(2-methyl-l-oxopropyl)- ⁇ -D-lyxo-hexopyranosyl]oxy]- 11 (5)-ethyl-8(S)-hydroxy-l 8(S)-( 1 (i?)-hydroxyethyl)-9, 13, 15-trimethyloxacyclooctadeca- 3, 5,9,13, 15-pentaene-2 -one.
• OPT-80 which is composed almost entirely of the R- Tiacumicin B, which is the most active component of OPT-80
• S-Tiacumicin B, Lipiarmycin A4, and C-19 ketone are summarized below.
• the structures of these compounds are shown in Formula VIII and Table 2 below.
• OPT-80 (which is composed almost entirely of the R-Tiacumicin B) and its related compounds were tested against C. difficile.
• the MIC values are reported below in Table 3. As we can see, OPT-80 was especially active when compared to S-Tiacumicin B and Lipiarmycin A4. Table 3: MIC /ml versus C. di cile strains
• OPT-80 (which is composed almost entirely of the R-Tiacumicin B) and its related compounds were tested against several other pathogens. The MIC values are reported below in Table 4. As we can see, OPT-80 was especially active when compared to S-Tiacumicin B and Lipiarmycin A4. Table 4: MIC ( ⁇ g/ml) against other microorganisms
• the post-antibiotic effect (PAE) of OPT-80 (which is composed almost entirely of the R-Tiacumicin B) was measured versus two strains of C. difficile, ATCC 43255 and a clinical isolate, LC3. Vancomycin and rifampin were tested additionally versus LC3. The PAE at 4x the MIC was observed to be extremely long: greater than 24 hours, for both strains. Because of the long duration of this effect, an exact PAE was not calculated. Vancomycin, on the other hand, had a more normal PAE of less than an hour when used at 4x the MIC versus strain LC3.
• OPT-80 which is composed almost entirely of the R-Tiacumicin B
• metronidazole metronidazole
• vancomycin vancomycin
• OPT-80 The in vitro activity of OPT-80 was determined against 350 anaerobes. The experimental procedure for which is outlined in Antimicrobial Agents and Chemotherapy , 2004, 48: 4430-4434, which is hereby incorporated by reference in its entirety. All organisms, including the 21 C. difficile strains, were separate isolates and not clonally related. All quality-control gram-negative and -positive strains recommended by NCCLS were included with each run: in every case, results (where available) were in range.
• Example 7 In Vitro Activities of OPT-80 Against Intestinal Bacteria
• Antimicrobial concentration ranges were selected to encompass or surpass the levels that would be achieved in the gut (to the extent that this information is available), subject to the limitations of solubility of the drugs in the testing medium.
• the range of concentration of OPT-80used during testing was 0.03 ⁇ g/ml to 1024 ⁇ g/ml.
• the bacteria tested were generally placed into genus, species, or other groups with at least 10 isolates.
• the ranges and the MICs at which 50 and 90% of isolates were inhibited were determined except for organisms with fewer than 10 strains tested, for which only the ranges are reported (Table 8).
• OPT-80 had good activity against most anaerobic gram-positive non-spore-forming rods and anaerobic gram-positive cocci. OPT-80 also showed good activity against enterococci and staphylococci.

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