Title of Invention	FLUORO SUBSTITUTED CYCLOALKANOINDOLES,COMPOSITIONS CONTAINING SUCH COMPOUNDS AND METHODS OF TREATMENT
Abstract	The present invention provides novel fluoro substituted cycloalkanoinadole derivatives which are prostaglandin receptor antagonists;more particularly,they are prostaglandin D2 receptor (DP receptor) antagonists.compounds of the present invention are useful for the tretment of various prostaglandin-mediated diseases and disorders. Accordingly,the present invention provides a method for the treatment of prostaglandin-mediated deseases using the novel compounds disclosed,as well as pharmaceutical compositions containing them.

Title of Invention

FLUORO SUBSTITUTED CYCLOALKANOINDOLES,COMPOSITIONS CONTAINING SUCH COMPOUNDS AND METHODS OF TREATMENT

Abstract

The present invention provides novel fluoro substituted cycloalkanoinadole derivatives which are prostaglandin receptor antagonists;more particularly,they are prostaglandin D2 receptor (DP receptor) antagonists.compounds of the present invention are useful for the tretment of various prostaglandin-mediated diseases and disorders. Accordingly,the present invention provides a method for the treatment of prostaglandin-mediated deseases using the novel compounds disclosed,as well as pharmaceutical compositions containing them.

Full Text	TREATMENT OF FUNGAL INFECTION WITH POLYENE OR BETA GLUCAN SYNTHASE INHIBITOR ANTIFUNGALS COMBINED WITH ANTI HSP90 ANTIBODIES The present invention relates to novel compositions and preparations that are effective antifungal agents, and a novel antibody which can be incorporated into the compositions and preparations. Fungal infections are a major cause of patient mortality in the intensive care unit and more generally in immunocompromised and debilitated patients (Gold, J.W.M., 1984, Am. J. Med 76:458-63; Klein, R,S. et al, 1984, N. Engl. J. Med ill: 354-357; Bumie, J.P., 1997, Current Anaesthesia & Critical Care 8: 180-183). The presence and persistence of fungal infections can be attributed to the selective pressure of broad-spectrum antifungals, frequently prolonged stay of patients in facilities such as an intensive care unit, problems in diagnosing the infections, and the lack of efficacy of the fungal agents used in therapy. While strict hygienic control may result in some prevention of fungal infections in a hospital or other environment, outbreaks of infections remain a serious problem and need to be addressed. Systemic fungal infections such as invasive candidiasis and invasive aspergillosis may be caused by a variety of fungal pathogens, for example the virulent Candida species C. albicans, C. tropicalis and C krusei and the less virulent species C parapsilosis and Torulopsis glabrata (the latter referred to in some texts as Candida glabrata). Although C albicans was once the most common fungal isolate obtained from intensive care units, recent studies indicate that C tropicalis^ C glabrata^ C parapsilosis and C krusei now account for about half of such isolates (Pfaller, M.A. et al^ 1998, J, Clin. Microbiol. M: 1886-1889; Pavese, P. et al, 1999, Pathol BioL 4^: 579-583). The rise of non-albicans species implies tiie emergence of Candida species resistant to conventional antifungal therapy (Walsh, TJ. et al., New Eng , J. Med. MQ: 764-771). Detection and diagnosis of the fungal pathogen responsible for an infection is critical for subsequent therapy because antifungal agents may be more effective agains certain strains- GB2240979 and EP0406029 (herein incorporated by reference in their entirety) disclosed a fungal stress protein and antibody thereto which could be used in a sensitive and highly specific test for detection of fungal pathogens. Traditionally, C albicans, C, tropicalis and C parapsilosis have been treated by the antifungal agent amphotericin B, regarded as the "gold standard" of systemic antifungal therapy (Bumie, J.P., 1997, supra). Unfortunately, amphotericin B is itself hihgly toxic and its use is tempered by side effects including chills, fever, myalgia or thrombophlebitis. Other antifungal agents include the oral azole drugs (miconazole, ketoconazole, itraconazole, fluconazole) and 5-fluorocytosine. However, fungal species such as C krusei and T. glabrata are resistant to fluconazole, and these species often occur in patients where this drug has been administered prophylactically- Furthermore, fluconazole-resistant strains of C albicans have been reported (Opportunistic Pathogens, 1997, ii 27-31). Thus despite the recent advances made in therapeutic drugs such as fluconazole, itraconazole and systemic liposomal-based variants of amphotericin B (Bumie, J.P., 1997, siq?ra\ the need for effective agents for treatment of fungal infections remains acute. The present invention addresses the above-identified need by providing a novel composition that is a significant improvement over prior art fungal agents for the treatment of human or animal fungal infections, and also a novel antibody which can be incorporated into the composition. The composition of the present invention comprises antibody which may bind one or more epitopes of a fungal stress protein, in combination with known antifungal agents. The inventors have found that, surprisingly, the efficacy of antifungal agents against fungal infections is significantly enhanced, allowing for either lower treatment dosages or more effective treatment at the same dose, which allows for reduction of unwanted side-effects. Furthermore, the composition of the present invention allows for effective treatment of fungal infections which are inherently resistant to the fungal agent used in the composition. According to the present invention there is provided the use of a composition comprising an antibody or an antigen binding fragment thereof specific for one or more epitopes of a fungal stress protein and an antifungal agent comprising at least one of the group consisting a polyene antifungal agent and an echinocandin. antifungal agent in a method of manufacture of a medicament for the treatment of fungal infections, wherein the fungus causing said fungal infection is resistant to said antifungal agent perse. Further provided according to the present invention is a combined preparation for simultaneous, separate or sequential use in the treatment of fungal infections, comprising an antibody or an antigen binding fragment thereof specific for one or more epitopes of a fungal stress protein and an antifungal agent comprising at least one of the group consisting a polyene antifungal agent and an echinocandin antifungal agent wherein the fungus causing said fungal infection is resistant to said antifungal agent perse. The antibody may be specific for a heat shock protein from a member of the Candida or Torulopsis genera. (The Candida and Torulopsis genera are generally deemed to be synonymous.) In particular, the antibody may be specific for the heat shock protein comprising hsp90 from Candida albicans, as described in GB2240979 and EP0406029- The antibody or an antigen binding fragment thereof may be specific for the epitope comprising the sequence of SEQ ID NO:1 Antibodies, their manufacture and uses are well known and disclosed in, for example, Harlow, E. and Lane, D., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1999. The antibodies may be generated using standard methods known in the art. Examples of antibodies include (but are not limited to) polyclonal, monoclonal, chimeric. single chain. Fab fragments, fragments produced by a Fab expression library, and antigen binding fragments of antibodies. Antibodies may be produced in a range of hosts, for example goats, rabbits, rats, mice, humans, and others. They may be immunized by injection with heat shock protein from the Candida genus, for example hsp90 from C. albicans, or any fragment or oligopeptide thereof which has immunogenic properties. Depending on the host species, various adjuvants may be used to increase an immunological response. Such adjuvants include, but are not limited to, Freund's, mineral gels such as aluminum hydroxide, and surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, and dinitrophenol. Among adjuvants used in humans, BCG (Bacille Calmette-Guerin) and Corynebacterivm parvum are particularly useful. Monoclonal antibodies to the heat shock protein from the Candida genus, for example hsp90 from C albicans, or any fragment or oligopeptide thereof may be prepared using any technique which provides for the production of antibody molecules by continuous cell lines in culture. These include, but are not limited to, the hybridoma technique, the human B-cell hybridoma technique, and the EBV-hybridoma technique (Koehler et aL, 1975, Nature, 256:495-497; Kosbor et aL, 1983, Immunol. Today 4:72; Cote et aL, 1983, PNAS USA, 80: 2026-2030; Cole et aL, 1985, Monoclonal Antibodies and Cancer Therapy, Alan R. Liss Inc., New York, pp. 17-96). In addition, techniques developed for the production of "chimeric antibodies", the splicing of mouse antibody genes to human antibody genes to obtain a molecule with appropriate antigen specificity and biological activity can be used (Morrison et aL, 1984, PNAS USA, 81: 6851-6855; Neuberger et aL, 1984, Nature, 212:604-608; Takeda et aL, 1985, Nature, 314: 452-454). Alternatively, techniques described for the production of single chain antibodies may be adapted, using methods known in the art, to produce The polyene antifungal agent may, for example, comprise amphotericin B, a derivative of amphotericin B, or nystatin. Derivatives of amphotericin B include formulations such as AmBisome (supplied for example by'NexStar Pharmaceuticals, Cambridge, UK), amphotericin-B lipid complex (Abelcet), amphotericin-B colloidal dispersion (Amphocil) and amphotericin-B intralipid emulsion (Bumie, J.P., 1997, supra), may be used. Amphotericin B may be used in combination with another antifungal agent, 5-fluorocytosine (Bumie, J.P., 1997, supra). The echinocandin antifungal agent may, for example, be Anidulafungin (LY303366; Eli Lilly & Co., Indianapolis, USA). Echinocandins are cyclic lipopeptides that inhibit synthesis of P-l,3-glucan in fungi (Redding, J.A, et al, 1998, Antimicrob. Agents Cherao. Ther. 42G): 1187-1194), The fungal infection which may be treated by the composition or combined preparation may be Mucormycosis, Blastomycosis, Coccidioidomycosis or Paracoccidioidomycosis, or the fungal infection may be caused by a Candida, Cryptococcus, Histoplasma, Aspergillus, or Torulopsis organism. The term "Coccidioidomycosis" is also referred to in the field as "Coccidiomycosis", and the term "Paracoccidioidomycosis" is likewise synonymous with "Paracoccidiomycosis". The fungal infection may be resistant to the antifungal agent perse, ie, fungal infections which are intrinsically untreatable by specific agents because that specific antifungal agent is ineffective as traditionally utilised on its own. Also provided is a composition or combined preparation as described herein for use in a method of treatment of fungal infections of the human or animal body. Also provided is a method of manufacture of a medicament for the treatment of fungal infections of the human or animal body characterised in the use of a composition or combined preparation as described in the present application. Methods of manufacture of medicaments are well known. For example, a medicament may additionally comprise a pharmaceutically acceptable carrier, diluent or excipient (Remington's Pharmaceutical Sciences and US Pharmacopoeia. 1984, Mack Publishing Company, Easton, PA, USA). Also provided is the use of a composition or combined preparation as described in the present application in a method of manufacture of a medicament for the treatment of fungal infections. The fungal infection may be resistant to the antifungal agent per se. Also provided is a method of treatment of fungal infections of the human or animal body comprising administering a composition or combined preparation according to the present application to a patient in need of same. The exact dose (i.e. a pharmaceutically acceptable dose) of the composition or combined preparation to be administered to a patient may be readily determined by one skilled in the art, for example by the use of simple dose-response experiments. The composition or combined preparation may be administered orally. Further provided according to the present invention is a kit comprising an antibody or an antigen binding fragment thereof specific for one or more epitopes of a fungal stress protein and an antifungal agent comprising any one of the group consisting a polyene antifungal agent and an echinocandin antifungal agent, for use in the treatment of fungal infections. The kit may be for use in the treatment of fungal infections, wherein the fungus causing the fungal infection is resistant to the antifungal agont per se. The antibody according to the invention may have a diagnostic use. Thus for diagnostic use the antibody may be employed to detect whether the stress protem is present in a host organism, to confirm whether the host has a particular fungal infection, for example Mucormycosis, Blastomycosis, Coccidioidomycosis or Paracoccidioidomycosis, or an infection due to a Candida, Cryptococcus, Histoplasma, AspergiIIus, or Torulopsis organism, or for example in the diagnosis of fungal abscesses, especially hepatic Candidiasis, and/or to monitor the progress of therapeutic treatment of such infections. Diagnostic methods of this type form a further aspect of the invention and may generally employ standard techniques, for example immunological methods such as enzyme-linked immunosorbent methods, radioimmuno-methods, latex agglutination methods or immunoblotting methods. The antibody according to the invention may be labelled with a detectable label or may be conjugated with effector molecule for example a drug e.g. an anti-fungal agent such as amphotericin B or fluorocytosine or a toxin, such as ricin, or an enzyme, using conventional procedures and the invention extends to such labelled antibodies or antibody conjugates. Also provided according to the present invention is the use of the antibody or antigen binding fragment according to the present invention in the preparation of a diagnostic for diagnosing one or more fungal infections. The diagnostic may be provided in a kit The kit may include instructions for use in diagnosing one or more fungal infections- The diagnostic kit as described herein is also provided according to the present invention. If desired, mixtures of antibodies may be used for diagnosis or treatment, for example mixtures of two or more antibodies recognising different epitopes of a fungal stress protein according to the invention, and/or mixtures of antibodies of a different class, e.g. mixtures of IgG and IgM antibodies recognising the same or different epitope(s) of the The contents of each of the references discussed herein, including the references cited therein, are herein incorporated by reference in their entirety. The present invention will be further apparent from the following description, which shows, by way of example only, specific embodiments of the composition and experimentation therewith. EXPERIMENTAL Experiments described below investigated the antifungal effect of antibody agamst an hsp90 antigen derived from Candida albicans used in combination with antifungals such as amphotericin B or fluconazole. Results show that, in some cases, the combination of antibody and antifungal agent causes an enhanced antifungal effect compared with each of the compounds on their own. A surprisingly strong synergistic effect is demonstrated for amphotericin B in combination with anti-Candida albicans hsp90 antibody against a variety of common problematic fungal pathogens. This synergistic effect has significant implications for clinical treatment of fungal infections. A preliminary clinical study involving four patients suffering from Candida infections demonstrated the effectiveness of the present mvention for humans. Material and Methods Strains: Non-Aspergillus yeast strains used (Table 1) were plated onto Sabouraud's dextrose agar (Oxoid, Basingstoke, UK) and incubated at 37oC for 24 hours. The strains were identified with the API 20C system (BioMerieux, Marcy L'Etoile, France). If needed, microscopical examinations of morphology on commeal agar (Oxoid) was used to confirm the identity. Isolates of Aspergillus spp (Table 1) were grown on Sabouraud's dextrose agar (Oxoid, Basingstoke, UK) at 35°C for 24 hours. For non-Aspergilliis strains, suspensions were prepared from individual colonies (diameter > 1mm) in 5 ml of sterile 0.85% saline to a density of 1 x 104 cells/mi as established by counting on a haemocytometer grid. For Aspergillus strains, see below. Antifungal agents: Amphotericin B was purchased from Sigma (Poole, Dorset) as a lyophilized powder for intravenous administration (Fungizone). Fluconazole was supplied as a solution for intravenous administration (Diflucan) by Pfizer, Amphotericin B was dissolved in dimethyl sulphoxide at a concentration of 1.2 mg/ml and fluconazole was dissolved in 0.85% saline also at a concentration of 1.2 rag/ml. Stock solutions were stored at -70oC until used. Abelcet (liposomal amphotericin B) manufactured by Bristol-Meyers Squib (USA) and prepared according to the manufacturers guidelines was used in the clinical study. Antibody: The DNA sequence of a former antibody specific for the Candida albicans hsp90 epitope disclosed in GB2240979 and EP0406029 was genetically modified by codon optimisation for expression in Escherichia coli (Operon Technologies Inc., Alameda, C A, USA) and inserted into an E. coli expression vector. The amino acid sequence of the anti-hsp90 antibody of the present invention comprises the sequence of SEQ ID NO: 2 (includes the heavy, light and spacer domains). The antibody according to the present invention recognises the epitope comprising the sequence of SEQ ID NO: 1. The anti-hsp90 antibody was expressed in an Escherichia coli host and then purified by affinity chromatography and an imidazole exchange column up to 95 % purity. Standard molecular biology protocols were employed (see, for example, Harlow & Lane, svpra; Sambrook, J, et al, 1989, Molecular Cloning: A Laboratory Manual 2nd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York; Sambrook, J. & Russell, D., 2001, Molecular Cloning: A Laboratory Manual. 3rd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor). Formulations of Mycograb (RTM) were prepared as follows: a vial containing 10 mg of pure auti-hsp90 antibody, 150 mg of pharmaceutical grade (Ph Eur) Urea and 174 mg L-Arginine (Ph Eur) were reconstituted in 5 ml water. Assay media: RPMI broth was prepared from RPMI1640 broth medium (Sigma R7880) supplemented with 0.3 g of glutamine per litre, buffered with 34.6 g of morpholine propanesulfonic acid (MOPS) per litre and adjusted to ph 7,0, The Broth microdilution test: Twofold dilutions (40 to 0.024 mg/ml for amphotericm B and 400 to 0,4 mg/ml for fluconazole) were prepared in RPMI broth starting from the two stock solutions. A 100 μl suspension of the inoculum diluted 1 in 10 (equivalent to 1 x 103 cfu) was added to the microtiter plates. To this was added 50 μl of the antifungal and then 50μl of the antibody. Antibody was either neat (0.4 mg/ml), or diluted to 1/10 or 1/100. When antibody was absent, the 50 μ1 volume was made up by RPMI. The total volume in each well was 200 μl. Final concentrations of antibody in the experiments were: 100 μ g/ml ("neat") 0,10 μg/ml ("1/10 antibody') or 1 μg/ml ("1/100 antibody"). Plates were incubated at 37oC overnight and the minimum inhibitory concentration (MIC) defined by the lower concentration inhibiting growth. Colony counts were determined for the wells where there was a visual reduction in yeast growth. Results were represented as colony forming units per ml of broth (cfu/ml). Aspergillus Studies: Isolates of Aspergillus fumigatus, Aspergillus flavus and Aspergillus niger were prepared in RPMI1640 medium. The suspensions were prepared to give a final inoculum of 2x10 conidia per ml and these were dispensed in 100 μl aliquots into flat-bottomed microtitre plates. A double dilution series of Amphotericin B ranging from 250 μg/ml to 0.75 μg/ml was prepared and dispensed to the appropriate wells. Mycograb was added to each of the wells at a final concentration of 100 μg/ml in formulation buffer. A control series was also prepared for each isolate which contained formulation buffer only. The plates were then incubated at 35*'Cy^00 rpm for 48 hours and MIC values for each isolate were determined by absence or presence of growth wells. Animal Synergy: Thirty CDl mice (each weighing about 25 g) were injected with 100μ1 of C albicans outbreak strain (equivalent to L5 x 10' cfu) and after 2 hours the mice were split into three groups and injected with: (A) Group 1 -100μl solution of 10 mM ammonium acetate (AAT; pH 9), followed by 100 Hl amphotericin B equivalent to 0.6 mg/kg in a 5% (w/v) glucose solution; (B) Group 2 - 100 ]i\ solution of 10 mM AAT (pH 9) solution containing 500 μg anti-hsp90 antibody, followed by 100 μl amphotericin B equivalent to 0,6 mg/kg in a 5 % (w/v) glucose solution; and (C) Group 3 - 100 μl solution of 10 mM AAT (pH 9) containing 50 μg anti-hsp90 antibody, followed by 100 μl amphotericin B equivalent to 0.6 mg/kg in a 5% (w/v) glucose solution. The animals were culled after 48 hours and yeast counts on Sabouraud's plates of liver, spleen and kidney tissue performed. Clinical study: An open-label safety and pharmacokinetics study of the anti-hsp90 antibody (in the form of Mycograb; see above) involving four patients suffering from Candida infections was conducted at the Central Manchester Health Care Trust Hospital and the Wythenshawe Hospital, both in Manchester, UK. Patients were examined for the signs of sepsis due to Candida, including: positive cultures of C albicans from multiple or deep sites; high or swimging temperature (pyrexia); high pulse rate (tachycardia) and high white cell count ("WBC"). Following conventional treatment with Abelcet (liposomal amphotericin B) and/or fluconazole, the patients were then additionally given various doses of Mycograb, including an optional test dose (0.1 mg/kg), and therapeutic dose(s) of 1 mg/kg. Patients were monitored for clinical and laboratory signs of infection (laboratory parameters tested include blood chemistry, haematology and clotting factors) and serum and urine levels of Mycograb tested. Results In vitro experiments examining the effect of combining an anti-Candida albicans hsp90 antibody ("antibody") and antifungal agents are presented m Tables 2 to 18. Animal experimental results are presented in Table 19. In vitro experiments: Compositions containing antibody and flaconazole: Table 2 shows the minimum inhibitory concentrations (MICs) of fluconazole against the test fungal pathogens, with or without the presence of the anti-C albicans hsp90 antibody at different dilutions, as assessed by the Broth microdilution test. In the presence of neat antibody and antibody diluted 10-fold, the MIC for the outbreak stram of C albicaiis was reduced four-fold (L56 μg/ml to 0.39 μg/ml fluconazole), whereas a IOC-fold dilution of the antibody resulted in a two-fold reduction of fluconazole MIC. A slight reduction in fluconazole MIC was observed for the fluconazole resistant strain of C. albicans and C. kmsei in the presence of neat antibody. At a dilution of 1/10 and 1/100, however, the antibody had no effect on the fluconazole MIC of these strains in comparison with no antibody. For the remaining fungal strains, ie. C. tropicalis, C parapsilosis and T, glabrata, the anti-C albicans hsp90 antibody had no discemable effect on the fluconazole MICs, Further experiments which quantified the number of cell colonies surviving at different fluconazole concentrations with different dilutions of the anti-C albicans hsp90 antibody were undertaken for each of the fungal strains represented in Table 2. At the fluconazole concentrations examined, the survival rate of C albicans (outbreak stram) was not reduced by the addition of neat antibody or antibody diluted 100-fold (Table 3). For the fluconazole-resistant strain of C. albicans, a two-fold reduction in colony survival was observed at 12,5μg/ml fluconazole in the presence of neat antibody (Table 4). Slight reductions in the survival rate of this stram were noticed at lower concentrations of fluconazole in the presence of neat antibody, but no effect was discemable at a 1/100 dilution of the antibody. Neat or diluted antibody had no significant antifungal effect against C krusei at the fluconazole concentrations tested (Table 5). For C. tropicalis, no marked effect on survival rate could be seen for each of the fluconazole concentrations examined in the presence or absence of antibody (Table 6), Table 7 shows that presence or absence of the antibody had no effect of the survival rate of T. glabrata colonies at each of the fluconazole concentrations tested- Presence or absence of the antibody had no notable effect on the survival rate of C parapsilosis colonies at the fluconazole concentrations as indicated in Table 8. Compositions with antibody and amphotericin B: Table 9 shows the minimum inhibitory concentrations (MICs) of amphotericin B against the test fungal pathogens, with or without the presence of the anti-C. albicans hsp90 antibody at different dilutions, as assessed by the Broth microdilution test. In contrast with the results obtained for fluconazole (see Tables 2-8 supra), all strains tested here showed at least a four-fold drop in MIC of amphotericin B when undiluted antibody was added to the incubation broth (Table 9). Furthennore, in all strains examined, there was at least a two-fold drop in amphotericin B MIC even when the antibody diluted 100-fold was added to the incubation broth (final antibody concentration: 1μg/ml). The greatest effect of the composition comprising antibody and amphotericin B at reducing the MIC of amphotericin B was observed with the fluconazole resistant strain of C. albicans. Neat antibody yielded a ten-fold reduction in the amphotericin B MC, and even at a 100-fold antibody dilution, the amphotericin B MIC was reduced by approximately 25 % (Table 9). Detailed experiments which quantified the number of cell colonies surviving at different amphotericin B concentrations with different dilutions of the anti-C albicans hsp90 antibody were undertaken for each of the fungal strains represented in Table 9. Table 10 shows survival rates for the outbreak strain of C albicans incubated with amphotericin B in the presence or absence of antibody. A dramatic reduction (at least 10-foid) m the number of surviving colonies was effected by the antibody at all the amphotericin concentrations tested. For example, at 0.078 μg/ml amphotericin B, the survival rate of C albicans (outbreak strain) was 0.2% in the presence of antibody diluted 100-fold compared with the survival rate of the strain without antibody. The inhibitory effect of the antibody diluted 100-fold at 0.078 μg/ml amphotericin B was equivalent to the survival rate of this strain at 0.156 μg/ml amphotericin B (without antibody). Therefore, even very diluted antibody is able to effect a reduction in the amount of amphotericin B required to achieve a specific mortality rate in this strain. Table 11 shows the survival rate of colonies of C albicans (fluconazole resistant strain) at different concentrations of amphotericin B and at different antibody dilutions. No noticeable effect of the antibody or amphotericin B could be seen at lower concentrations of the antifungal agent. However, at amphotericin B levels approachmg the MIC of the antifungal agent (see Table 9, supra), the antibody was observed to have a marked effect on colony survival. For example, at 0,078 μg/ml amphotericin B, antibody at a 100-fold dilution effected a survival rate of this strain of 0.1 % compared with no antibody. Colony survival rates of C knisei in the presence of amphotericin B and different amounts of antibody are represented in Table 12. The antibody can be seen to be very effective against this strain at the higher concentrations of amphotericin B examined. Even at a 100-fold dilution, the number of C krusei colonies detected in the presence of 0312 fig/ml amphotericin B v/as 0.01 % of those surviving without antibody. For all concentrations of amphotericin B tested (range from 0.019 - 0.156 μg/ml), the antibody was seen to be effective at reducing the colony survival rate of the strain C tropicalis (Table 13). The effect was enhanced at higher antibody and amphotericin B concentrations. Table 14 shows the survival rate for T. glabrata in the presence of various concentrations of amphotericin B and antibody. The antibody was observed to be highly effective at inhibiting growth of T. glabrata at all concentrations of amphotericin B tested. For example, at a 100-fold dilution of the antibody, the growth of this strain was inhibited by 99.2 % at 0.009 μg/ml amphotericin B, 99.99% at 0.019μg/ml amphotericin B and 99.91 % at 0.039μg/ml amphotericin B. The survival of the fungal strain C parapsilosis at different levels of antibody and amphotericin B is shown in Table 15. Neat antibody was observed to achieve a reduction in the survival rate of this strain at all levels of amphotericin B tested. At lower concentrations of antibody, the effect was less dramatic than for the other strains examined. Compositions with antibody but without antifungal agent: In a further experiment, the effect of different concentrations of the anti-C albicans hsp90 antibody alone (no antifungal agent) on the different fungal strains (used in Tables 2-15, supra) was tested. The results shown in Table 16 reveal that for the most of the strains used, the antibody itself had no effect on their survival. However, some diminution in survival rate which can be attributed to the antibody alone was observed in the strains L glabrata, C tropicalis and C parapsilosis. Experiments with Aspergillus spp: The MIC of Aspergillus fumigatus to Amphotericin B was 2.5μg/ml. With the addition of Mycograb, the MIC shifted to 0.125μg/ml (two-fold decrease). The MIC of Aspergillus flavus to Amphotericin B was 2.5μg/ml. With the addition of 100 μLg/ml of Mycograb the MIC shifted to 0.125 μg/ml (two-fold decrease). The MIC of Aspergillus niger to Amphotericin B was 2.5 μg/ml. With addition of 100 μg/ml of Mycograb the MIC shifted to 0.125 μg/ml (two-fold decrease). Summary of in vitro results: The results shown in Tables 2-16 reveal that while the anti-C albicans hsp90 antibody on its own was able to inhibit growth of certain fungal strains, a surprisingly high level of antifungal activity against all the strains examined was observed when the antibody was used in combination with amphotericm B- This surprising effect between the antibody and amphotericin B is not observed with other antifungal agents examined: fluconazole combined with the antibody did not produce a significant and potentially useful outcome. Using the cut-off criterion of a four-fold difference in MIC improvement, data in Table 2 reveal that fluconazole combined with neat antibody (final concentration 100 μg/ml) or a 10-fold dilution of antibody (final concentration 10 μg/ml) was effective only against the outbreak strain of C albicans. However, using the same criterion, it can be seen from Table 9 that amphotericin B combined with neat antibody or a 10-fold dilution of the antibody was effective agamst all the fungal strains tested. It can therefore be concluded that there is a strong synergy between amphotericin B and the anti-C albicans hsp90 antibody as measured by improvement in MIC, For the experiments in which fungal colonies were quantified for different antifungal and antibody treatments (see Tables 3-8 and 10-15, supra), a different cut-off criterion which defines a two log drop (100-fold) drop in surviving colonies can be employed to assess potentially useful combinations of treatments. A summary of the results for fluconazole in combination with the anti-C albicans hsp90 antibody is presented in Table 17. Here, the lowest concentration of fluconazole resulting in the desired effect (or the highest concentration used in the experiment) used is shown, together with an mdication of the cut-off criterion of at least a 100-fold drop is fungal survival rate was achieved. The results show that only the fluconazole resistant strain of C albicans when combined with fluconazole and neat antibody produced a significant effect. A summary of the results for amphotericin B in combination with the anti-C albicans hsp90 antibody is presented in Table 18. It can be seen that the cut-off criterion (100-fold reduction in fungal colony growth) is satisfied with neat antibody for all fungal strains examined, with a 10-fold dilution of the antibody for C albicans (outbreak strain and fluconazole resistant stram), C krusei and T. glabrata, and with a 100-fold reduction in antibody for C. albicans (outbreak strain) and T glabrata. It is noteworthy that synergy between amphotericin B and the antibody was observed not only against fluconazole sensitive strains of C albicans but also fluconazole resistant strains of C albicans and yeasts such as Candida krusei and T. glabrata which are intrinsically resistant to fluconazoles. (+ indicates cut-off criterion satisfied; - indicates cut-off criterion not satisfied; cut-ofif criterion is at least 100-fold reduction in colony count) The results with Aspergillus spp show that there was synergy between Amphotericin B and Mycograb in vitro against the commonest aspergillus spp. (2) Animal experiments: Mice infected with the outbreak strain of Candida albicans were treated with amphotericin B only (Group 1), amphotericin B and 500 μg anti-hsp90 antibody (Group 2) and amphotericin B and 50μg anti-hsp90 antibody (Group 3). Yeast colony counts for various tissues from the mice after a treatment period of 48 hours are shown in Table 19. The results show that animals treated with amphotericin B and 500μg antibody (Group 2) showed a significant reduction (at least one order of magnitude) in the number of yeast counts compared with animals treated with amphotericin B only (Group 1). Animals treated with amphotericin B and 50 fig antibody (Group 3) also showed diminished yeast counts compared with the animals treated with amphotericin B only (Group 1). The in vivo data therefore corroborates the in vitro data and confirms the synergy between the anti-hsp90 antibody and the antifungal agent amphotericin B for the treatment of fungal infections. (3) Clinical Study: Four patients with evidence of Candida infection and who were not responding to conventional antifungal treatment were given a combined treatment of antifungals and anti-hsp90 antibody in the form of Mycograb (see above) and their condition monitored. Patient 1 was given a primary diagnosis of acute pancreatitis and the patient had postoperative adult respiratory distress syndrome (ARDS) requiring ventilation. C albicans was grown in vitro from multiple sites including pancreatic bed. The patient had a very high white cell count (WBC) (78,4), although this was highly variable and may not have been caused by the sepsis alone. Abelcet treatment at 3 mg/kg was initiated. Five days after initiation of Abelcet treatment, Patient 1 was additionally given a first test dose of Mycograb at 0.1 mg/kg (Day 1). On Day 3, the patient was given a clincal dose of Mycograb at 1 mg/kg. Due to several factors, for example a worsening platelet count which had been low for at least four days, the Abelcet and Mycograb treatments were discontinued on Day 3 after the clinical dose of Mycograb had been given. However, Patient 1 regrew C. albicans from ascites six days later (Day 9) and was put on fluconazole (400 mg). The following day (Day 10), the patient was given the final two clinical doses of Mycograb at 1 mg/kg per dose. Although Patient 1 had been on Abelcet for seven days, before the patient received the clinical dose of Mycograb on Day 3, C albicans was still being grown firom the patient's trachyostomy site and the patient had a tachycardia. The combined treatment with Abelcet and Mycograb on Day 3 resulted in a period of five days during which C. albicans was not grown. No Mycograb-related changes in blood chemistry, haematology and clotting factors were observed during treatment with Mycograb, Treatment of the subsequent recurrence with fluconazole and Mycograb (two doses on Day 10) was less successful, as would be expected firom the in vitro synergy results (see for example Tables 2 and 17), but the patient did eventually recover firom the candidosis. Serum levels of Mycograb in Patient 1 at different time intervals following administration of Mycograb doses are shown in Table 20. The test dose at Day 1 did not give measurable serum levels. The 1.0 mg/kg doses at Day 3 and Day 10 did give detectable serum levels, and these levels were comparable with those at which synergy with amphotericin B was demonstrable in vitro (see Tables 9 and 18). Following the second dose on Day 10, serum levels of Mycograb improved, indicating some tissue accumulation following the first dose. Mycograb was detectable in the urine at the 1.0 mg/ml doses (data not shown). Table 20. Serum levels (in μg/ml) of Mycograb in Patient 1 Patient 2 was diagnosed as having a small bowel constriction due to adhesions and had ARDS requiring ventilation. C albicans was grown from multiple sites including ascitic fluid, with the infection associated with a fluctuating temperature (35.8-38.2oC), raised WBC (11.4) and occasional tachycardia (110). The patient was started on Abelcet at 3 mg/kg. Four days after the commencement of Abelcet treatment, Patient 2 still retained a fluctuating temperature, raised WBC and occasional tachycardia. The patient was given a 0.1 mg/kg test dose of Mycograb (Day 1). The following day, the patient was given a clinical dose of 1 mg/kg Mycograb (Day 2), The last dose of Abelcet was also given on Day 2 due to completion of a 5 day treatment program. Two days later (Day 4), the patient received the final two clinical doses of Mycograb. The Mycograb was well tolerated by the patient. The clinical dose of Mycograb on Day 2 was associated with a falling and stabilising temperature (38,2 to 36.7oC on Day 2 after receiving the clinical dose, staying at 36.7-37.4°C through to Day 3) and a falling WBC (from 11.9 to 9.6). On Day 4, the patient was looking clinically better and no C albicans was grown from ascites, blood cultures or urine. No Mycograb-related changes in blood chemistry, haematology and dotting factors were observed during treatment. Subsequent recovery was complicated by an episode of bacterial sepsis but this responded to antibiotics and the patient made a full recovery. Serum levels of Mycograb in Patient 2 at different time intervals following administration of Mycograb doses are shown in Table 21. The test dose at Day 1 did not give measurable serum levels. The 1.0 mg/kg dose at Day 2 did give detectable serum levels, and these levels were compatible with those at which synergy with amphotericin B was demonstrable in vitro (see Tables 9 and 18). Mycograb was detectable in the urine at the 1.0 mg/kg doses (data not shown). Patient 3 had a six week history of pancreatitis which led to an 80% pancreatectomy. The patient had moderately raised LFT (liver function test) levels, possibly related to alcohol abuse, and was an MRSA (Methicillin-resistant Staphylococcus avreus) carrier. C albicans were grown from multiple sites so the patient was treated with intravenous fluconazole. Twelve days later, after failing to respond to fluconazole, the patient was changed to 300 mg Abelcet. Three days later, the patient was still pyrexial (38.5oC) and C albicans was still growing from multiple sites (abdominal drains and gastroscopy tube), and was therefore given a clinical dose (1 mg/kg) of Mycograb (Day 1) in addition to the Abelcet. On the same day as the Mycograb dose was given, Patient 3 suffered an acute episode of Gram-negative-type septic shock (high temperature of 39.5oC, hypotensive), probably due to Pseudomonas aeruginosa, which was subsequently grown from his pancreatic drain, although he also grow Enterococcus faecalis from blood cultures. Due to this episode, no further Mycograb doses were given. The patient subsequently responded to antibiotic therapy (vancomycin and ceftazidime). Due to the bacterial complications, it was difficult to assess the impact of the single dose of Mycograb on Patient 3. However, it was noted that he stopped growing C. albicans (for example, from his gastrostomy tube and wound drain) for 48 hours after the dose and that he became apyrexial on Day 2 and Day 3. No Mycograb-related changes in laboratory parameters (blood chemistry, haematology and clotting factors) were observed. On Day 4, the patient had a recurrence of C albicans while he was still on Abelcet but a full recovery was made subsequently. Serum levels of Mycograb in Patient 3 at different time intervals following administration of the Mycograb dose are shown in Table 22. The single 1.0 mg/kg dose at Day 1 gave detectable serum levels, and these levels were compatible with those at which synergy with amphotericin B was demonstrable in vitro (see Tables 9 and 18). Mycograb was also detectable in the urine following the 1.0 mg/kg dose (data not shovra). Patient 4 was diagnosed with C albicans empyema, although the patient was originally admitted to ITU (Intensive Treatment Unit) with a lung abscess due to Streptococcus milleri (isolated from blood cultures), C albicans was grown from two bronchial lavage specimens (right and left lung) and three and four days later from two empyema fluid specimens. Treatment was started the following day with Abelcet (5 mg/kg). Five days after commencement of Abelcet treatment, some clinical deterioration was noted and the following moming (Day 1) this was associated with high WBC (15.7) and C albicans regrown from an intercostal drain fluid, Mycograb (1 mg/kg bd) was given to Patient 4 at 8.30 am and 8.30 pm on Day 1. Apart from a temporary rise in temperature on the night of Day 1, the patient improved clinically, C albicans was not grown from empyema fluid specimen cultured on Day 3, and the patient became progressively better. The Mycograb was well tolerated by Patient 4, No Mycograb-related changes in laboratory parameters (blood chemistry, haematology and clotting factors) were observed. Thus the patient was still growing C albicans from a chest drain six days after commencing Abelcet treatment and his WBC was high (15.7) just before receiving the first Mycograb dose, but thereafter the patient steadily improved and stopped growing C. albicans. Serum levels of Mycograb in Patient 4 at different time intervals following administration of Mycograb doses are shown in Table 23, The 1.0 mg/kg doses given on Day 1 gave detectable serum levels which were compatible with those at which synergy with amphotericin B was demonstrable in vitro (see Tables 9 and 18). Following the second dose on Day 1, semm levels of Mycograb improved, indicating some tissue accumulation following the first dose. Mycograb was detectable in the urine at the 1.0 mg/ml doses (data not shown). Conclusions The data presented here clearly demonstrates that there is a surprising synergism between the znii-Candida hsp90 antibody and the antifungal agent amphotericin B which effects enhanced antifungal activity against a wide variety of pathologically important fungal strains. These results allows for the use of novel, highly effective compositions for the treatment of human or animal fungal infections, and also a novel antibody which can be incorporated into the composition. The present invention allows for either lower treatment dosages or more effective treatment at the same dosages, thereby reducing unwanted side-effects. Clinical implications of the present invention include: (i) the production of a synergistic combination of amphotericin B and anti-hsp90 antibody in the treatment of disseminated yeast infection should become the treatment of choice. This would lead to a reduction in mortality and morbidity for these infections. The preliminary clinical study results provided herewith confirm the efficacy of the present invention in comparison with existing methods of treatment; (ii) amphotericin B is a toxic, particularly nephrotoxic, drug. The synergy provided by the present invention means that a lower dose of amphotericin B could be used while maintaining efficacy and concomitantly reducing toxicity; and (iii) the toxicity sparing effect of the anti-hsp90 antibody would allow the clinical efficacy of higher doses of amphotericin B to be explored and further contribute to an improved clinical outcome. We claim: and pharmaceutically acceptable salts thereof, wherein n is 0 or 1; m is 1, 2 or 3; R1 is H, C1-C3 alkyl, halogenated C1-C3 alkyl or cyclopropyl; R2 is 4-chlorophenyl or 2,4,6-trichlorophenyl. 2. The compound as claimed in as claimed in claim 1 wherein n is 0. 3. The compound as claimed in claim 1 wherein n is 1. 4. The compound as claimed in claim 1 wherein m is 1. 5. The compound as claimed in claim 1 wherein m is 2. 6. The compound as claimed in claim 1 wherein R] is H. 7. The compound as claimed in claim 1 wherein R] is CH3. 8. The compound as claimed in claim 1 wherein R2 is 4-chlorophenyl. 9. The compound as claimed in claim 1 wherein R2 is 2,4,6-trichlorophenyl. 10. The compound as claimed in claim 1 having the stereoconfiguration shown below: 11. The compound as claimed in claim 10 wherein n is 0. 12. The compound as claimed in claim 10 wherein n is 1. 13. The compound as claimed in claim 10 wherein m is 1. 14. The compound as claimed in claim 10 wherein m is 2. 15. The compound as claimed in claim 10 wherein R1 is H. 16. The compound as claimed in claim 10 wherein R1 is CH3. 17. The compound as claimed in claim 10 wherein R2 is 4-chloro-phenyl. 18. The compound as claimed in claim 10 wherein R2 is 2,4,6-trichlorophenyl. 19. A compound according to claim 1 or 10 selected from [4-(4-chlorobenzyl)-7-fluoro-5-(methanesulfonyl)-l,2,3,4-tetrahydrocyclopenta[b]-indol-3-yl]acetic acid, {4-[ 1 -(4-chlorophenyl)ethyl]-7-fluoro-5-methanesulfonyl-1,2,3,4-tetrahydrocyclo-penta[b]indol-3-yl}acetic acid, [9-(4-chlorobenzyl)-6-fluoro-8-methanesulfonyl-2,3,4,9-tetrahydro-lH-carbazol-l-yl]- acetic acid, and [4-(4-chlorobenzyl)-7-fluoro-5-methanesulfonyl-l-OXO-1,2,3,4-tetrahydro-cyclopenta- [b]indol-3-yl]acetic acid, or a pharmaceutically acceptable salt thereof. 20. The compound according to claim 19, wherein the compound is (-)-[4-(4-chlorobenzyl)-7-fluoro-5-(methanesulfonyl)-l,2,3,4-tetrahydrocyclopenta[b]-indo]-3-yl]acetic acid and pharmaceutically acceptable salts thereof. 21. The compound according to claim 19, wherein the compound is (+/-)-4-[l-(4-chlorophenyl)ethyl]-7-fluoro-5-methanesulfonyM,2,3,4-tetrahydro-cyclopenta[b]indol-3-yl}acetic acid and pharmaceutically acceptable salts thereof. 22. The compound according to claim 19, wherein the compound is (+/-)-[9-(4-chlorobenzyl)-6-fluoro-8-methanesulfonyl-2,3,4,9-tetrahydro-lH-carbazol-l-yl]acetic acid and pharmaceutically acceptable salts thereof. 23. The compound according to claim 19, wherein the compound is [4-(4-chlorobenzyl)-7-fluoro-5-methanesulfonyl-l-oxo-l,2,3,4-tetrahydro-cyclopenta[b]indol-3-yl]acetic acid and pharmaceutically acceptable salts thereof. 24. A pharmaceutical composition comprising a compound of claim 1 and a pharmaceutically acceptable carrier. 25. A pharmaceutical composition comprising a compound of any one of the claims 1 to 23, a second pharmaceutical agent selected from an antihistamine, a leukotrine antagonist and a leukotriene biosynthesis inhibitor and a pharmaceutically acceptable carrier. 26. A pharmaceutically acceptable salt of a compound of formula I as defined in any one of claims 1 to 18. Dated this 21st day of July 2004 T.SRINIVASAN Agent for the Applicant To The Controller of Patents The Patent Office at Chennai

Full Text

TREATMENT OF FUNGAL INFECTION WITH POLYENE OR BETA GLUCAN SYNTHASE INHIBITOR ANTIFUNGALS COMBINED WITH ANTI HSP90 ANTIBODIES
The present invention relates to novel compositions and preparations that are effective antifungal agents, and a novel antibody which can be incorporated into the compositions and preparations.
Fungal infections are a major cause of patient mortality in the intensive care unit and more generally in immunocompromised and debilitated patients (Gold, J.W.M., 1984, Am. J. Med 76:458-63; Klein, R,S. et al, 1984, N. Engl. J. Med ill: 354-357; Bumie, J.P., 1997, Current Anaesthesia & Critical Care 8: 180-183). The presence and persistence of fungal infections can be attributed to the selective pressure of broad-spectrum antifungals, frequently prolonged stay of patients in facilities such as an intensive care unit, problems in diagnosing the infections, and the lack of efficacy of the fungal agents used in therapy. While strict hygienic control may result in some prevention of fungal infections in a hospital or other environment, outbreaks of infections remain a serious problem and need to be addressed.
Systemic fungal infections such as invasive candidiasis and invasive aspergillosis may be caused by a variety of fungal pathogens, for example the virulent Candida species C. albicans, C. tropicalis and C krusei and the less virulent species C parapsilosis and Torulopsis glabrata (the latter referred to in some texts as Candida glabrata). Although C albicans was once the most common fungal isolate obtained from intensive care units, recent studies indicate that C tropicalis^ C glabrata^ C parapsilosis and C krusei now account for about half of such isolates (Pfaller, M.A. et al^ 1998, J, Clin. Microbiol. M: 1886-1889; Pavese, P. et al, 1999, Pathol BioL 4^: 579-583). The rise of non-albicans species implies tiie emergence of Candida species resistant to conventional antifungal therapy (Walsh, TJ. et al., New Eng , J. Med. MQ: 764-771).

Detection and diagnosis of the fungal pathogen responsible for an infection is critical for subsequent therapy because antifungal agents may be more effective agains certain strains- GB2240979 and EP0406029 (herein incorporated by reference in their entirety) disclosed a fungal stress protein and antibody thereto which could be used in a sensitive and highly specific test for detection of fungal pathogens.
Traditionally, C albicans, C, tropicalis and C parapsilosis have been treated by the antifungal agent amphotericin B, regarded as the "gold standard" of systemic antifungal therapy (Bumie, J.P., 1997, supra). Unfortunately, amphotericin B is itself hihgly toxic and its use is tempered by side effects including chills, fever, myalgia or thrombophlebitis. Other antifungal agents include the oral azole drugs (miconazole, ketoconazole, itraconazole, fluconazole) and 5-fluorocytosine. However, fungal species such as C krusei and T. glabrata are resistant to fluconazole, and these species often occur in patients where this drug has been administered prophylactically- Furthermore, fluconazole-resistant strains of C albicans have been reported (Opportunistic Pathogens, 1997, ii 27-31). Thus despite the recent advances made in therapeutic drugs such as fluconazole, itraconazole and systemic liposomal-based variants of amphotericin B (Bumie, J.P., 1997, siq?ra\ the need for effective agents for treatment of fungal infections remains acute.
The present invention addresses the above-identified need by providing a novel composition that is a significant improvement over prior art fungal agents for the treatment of human or animal fungal infections, and also a novel antibody which can be incorporated into the composition. The composition of the present invention comprises antibody which may bind one or more epitopes of a fungal stress protein, in combination with known antifungal agents. The inventors have found that, surprisingly, the efficacy of antifungal agents against fungal infections is significantly enhanced, allowing for either lower treatment dosages or more effective treatment at the same dose, which allows for reduction of unwanted side-effects. Furthermore, the composition of the present invention

allows for effective treatment of fungal infections which are inherently resistant to the fungal agent used in the composition.
According to the present invention there is provided the use of a composition comprising an antibody or an antigen binding fragment thereof specific for one or more epitopes of a fungal stress protein and an antifungal agent comprising at least one of the group consisting a polyene antifungal agent and an echinocandin. antifungal agent in a method of manufacture of a medicament for the treatment of fungal infections, wherein the fungus causing said fungal infection is resistant to said antifungal agent perse.
Further provided according to the present invention is a combined preparation for simultaneous, separate or sequential use in the treatment of fungal infections, comprising an antibody or an antigen binding fragment thereof specific for one or more epitopes of a fungal stress protein and an antifungal agent comprising at least one of the group consisting a polyene antifungal agent and an echinocandin antifungal agent wherein the fungus causing said fungal infection is resistant to said antifungal agent perse.
The antibody may be specific for a heat shock protein from a member of the Candida or Torulopsis genera. (The Candida and Torulopsis genera are generally deemed to be synonymous.) In particular, the antibody may be specific for the heat shock protein comprising hsp90 from Candida albicans, as described in GB2240979 and EP0406029-
The antibody or an antigen binding fragment thereof may be specific for the epitope comprising the sequence of SEQ ID NO:1
Antibodies, their manufacture and uses are well known and disclosed in, for example, Harlow, E. and Lane, D., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1999.

The antibodies may be generated using standard methods known in the art. Examples of antibodies include (but are not limited to) polyclonal, monoclonal, chimeric.

single chain. Fab fragments, fragments produced by a Fab expression library, and antigen
binding fragments of antibodies.
Antibodies may be produced in a range of hosts, for example goats, rabbits, rats, mice, humans, and others. They may be immunized by injection with heat shock protein from the Candida genus, for example hsp90 from C. albicans, or any fragment or oligopeptide thereof which has immunogenic properties. Depending on the host species, various adjuvants may be used to increase an immunological response. Such adjuvants include, but are not limited to, Freund's, mineral gels such as aluminum hydroxide, and surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, and dinitrophenol. Among adjuvants used in humans, BCG (Bacille Calmette-Guerin) and Corynebacterivm parvum are particularly useful.
Monoclonal antibodies to the heat shock protein from the Candida genus, for example hsp90 from C albicans, or any fragment or oligopeptide thereof may be prepared using any technique which provides for the production of antibody molecules by continuous cell lines in culture. These include, but are not limited to, the hybridoma technique, the human B-cell hybridoma technique, and the EBV-hybridoma technique (Koehler et aL, 1975, Nature, 256:495-497; Kosbor et aL, 1983, Immunol. Today 4:72; Cote et aL, 1983, PNAS USA, 80: 2026-2030; Cole et aL, 1985, Monoclonal Antibodies and Cancer Therapy, Alan R. Liss Inc., New York, pp. 17-96).
In addition, techniques developed for the production of "chimeric antibodies", the splicing of mouse antibody genes to human antibody genes to obtain a molecule with appropriate antigen specificity and biological activity can be used (Morrison et aL, 1984, PNAS USA, 81: 6851-6855; Neuberger et aL, 1984, Nature, 212:604-608; Takeda et aL, 1985, Nature, 314: 452-454). Alternatively, techniques described for the production of single chain antibodies may be adapted, using methods known in the art, to produce

The polyene antifungal agent may, for example, comprise amphotericin B, a derivative of amphotericin B, or nystatin. Derivatives of amphotericin B include formulations such as AmBisome (supplied for example by'NexStar Pharmaceuticals, Cambridge, UK), amphotericin-B lipid complex (Abelcet), amphotericin-B colloidal dispersion (Amphocil) and amphotericin-B intralipid emulsion (Bumie, J.P., 1997, supra), may be used. Amphotericin B may be used in combination with another antifungal agent, 5-fluorocytosine (Bumie, J.P., 1997, supra).
The echinocandin antifungal agent may, for example, be Anidulafungin (LY303366; Eli Lilly & Co., Indianapolis, USA). Echinocandins are cyclic lipopeptides that inhibit synthesis of P-l,3-glucan in fungi (Redding, J.A, et al, 1998, Antimicrob. Agents Cherao. Ther. 42G): 1187-1194),
The fungal infection which may be treated by the composition or combined preparation may be Mucormycosis, Blastomycosis, Coccidioidomycosis or Paracoccidioidomycosis, or the fungal infection may be caused by a Candida, Cryptococcus, Histoplasma, Aspergillus, or Torulopsis organism. The term "Coccidioidomycosis" is also referred to in the field as "Coccidiomycosis", and the term "Paracoccidioidomycosis" is likewise synonymous with "Paracoccidiomycosis". The fungal infection may be resistant to the antifungal agent perse, ie, fungal infections which are intrinsically untreatable by specific agents because that specific antifungal agent is ineffective as traditionally utilised on its own.
Also provided is a composition or combined preparation as described herein for use in a method of treatment of fungal infections of the human or animal body.
Also provided is a method of manufacture of a medicament for the treatment of fungal infections of the human or animal body characterised in the use of a composition

or combined preparation as described in the present application. Methods of manufacture of medicaments are well known. For example, a medicament may additionally comprise a pharmaceutically acceptable carrier, diluent or excipient (Remington's Pharmaceutical Sciences and US Pharmacopoeia. 1984, Mack Publishing Company, Easton, PA, USA).
Also provided is the use of a composition or combined preparation as described in the present application in a method of manufacture of a medicament for the treatment of fungal infections. The fungal infection may be resistant to the antifungal agent per se.
Also provided is a method of treatment of fungal infections of the human or animal body comprising administering a composition or combined preparation according to the present application to a patient in need of same. The exact dose (i.e. a pharmaceutically acceptable dose) of the composition or combined preparation to be administered to a patient may be readily determined by one skilled in the art, for example by the use of simple dose-response experiments. The composition or combined preparation may be administered orally.
Further provided according to the present invention is a kit comprising an antibody or an antigen binding fragment thereof specific for one or more epitopes of a fungal stress protein and an antifungal agent comprising any one of the group consisting a polyene antifungal agent and an echinocandin antifungal agent, for use in the treatment of fungal infections. The kit may be for use in the treatment of fungal infections, wherein the fungus causing the fungal infection is resistant to the antifungal agont per se.
The antibody according to the invention may have a diagnostic use. Thus for diagnostic use the antibody may be employed to detect whether the stress protem is present

in a host organism, to confirm whether the host has a particular fungal infection, for example Mucormycosis, Blastomycosis, Coccidioidomycosis or Paracoccidioidomycosis, or an infection due to a Candida, Cryptococcus, Histoplasma, AspergiIIus, or Torulopsis organism, or for example in the diagnosis of fungal abscesses, especially hepatic Candidiasis, and/or to monitor the progress of therapeutic treatment of such infections. Diagnostic methods of this type form a further aspect of the invention and may generally employ standard techniques, for example immunological methods such as enzyme-linked immunosorbent methods, radioimmuno-methods, latex agglutination methods or immunoblotting methods.
The antibody according to the invention may be labelled with a detectable label or may be conjugated with effector molecule for example a drug e.g. an anti-fungal agent such as amphotericin B or fluorocytosine or a toxin, such as ricin, or an enzyme, using conventional procedures and the invention extends to such labelled antibodies or antibody conjugates.
Also provided according to the present invention is the use of the antibody or antigen binding fragment according to the present invention in the preparation of a diagnostic for diagnosing one or more fungal infections. The diagnostic may be provided in a kit The kit may include instructions for use in diagnosing one or more fungal infections- The diagnostic kit as described herein is also provided according to the present invention.
If desired, mixtures of antibodies may be used for diagnosis or treatment, for example mixtures of two or more antibodies recognising different epitopes of a fungal stress protein according to the invention, and/or mixtures of antibodies of a different class, e.g. mixtures of IgG and IgM antibodies recognising the same or different epitope(s) of the

The contents of each of the references discussed herein, including the references cited therein, are herein incorporated by reference in their entirety.
The present invention will be further apparent from the following description, which shows, by way of example only, specific embodiments of the composition and experimentation therewith.
EXPERIMENTAL
Experiments described below investigated the antifungal effect of antibody agamst an hsp90 antigen derived from Candida albicans used in combination with antifungals such as amphotericin B or fluconazole. Results show that, in some cases, the combination of antibody and antifungal agent causes an enhanced antifungal effect compared with each of the compounds on their own. A surprisingly strong synergistic effect is demonstrated for amphotericin B in combination with anti-Candida albicans hsp90 antibody against a variety of common problematic fungal pathogens. This synergistic effect has significant implications for clinical treatment of fungal infections. A preliminary clinical study involving four patients suffering from Candida infections demonstrated the effectiveness of the present mvention for humans.
Material and Methods
Strains:
Non-Aspergillus yeast strains used (Table 1) were plated onto Sabouraud's dextrose agar (Oxoid, Basingstoke, UK) and incubated at 37oC for 24 hours. The strains were identified with the API 20C system (BioMerieux, Marcy L'Etoile, France). If needed, microscopical examinations of morphology on commeal agar (Oxoid) was used to confirm the identity.

Isolates of Aspergillus spp (Table 1) were grown on Sabouraud's dextrose agar (Oxoid, Basingstoke, UK) at 35°C for 24 hours.

For non-Aspergilliis strains, suspensions were prepared from individual colonies (diameter > 1mm) in 5 ml of sterile 0.85% saline to a density of 1 x 104 cells/mi as established by counting on a haemocytometer grid. For Aspergillus strains, see below.
Antifungal agents:
Amphotericin B was purchased from Sigma (Poole, Dorset) as a lyophilized powder for intravenous administration (Fungizone). Fluconazole was supplied as a solution for intravenous administration (Diflucan) by Pfizer, Amphotericin B was dissolved in dimethyl sulphoxide at a concentration of 1.2 mg/ml and fluconazole was dissolved in 0.85% saline also at a concentration of 1.2 rag/ml. Stock solutions were stored at -70oC until used. Abelcet (liposomal amphotericin B) manufactured by Bristol-Meyers Squib (USA) and prepared according to the manufacturers guidelines was used in the clinical study.
Antibody:
The DNA sequence of a former antibody specific for the Candida albicans hsp90 epitope disclosed in GB2240979 and EP0406029 was genetically modified by codon optimisation for expression in Escherichia coli (Operon Technologies Inc., Alameda, C A, USA) and inserted into an E. coli expression vector. The amino acid sequence of the anti-hsp90 antibody of the present invention comprises the sequence of SEQ ID NO: 2 (includes the heavy, light and spacer domains). The antibody according to the present invention recognises the epitope comprising the sequence of SEQ ID NO: 1.
The anti-hsp90 antibody was expressed in an Escherichia coli host and then purified by affinity chromatography and an imidazole exchange column up to 95 % purity. Standard molecular biology protocols were employed (see, for example, Harlow & Lane, svpra; Sambrook, J, et al, 1989, Molecular Cloning: A Laboratory Manual 2nd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York; Sambrook, J. &

Russell, D., 2001, Molecular Cloning: A Laboratory Manual. 3rd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor).
Formulations of Mycograb (RTM) were prepared as follows: a vial containing 10 mg of pure auti-hsp90 antibody, 150 mg of pharmaceutical grade (Ph Eur) Urea and 174 mg L-Arginine (Ph Eur) were reconstituted in 5 ml water.
Assay media:
RPMI broth was prepared from RPMI1640 broth medium (Sigma R7880) supplemented with 0.3 g of glutamine per litre, buffered with 34.6 g of morpholine propanesulfonic acid (MOPS) per litre and adjusted to ph 7,0,
The Broth microdilution test:
Twofold dilutions (40 to 0.024 mg/ml for amphotericm B and 400 to 0,4 mg/ml for fluconazole) were prepared in RPMI broth starting from the two stock solutions. A 100 μl suspension of the inoculum diluted 1 in 10 (equivalent to 1 x 103 cfu) was added to the microtiter plates. To this was added 50 μl of the antifungal and then 50μl of the antibody. Antibody was either neat (0.4 mg/ml), or diluted to 1/10 or 1/100. When antibody was absent, the 50 μ1 volume was made up by RPMI. The total volume in each well was 200 μl. Final concentrations of antibody in the experiments were: 100 μ g/ml ("neat") 0,10 μg/ml ("1/10 antibody') or 1 μg/ml ("1/100 antibody").
Plates were incubated at 37oC overnight and the minimum inhibitory concentration (MIC) defined by the lower concentration inhibiting growth.
Colony counts were determined for the wells where there was a visual reduction in yeast growth. Results were represented as colony forming units per ml of broth (cfu/ml).

Aspergillus Studies:
Isolates of Aspergillus fumigatus, Aspergillus flavus and Aspergillus niger were prepared in RPMI1640 medium. The suspensions were prepared to give a final inoculum of 2x10 conidia per ml and these were dispensed in 100 μl aliquots into flat-bottomed microtitre plates. A double dilution series of Amphotericin B ranging from 250 μg/ml to 0.75 μg/ml was prepared and dispensed to the appropriate wells. Mycograb was added to each of the wells at a final concentration of 100 μg/ml in formulation buffer. A control series was also prepared for each isolate which contained formulation buffer only. The plates were then incubated at 35*'Cy^00 rpm for 48 hours and MIC values for each isolate were determined by absence or presence of growth wells.
Animal Synergy:
Thirty CDl mice (each weighing about 25 g) were injected with 100μ1 of C albicans outbreak strain (equivalent to L5 x 10' cfu) and after 2 hours the mice were split into three groups and injected with:
(A) Group 1 -100μl solution of 10 mM ammonium acetate (AAT; pH 9), followed by 100 Hl amphotericin B equivalent to 0.6 mg/kg in a 5% (w/v) glucose solution;
(B) Group 2 - 100 ]i\ solution of 10 mM AAT (pH 9) solution containing 500 μg anti-hsp90 antibody, followed by 100 μl amphotericin B equivalent to 0,6 mg/kg in a 5 % (w/v) glucose solution; and
(C) Group 3 - 100 μl solution of 10 mM AAT (pH 9) containing 50 μg anti-hsp90 antibody, followed by 100 μl amphotericin B equivalent to 0.6 mg/kg in a 5% (w/v) glucose solution.
The animals were culled after 48 hours and yeast counts on Sabouraud's plates of liver, spleen and kidney tissue performed.

Clinical study:
An open-label safety and pharmacokinetics study of the anti-hsp90 antibody (in the form of Mycograb; see above) involving four patients suffering from Candida infections was conducted at the Central Manchester Health Care Trust Hospital and the Wythenshawe Hospital, both in Manchester, UK. Patients were examined for the signs of sepsis due to Candida, including: positive cultures of C albicans from multiple or deep sites; high or swimging temperature (pyrexia); high pulse rate (tachycardia) and high white cell count ("WBC").
Following conventional treatment with Abelcet (liposomal amphotericin B) and/or fluconazole, the patients were then additionally given various doses of Mycograb, including an optional test dose (0.1 mg/kg), and therapeutic dose(s) of 1 mg/kg. Patients were monitored for clinical and laboratory signs of infection (laboratory parameters tested include blood chemistry, haematology and clotting factors) and serum and urine levels of Mycograb tested.
Results
In vitro experiments examining the effect of combining an anti-Candida albicans hsp90 antibody ("antibody") and antifungal agents are presented m Tables 2 to 18. Animal experimental results are presented in Table 19.
In vitro experiments:
Compositions containing antibody and flaconazole:
Table 2 shows the minimum inhibitory concentrations (MICs) of fluconazole against the test fungal pathogens, with or without the presence of the anti-C albicans hsp90 antibody at different dilutions, as assessed by the Broth microdilution test. In the

presence of neat antibody and antibody diluted 10-fold, the MIC for the outbreak stram of C albicaiis was reduced four-fold (L56 μg/ml to 0.39 μg/ml fluconazole), whereas a IOC-fold dilution of the antibody resulted in a two-fold reduction of fluconazole MIC.
A slight reduction in fluconazole MIC was observed for the fluconazole resistant strain of C. albicans and C. kmsei in the presence of neat antibody. At a dilution of 1/10 and 1/100, however, the antibody had no effect on the fluconazole MIC of these strains in comparison with no antibody.
For the remaining fungal strains, ie. C. tropicalis, C parapsilosis and T, glabrata, the anti-C albicans hsp90 antibody had no discemable effect on the fluconazole MICs,

Further experiments which quantified the number of cell colonies surviving at different fluconazole concentrations with different dilutions of the anti-C albicans hsp90 antibody were undertaken for each of the fungal strains represented in Table 2.
At the fluconazole concentrations examined, the survival rate of C albicans (outbreak stram) was not reduced by the addition of neat antibody or antibody diluted 100-fold (Table 3).

For the fluconazole-resistant strain of C. albicans, a two-fold reduction in colony survival was observed at 12,5μg/ml fluconazole in the presence of neat antibody (Table 4). Slight reductions in the survival rate of this stram were noticed at lower concentrations of fluconazole in the presence of neat antibody, but no effect was discemable at a 1/100 dilution of the antibody.

Neat or diluted antibody had no significant antifungal effect against C krusei at the fluconazole concentrations tested (Table 5).

For C. tropicalis, no marked effect on survival rate could be seen for each of the fluconazole concentrations examined in the presence or absence of antibody (Table 6),

Table 7 shows that presence or absence of the antibody had no effect of the survival rate of T. glabrata colonies at each of the fluconazole concentrations tested-

Presence or absence of the antibody had no notable effect on the survival rate of C parapsilosis colonies at the fluconazole concentrations as indicated in Table 8.

Compositions with antibody and amphotericin B:
Table 9 shows the minimum inhibitory concentrations (MICs) of amphotericin B against the test fungal pathogens, with or without the presence of the anti-C. albicans hsp90 antibody at different dilutions, as assessed by the Broth microdilution test.
In contrast with the results obtained for fluconazole (see Tables 2-8 supra), all strains tested here showed at least a four-fold drop in MIC of amphotericin B when undiluted antibody was added to the incubation broth (Table 9). Furthennore, in all strains examined, there was at least a two-fold drop in amphotericin B MIC even when the antibody diluted 100-fold was added to the incubation broth (final antibody concentration: 1μg/ml).
The greatest effect of the composition comprising antibody and amphotericin B at reducing the MIC of amphotericin B was observed with the fluconazole resistant strain of C. albicans. Neat antibody yielded a ten-fold reduction in the amphotericin B

MC, and even at a 100-fold antibody dilution, the amphotericin B MIC was reduced by approximately 25 % (Table 9).

Detailed experiments which quantified the number of cell colonies surviving at different amphotericin B concentrations with different dilutions of the anti-C albicans hsp90 antibody were undertaken for each of the fungal strains represented in Table 9.

Table 10 shows survival rates for the outbreak strain of C albicans incubated with amphotericin B in the presence or absence of antibody. A dramatic reduction (at least 10-foid) m the number of surviving colonies was effected by the antibody at all the amphotericin concentrations tested. For example, at 0.078 μg/ml amphotericin B, the survival rate of C albicans (outbreak strain) was 0.2% in the presence of antibody diluted 100-fold compared with the survival rate of the strain without antibody. The inhibitory effect of the antibody diluted 100-fold at 0.078 μg/ml amphotericin B was equivalent to the survival rate of this strain at 0.156 μg/ml amphotericin B (without antibody). Therefore, even very diluted antibody is able to effect a reduction in the amount of amphotericin B required to achieve a specific mortality rate in this strain.

Table 11 shows the survival rate of colonies of C albicans (fluconazole resistant strain) at different concentrations of amphotericin B and at different antibody dilutions. No noticeable effect of the antibody or amphotericin B could be seen at lower concentrations of the antifungal agent. However, at amphotericin B levels approachmg the MIC of the antifungal agent (see Table 9, supra), the antibody was observed to have a marked effect on colony survival. For example, at 0,078 μg/ml amphotericin B, antibody at a 100-fold dilution effected a survival rate of this strain of 0.1 % compared with no antibody.

Colony survival rates of C knisei in the presence of amphotericin B and different amounts of antibody are represented in Table 12. The antibody can be seen to be very effective against this strain at the higher concentrations of amphotericin B examined. Even at a 100-fold dilution, the number of C krusei colonies detected in the presence of 0312 fig/ml amphotericin B v/as 0.01 % of those surviving without antibody.

For all concentrations of amphotericin B tested (range from 0.019 - 0.156 μg/ml), the antibody was seen to be effective at reducing the colony survival rate of the strain C tropicalis (Table 13). The effect was enhanced at higher antibody and amphotericin B concentrations.

Table 14 shows the survival rate for T. glabrata in the presence of various concentrations of amphotericin B and antibody. The antibody was observed to be highly effective at inhibiting growth of T. glabrata at all concentrations of amphotericin B tested. For example, at a 100-fold dilution of the antibody, the growth of this strain was inhibited by 99.2 % at 0.009 μg/ml amphotericin B, 99.99% at 0.019μg/ml amphotericin B and 99.91 % at 0.039μg/ml amphotericin B.

The survival of the fungal strain C parapsilosis at different levels of antibody and amphotericin B is shown in Table 15. Neat antibody was observed to achieve a reduction in the survival rate of this strain at all levels of amphotericin B tested. At lower concentrations of antibody, the effect was less dramatic than for the other strains examined.

Compositions with antibody but without antifungal agent:
In a further experiment, the effect of different concentrations of the anti-C albicans hsp90 antibody alone (no antifungal agent) on the different fungal strains (used in Tables 2-15, supra) was tested. The results shown in Table 16 reveal that for the most of the strains used, the antibody itself had no effect on their survival. However, some diminution in survival rate which can be attributed to the antibody alone was observed in the strains L glabrata, C tropicalis and C parapsilosis.

Experiments with Aspergillus spp:
The MIC of Aspergillus fumigatus to Amphotericin B was 2.5μg/ml. With the addition of Mycograb, the MIC shifted to 0.125μg/ml (two-fold decrease). The MIC of Aspergillus flavus to Amphotericin B was 2.5μg/ml. With the addition of 100 μLg/ml of Mycograb the MIC shifted to 0.125 μg/ml (two-fold decrease). The MIC of Aspergillus niger to Amphotericin B was 2.5 μg/ml. With addition of 100 μg/ml of Mycograb the MIC shifted to 0.125 μg/ml (two-fold decrease).
Summary of in vitro results:
The results shown in Tables 2-16 reveal that while the anti-C albicans hsp90 antibody on its own was able to inhibit growth of certain fungal strains, a surprisingly high level of antifungal activity against all the strains examined was observed when the antibody was used in combination with amphotericm B- This surprising effect between the antibody and amphotericin B is not observed with other antifungal agents examined: fluconazole combined with the antibody did not produce a significant and potentially useful outcome.
Using the cut-off criterion of a four-fold difference in MIC improvement, data in Table 2 reveal that fluconazole combined with neat antibody (final concentration 100 μg/ml) or a 10-fold dilution of antibody (final concentration 10 μg/ml) was effective only against the outbreak strain of C albicans. However, using the same criterion, it can be seen from Table 9 that amphotericin B combined with neat antibody or a 10-fold dilution of the antibody was effective agamst all the fungal strains tested. It can therefore be concluded

that there is a strong synergy between amphotericin B and the anti-C albicans hsp90 antibody as measured by improvement in MIC,
For the experiments in which fungal colonies were quantified for different antifungal and antibody treatments (see Tables 3-8 and 10-15, supra), a different cut-off criterion which defines a two log drop (100-fold) drop in surviving colonies can be employed to assess potentially useful combinations of treatments.
A summary of the results for fluconazole in combination with the anti-C albicans hsp90 antibody is presented in Table 17. Here, the lowest concentration of fluconazole resulting in the desired effect (or the highest concentration used in the experiment) used is shown, together with an mdication of the cut-off criterion of at least a 100-fold drop is fungal survival rate was achieved. The results show that only the fluconazole resistant strain of C albicans when combined with fluconazole and neat antibody produced a significant effect.

A summary of the results for amphotericin B in combination with the anti-C albicans hsp90 antibody is presented in Table 18. It can be seen that the cut-off criterion (100-fold reduction in fungal colony growth) is satisfied with neat antibody for all fungal strains examined, with a 10-fold dilution of the antibody for C albicans (outbreak strain and fluconazole resistant stram), C krusei and T. glabrata, and with a 100-fold reduction in antibody for C. albicans (outbreak strain) and T glabrata.
It is noteworthy that synergy between amphotericin B and the antibody was observed not only against fluconazole sensitive strains of C albicans but also fluconazole resistant strains of C albicans and yeasts such as Candida krusei and T. glabrata which are intrinsically resistant to fluconazoles.

(+ indicates cut-off criterion satisfied; - indicates cut-off criterion not satisfied; cut-ofif criterion is at least 100-fold reduction in colony count)
The results with Aspergillus spp show that there was synergy between Amphotericin B and Mycograb in vitro against the commonest aspergillus spp.
(2) Animal experiments:
Mice infected with the outbreak strain of Candida albicans were treated with amphotericin B only (Group 1), amphotericin B and 500 μg anti-hsp90 antibody (Group 2) and amphotericin B and 50μg anti-hsp90 antibody (Group 3). Yeast colony counts for various tissues from the mice after a treatment period of 48 hours are shown in Table 19. The results show that animals treated with amphotericin B and 500μg antibody (Group 2) showed a significant reduction (at least one order of magnitude) in the number of yeast counts compared with animals treated with amphotericin B only (Group 1). Animals treated with amphotericin B and 50 fig antibody (Group 3) also showed diminished yeast

counts compared with the animals treated with amphotericin B only (Group 1). The in vivo data therefore corroborates the in vitro data and confirms the synergy between the anti-hsp90 antibody and the antifungal agent amphotericin B for the treatment of fungal infections.

(3) Clinical Study:
Four patients with evidence of Candida infection and who were not responding to conventional antifungal treatment were given a combined treatment of antifungals and anti-hsp90 antibody in the form of Mycograb (see above) and their condition monitored.
Patient 1 was given a primary diagnosis of acute pancreatitis and the patient had postoperative adult respiratory distress syndrome (ARDS) requiring ventilation. C albicans was grown in vitro from multiple sites including pancreatic bed. The patient had a very high white cell count (WBC) (78,4), although this was highly variable and may not have been caused by the sepsis alone. Abelcet treatment at 3 mg/kg was initiated.

Five days after initiation of Abelcet treatment, Patient 1 was additionally given a first test dose of Mycograb at 0.1 mg/kg (Day 1). On Day 3, the patient was given a clincal dose of Mycograb at 1 mg/kg. Due to several factors, for example a worsening platelet count which had been low for at least four days, the Abelcet and Mycograb treatments were discontinued on Day 3 after the clinical dose of Mycograb had been given. However, Patient 1 regrew C. albicans from ascites six days later (Day 9) and was put on fluconazole (400 mg). The following day (Day 10), the patient was given the final two clinical doses of Mycograb at 1 mg/kg per dose.
Although Patient 1 had been on Abelcet for seven days, before the patient received the clinical dose of Mycograb on Day 3, C albicans was still being grown firom the patient's trachyostomy site and the patient had a tachycardia. The combined treatment with Abelcet and Mycograb on Day 3 resulted in a period of five days during which C. albicans was not grown. No Mycograb-related changes in blood chemistry, haematology and clotting factors were observed during treatment with Mycograb, Treatment of the subsequent recurrence with fluconazole and Mycograb (two doses on Day 10) was less successful, as would be expected firom the in vitro synergy results (see for example Tables 2 and 17), but the patient did eventually recover firom the candidosis.
Serum levels of Mycograb in Patient 1 at different time intervals following administration of Mycograb doses are shown in Table 20. The test dose at Day 1 did not give measurable serum levels. The 1.0 mg/kg doses at Day 3 and Day 10 did give detectable serum levels, and these levels were comparable with those at which synergy with amphotericin B was demonstrable in vitro (see Tables 9 and 18). Following the second dose on Day 10, serum levels of Mycograb improved, indicating some tissue accumulation following the first dose. Mycograb was detectable in the urine at the 1.0 mg/ml doses (data not shown).
Table 20. Serum levels (in μg/ml) of Mycograb in Patient 1

Patient 2 was diagnosed as having a small bowel constriction due to adhesions and had ARDS requiring ventilation. C albicans was grown from multiple sites including ascitic fluid, with the infection associated with a fluctuating temperature (35.8-38.2oC), raised WBC (11.4) and occasional tachycardia (110). The patient was started on Abelcet at 3 mg/kg.
Four days after the commencement of Abelcet treatment, Patient 2 still retained a fluctuating temperature, raised WBC and occasional tachycardia. The patient was given a 0.1 mg/kg test dose of Mycograb (Day 1). The following day, the patient was given a clinical dose of 1 mg/kg Mycograb (Day 2), The last dose of Abelcet was also given on Day 2 due to completion of a 5 day treatment program. Two days later (Day 4), the patient received the final two clinical doses of Mycograb.

The Mycograb was well tolerated by the patient. The clinical dose of Mycograb on Day 2 was associated with a falling and stabilising temperature (38,2 to 36.7oC on Day 2 after receiving the clinical dose, staying at 36.7-37.4°C through to Day 3) and a falling WBC (from 11.9 to 9.6). On Day 4, the patient was looking clinically better and no C albicans was grown from ascites, blood cultures or urine. No Mycograb-related changes in blood chemistry, haematology and dotting factors were observed during treatment. Subsequent recovery was complicated by an episode of bacterial sepsis but this responded to antibiotics and the patient made a full recovery.
Serum levels of Mycograb in Patient 2 at different time intervals following administration of Mycograb doses are shown in Table 21. The test dose at Day 1 did not give measurable serum levels. The 1.0 mg/kg dose at Day 2 did give detectable serum levels, and these levels were compatible with those at which synergy with amphotericin B was demonstrable in vitro (see Tables 9 and 18). Mycograb was detectable in the urine at the 1.0 mg/kg doses (data not shown).

Patient 3 had a six week history of pancreatitis which led to an 80% pancreatectomy. The patient had moderately raised LFT (liver function test) levels, possibly related to alcohol abuse, and was an MRSA (Methicillin-resistant Staphylococcus avreus) carrier. C albicans were grown from multiple sites so the patient was treated with intravenous fluconazole. Twelve days later, after failing to respond to fluconazole, the patient was changed to 300 mg Abelcet. Three days later, the patient was still pyrexial (38.5oC) and C albicans was still growing from multiple sites (abdominal drains and gastroscopy tube), and was therefore given a clinical dose (1 mg/kg) of Mycograb (Day 1) in addition to the Abelcet.
On the same day as the Mycograb dose was given, Patient 3 suffered an acute episode of Gram-negative-type septic shock (high temperature of 39.5oC, hypotensive), probably due to Pseudomonas aeruginosa, which was subsequently grown from his pancreatic drain, although he also grow Enterococcus faecalis from blood cultures. Due to this episode, no further Mycograb doses were given. The patient subsequently responded to antibiotic therapy (vancomycin and ceftazidime).
Due to the bacterial complications, it was difficult to assess the impact of the single dose of Mycograb on Patient 3. However, it was noted that he stopped growing C. albicans (for example, from his gastrostomy tube and wound drain) for 48 hours after the dose and that he became apyrexial on Day 2 and Day 3. No Mycograb-related changes in laboratory parameters (blood chemistry, haematology and clotting factors) were observed.

On Day 4, the patient had a recurrence of C albicans while he was still on Abelcet but a full recovery was made subsequently.
Serum levels of Mycograb in Patient 3 at different time intervals following administration of the Mycograb dose are shown in Table 22. The single 1.0 mg/kg dose at Day 1 gave detectable serum levels, and these levels were compatible with those at which synergy with amphotericin B was demonstrable in vitro (see Tables 9 and 18). Mycograb was also detectable in the urine following the 1.0 mg/kg dose (data not shovra).

Patient 4 was diagnosed with C albicans empyema, although the patient was originally admitted to ITU (Intensive Treatment Unit) with a lung abscess due to Streptococcus milleri (isolated from blood cultures), C albicans was grown from two bronchial lavage specimens (right and left lung) and three and four days later from two empyema fluid specimens. Treatment was started the following day with Abelcet (5

mg/kg). Five days after commencement of Abelcet treatment, some clinical deterioration was noted and the following moming (Day 1) this was associated with high WBC (15.7) and C albicans regrown from an intercostal drain fluid,
Mycograb (1 mg/kg bd) was given to Patient 4 at 8.30 am and 8.30 pm on Day 1. Apart from a temporary rise in temperature on the night of Day 1, the patient improved clinically, C albicans was not grown from empyema fluid specimen cultured on Day 3, and the patient became progressively better.
The Mycograb was well tolerated by Patient 4, No Mycograb-related changes in laboratory parameters (blood chemistry, haematology and clotting factors) were observed. Thus the patient was still growing C albicans from a chest drain six days after commencing Abelcet treatment and his WBC was high (15.7) just before receiving the first Mycograb dose, but thereafter the patient steadily improved and stopped growing C. albicans.
Serum levels of Mycograb in Patient 4 at different time intervals following administration of Mycograb doses are shown in Table 23, The 1.0 mg/kg doses given on Day 1 gave detectable serum levels which were compatible with those at which synergy with amphotericin B was demonstrable in vitro (see Tables 9 and 18). Following the second dose on Day 1, semm levels of Mycograb improved, indicating some tissue accumulation following the first dose. Mycograb was detectable in the urine at the 1.0 mg/ml doses (data not shown).

Conclusions
The data presented here clearly demonstrates that there is a surprising synergism between the znii-Candida hsp90 antibody and the antifungal agent amphotericin B which effects enhanced antifungal activity against a wide variety of pathologically important fungal strains. These results allows for the use of novel, highly effective compositions for the treatment of human or animal fungal infections, and also a novel antibody which can be incorporated into the composition. The present invention allows for either lower treatment dosages or more effective treatment at the same dosages, thereby reducing unwanted side-effects.
Clinical implications of the present invention include: (i) the production of a synergistic combination of amphotericin B and anti-hsp90 antibody in the treatment of

disseminated yeast infection should become the treatment of choice. This would lead to a reduction in mortality and morbidity for these infections. The preliminary clinical study results provided herewith confirm the efficacy of the present invention in comparison with existing methods of treatment; (ii) amphotericin B is a toxic, particularly nephrotoxic, drug. The synergy provided by the present invention means that a lower dose of amphotericin B could be used while maintaining efficacy and concomitantly reducing toxicity; and (iii) the toxicity sparing effect of the anti-hsp90 antibody would allow the clinical efficacy of higher doses of amphotericin B to be explored and further contribute to an improved clinical outcome.

We claim:

and pharmaceutically acceptable salts thereof, wherein
n is 0 or 1; m is 1, 2 or 3; R1 is H, C1-C3 alkyl, halogenated C1-C3 alkyl or
cyclopropyl; R2 is 4-chlorophenyl or 2,4,6-trichlorophenyl.
2. The compound as claimed in as claimed in claim 1 wherein n is 0.
3. The compound as claimed in claim 1 wherein n is 1.
4. The compound as claimed in claim 1 wherein m is 1.
5. The compound as claimed in claim 1 wherein m is 2.
6. The compound as claimed in claim 1 wherein R] is H.
7. The compound as claimed in claim 1 wherein R] is CH3.
8. The compound as claimed in claim 1 wherein R2 is 4-chlorophenyl.
9. The compound as claimed in claim 1 wherein R2 is 2,4,6-trichlorophenyl.

10. The compound as claimed in claim 1 having the
stereoconfiguration shown below:

11. The compound as claimed in claim 10 wherein n is 0.
12. The compound as claimed in claim 10 wherein n is 1.
13. The compound as claimed in claim 10 wherein m is 1.
14. The compound as claimed in claim 10 wherein m is 2.
15. The compound as claimed in claim 10 wherein R1 is H.
16. The compound as claimed in claim 10 wherein R1 is CH3.
17. The compound as claimed in claim 10 wherein R2 is 4-chloro-phenyl.
18. The compound as claimed in claim 10 wherein R2 is 2,4,6-trichlorophenyl.
19. A compound according to claim 1 or 10 selected from [4-(4-chlorobenzyl)-7-fluoro-5-(methanesulfonyl)-l,2,3,4-tetrahydrocyclopenta[b]-indol-3-yl]acetic acid,
{4-[ 1 -(4-chlorophenyl)ethyl]-7-fluoro-5-methanesulfonyl-1,2,3,4-tetrahydrocyclo-penta[b]indol-3-yl}acetic acid, [9-(4-chlorobenzyl)-6-fluoro-8-methanesulfonyl-2,3,4,9-tetrahydro-lH-carbazol-l-yl]-
acetic acid, and

[4-(4-chlorobenzyl)-7-fluoro-5-methanesulfonyl-l-OXO-1,2,3,4-tetrahydro-cyclopenta-
[b]indol-3-yl]acetic acid, or
a pharmaceutically acceptable salt thereof.
20. The compound according to claim 19, wherein the compound is (-)-[4-(4-chlorobenzyl)-7-fluoro-5-(methanesulfonyl)-l,2,3,4-tetrahydrocyclopenta[b]-indo]-3-yl]acetic acid and pharmaceutically acceptable salts thereof.
21. The compound according to claim 19, wherein the compound is
(+/-)-4-[l-(4-chlorophenyl)ethyl]-7-fluoro-5-methanesulfonyM,2,3,4-tetrahydro-cyclopenta[b]indol-3-yl}acetic acid and pharmaceutically acceptable salts thereof.
22. The compound according to claim 19, wherein the compound is (+/-)-[9-(4-chlorobenzyl)-6-fluoro-8-methanesulfonyl-2,3,4,9-tetrahydro-lH-carbazol-l-yl]acetic acid and pharmaceutically acceptable salts thereof.
23. The compound according to claim 19, wherein the compound is [4-(4-chlorobenzyl)-7-fluoro-5-methanesulfonyl-l-oxo-l,2,3,4-tetrahydro-cyclopenta[b]indol-3-yl]acetic acid and pharmaceutically acceptable salts thereof.
24. A pharmaceutical composition comprising a compound of claim 1 and a pharmaceutically acceptable carrier.
25. A pharmaceutical composition comprising a compound of any one of the claims 1 to 23, a second pharmaceutical agent selected from an antihistamine, a leukotrine antagonist and a leukotriene biosynthesis inhibitor and a pharmaceutically acceptable carrier.
26. A pharmaceutically acceptable salt of a compound of formula I as defined in any one of claims 1 to 18.
Dated this 21st day of July 2004
T.SRINIVASAN
Agent for the Applicant
To
The Controller of Patents
The Patent Office at Chennai

Documents:

1609-chenp-2004-abstract.pdf

1609-chenp-2004-claims duplicate.pdf

1609-chenp-2004-claims original.pdf

1609-chenp-2004-correspondnece-others.pdf

1609-chenp-2004-correspondnece-po.pdf

1609-chenp-2004-description(complete) duplicate.pdf

1609-chenp-2004-description(complete) original.pdf

1609-chenp-2004-form 1.pdf

1609-chenp-2004-form 13.pdf

1609-chenp-2004-form 26.pdf

1609-chenp-2004-form 3.pdf

1609-chenp-2004-form 5.pdf

1609-chenp-2004-pct.pdf

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Patent Number

205687

Indian Patent Application Number

1609/CHENP/2004

PG Journal Number

26/2007

Publication Date

29-Jun-2007

Grant Date

09-Apr-2007

Date of Filing

21-Jul-2004

Name of Patentee

M/S. MERCK FROSST CANADA & CO

Applicant Address

16711 Trans-Canada Highway, Kirkland, Québec H9H 3L1

Inventors:

#	Inventor's Name	Inventor's Address
1	BERTHELETTE, Carl	16711 Trans-Canada Highway, Kirkland, Québec H9H 3L1
2	LACHANCE, Nicolas	16711 Trans-Canada Highway, Kirkland, Québec H9H 3L1
3	LI, Lianhai	16711 Trans-Canada Highway, Kirkland, Québec H9H 3L1
4	STURINO, Claudio	16711 Trans-Canada Highway, Kirkland, Québec H9H 3L1
5	WANG, Zhaoyin	16711 Trans-Canada Highway, Kirkland, Québec H9H 3L1

PCT International Classification Number

C07D 209/00

PCT International Application Number

PCT/CA2003/000084

PCT International Filing date

2003-01-22

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1	60/351,384	2002-01-24	U.S.A.