||||||||||||||| US005434163A United States Patent 19 11) Patent Number: 5,434,163 Edlind et al. 45 Date of Patent: Jul.18, 1995
54 TREATMENT OF CRYPTOCOCCUS 56 References Cited NEOFORMANS INFECTION U.S. PATENT DOCUMENTS Edlin 3,745,187 7/1973 Naguchi et al...... 514/310 75 Inventors: PE F.Y.Eth of 3,769,308 10/1973 Kohimoto et al...... 514/310 Pa 9 s 4,725,605 2/1988 Crossley et al...... 514/338 4,794,123 12/1988 Crossley et al...... 514/232 4,863,945 9/1989 Crossley et al...... 514/393 73 Assignee: The Medical College of Pennsylvania, 4,952,589 8/1990 Brown ...... 514/310 Philadelphia, Pa. 5,061,715 10/1991 Sunkara ...... 514/34 5,151,426 8/1991 Bellene ...... 514/262 21 Appl. No.: 62,444 5,200,417 4/1993 Brown et al...... 514/310 Primary Examiner-Werren B. Lone 22 Filed: May 14, 1993 Attorney, Agent, or Firm-Janice E. Williams; Edward T. Lentz 51 Int. Cl...... A61K 3/47 I52) U.S. Cl...... sia/30.5/268, 7 ABSTRACT 514/393; 514/395; 514/365; 514/31; 546/143; A method of treatment of Cryptococcus neoformans in 546/144; 546/145; 546/194; 546/201; 546/210; fection by administering to a patient in need thereof an 546/300 effective amount of a benzimidazile compound. 58 Field of Search ...... 514/310, 232, 226.8, 514/393; 546/143, 144, 145, 194, 201, 210, 300 6 Claims, No Drawings 5,434,163 1. 2 maceutical composition such as a tablet using standard TREATMENT OF CRYPTOCOCCUS formulation methods and techniques. NEOFORMANS INFECTION It is expected that the effective dosage of the benz imidazole anthelmintic compound when used to treat BACKGROUND OF THE INVENTION 5 the conditions disclosed herein will be in the range of The present invention relates to the use of benzimid from 10 to 5000 mg per day, the compound being ad azole anthelmintics in the treatment of cryptococcal ministered in one or more discrete dosage units, one or infection including meningitis in particular in AIDS more times to achieve effective therapeutic levels in the patients, including individuals exhibiting HIV infection patient's cerebral spinal fluid, blood or other infection as well as auto-immune deficiency syndrome. 10 site for as long as is necessary to treat the condition and Cryptococcus neoformans is an encapsulated yeast that maintain the patient free of infection. The precise size, causes meningitis in AIDS patients. Currently recom per dose strength, frequency and duration of the dosage mended therapy includes amphotericin B--/-flucyto regimen will of course depend on the severity of the condition. sine. Toxicity is high and relapses are common. Benz 5 imidazoles are a large group of drugs used clinically for SPECIFIC EMBODIMENT OF THE INVENTION helminth infections and agriculturally as antifungal agents. There exists a need for additional or better ther The following example serves to illustrate specific aspects of the invention. Those skilled in the an with apies for treatment of Crytococcus neoformans. readily appreciate that the specific experiments detailed DETAILED DESCRIPTION OF THE 20 are only illustrative of the invention as described more INVENTION fully in the claims which follow thereafter. It has now been found that certain benzimidazole Eleven benzimidazole derivatives and amphotericin anthelmintics also have in vitro activity against Crypto B were tested in vitro against three isolates of Cryptococ coccus neoformans and as such are expected to be of use 25 cus neoformans. Drug concentrations inhibiting growth in the treatment of infections caused by this organism. 50% (IC50) were determined. More specifically the compounds are expected to be of Activity of benzimidazole derivatives and amphoteri use in the treatment of meningitis caused by this organ cin B against in vitro growth of three C. neoformans ism in particular in patients having AIDS. isolates are shown in Table I. Inhibitory concentrations In particular, fenbendazole, albendazole and meben 30 were determined by a broth dilution method. All values dazole have been found to exhibit high levels of activity are in pg/ml. Yeast were diluted from fresh overnight against the organism and are the preferred benzimid cultures grown in YEPD at 30° C. to a destiny of azole derivatives of the present invention. Those skilled 1 x 104 cells per ml YEPD, and 1 ml portions were in the an will recognize that other benzimidazole anthel aliquoted to culture tubes. Drags were added by two mintics and their derivatives may exhibit activity 35 fold serial dilution. Cultures were incubated at 30° C. against Cryptococcus neoformans as well and may be with shaking for 20 hrs. Cell numbers were determined substituted for the preferred embodiment of the present with a hemacytometer. invention. TABLE I The presently most preferred benzimidazoles for the U4 IU4 IU4 composition of the invention are fenbendazole methyl DRUG IC50 IC90 IC50 IC90 IC50 IC90 5-(phenylthio)-1H-benzimidazol-2-ylcarbamate), alben Fenbendazole 0.019 O.028 0.01 0.014 (0.06 (0.016 dazole methyl 5-(n-propylthio)-1H-benzimidazol-2- Nocodazole 0.3 0.22 0.09 0.12 0.20 0.29 ylcarbamate and mebendazole. However, it is possible Parbendazole 0.16 0.23 0.16 0.23 0.16 0.22 Mebendazole 0.23 0.43 0.18 0.32 0.19 0.40 to substitute other known and commercially available 45 Albendazole 0.16 0.25 0.3 0.45 0.30 O.45 benzimidazoles or prodrugs which metabolize into a Oxibendazole 2.3 3.2 1.0 2. 1.4 2. suitable benzimidazole. Among such compounds are Carbendazim 2.0 3.6 1.5 2.0 1.6 2.3 oxibendazole methyl 5-(n-propoxy)-1H-benzimidazol Benomyl 2.3 3.7 2.8 3.8 2.0 3.5 2-ylcarbamate), oxfendazole methyl 5-(phenylsulfinyl)- Oxfendazole 2.5 > 4.0 3.2 > 4.0 2.8 > 4.0 Thiabendazole > 4.0 4.0 d 4.0 > 4.0 > 4.0 > 4.0 1H-benzimidazole-2-ylcarbamate, parbendazole, cam 50 Benzinidazole > 4.0 4.0 4.0 > 4.0 4.0 >40 bendazole, flubendazole, ricobendazole, luxabendazole Amphotericin 0.035 0.064 0.024 0.065 0.011 0.045 and others. These benzinidazole are commercially B available. Prodrugs which metabolize in vitro into selected Four derivatives were found to have moderately high benzimidazoles may also be employed to replace the 55 activity (IC90=0.2-0.5 ug/ml, IC50=0.1-0.3 ug/ml), preferred benzimidazoles in the compositions of this while one, fenbendazole was highly active (IC90=0.- invention. Among the known prodrugs are thiopha 02-0.03 ug/ml, ICso=0.1-0.2 pg/ml). This was 2 fold nates 1,2-phenylenebis (iminocarbonothioyl) biscar more active than amphotericin B. Ten additional clini bamic acid diethyl esters. See, for example, U.S. Pat. cal isolates of Cryptococcus neoformans were tested Nos. 3,745,187 and 3,769,308. Another prodrug which 60 against fenbendazole, mebendazole and albendazole; may be useful is Febantel, i.e., 2-(methoxyacetyl) ani no-4-(phenylthio)-phenyl)carbonimidoyl biscarbamic similar sensitivities were observed. acid dimethyl ester. Also useful may be the Netobimin TABLE II esters. These known compounds may also be prepared DRUG IC50 (range) IC90 (range) by known and conventional methods by one of skill in 65 fenbendazole 0.012-0.024 0.05-0.033 the art. albendazole 0.2-0.18 0.22-0.45 When used in the method of the invention, the benz mebendazole 0.12-0.31 0.22-0.45 imidazole compound is formulated in a standard phar 5,434,163 3 4 Activity of fenbendazole, albendazole, and meben TABLE III LC90 (ug/ml) dazole against in vitro growth often C. neoformans clini amphotericin cal isolates is shown in Table 2. All values are in ug/ml. 5 Isolate fenbendazole albendazole mebendazole B 1KR 0.06 0.92 1.3 0.51 IC50 and IC90 values were comparable to those pres 14116 0.07 2.1 2.0 0.54 ented in Table 1, and varied over a narrow range (ap In vitrocidal activities offenbendazole, albendazole, proximately two-fold). 10 mebendazole, and amphotericin B against two C. neo formans isolates are shown in Table 3. Cidal activity Cidal activity was tested for two isolates. LC90 values was estimated by plating cultures on drug-free medium following a 20 h. incubation in the presence of different were 1.2 ug/ml, LC50=0.3 to 1.4 ug/ml for alben drug concentrations. Activity is expressed as LC90, the dazole and mebendazole and 0.06-0.1 g/ml, 15 concentration resulting in 90% reduction in cell number relative to the starting (t=0) number. LCso=0.03 to 0.04 g/ml for fenbendazole; the latter Spontaneous resistance to 1 ug/ml fenbendazole oc curred at a frequency of <1X 10-7; the corresponding are about 9 to 13 fold lower than the values obtained for frequency for amphotericin B was approximately amphotericin B. 20 2X 10-6.
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5,434,163 7 8 Alignment of 6-tubulin residues 107-260 is shown in The high inhibitory and cidal activities of certain Table 4. Dots represent identity to the CNBTB se benzimidazoles against C. neoformans suggest that this quence. Arrows indicate residues responsible for beno group may provide a new therapeutic approach to cryp myl or thiabendazole resistance in S. cerevisiae (R241 to tococcocal meningitis. The route of administration of H), N. crassa (F167 to Y), and A. nidulans (A165 to V; benzimidazoles is oral, a clear advantage over ampho E198 to D, N, or K; F200 to Y). The sequences are from tericin B. However, benzimidazoles derivatives vary in C. neoformans (CNBTB and CNBTA), P. carini their intestinal absorption. Penetration of benzimida (PCBT), A. nidulans (ANBENA and ANTUBB), a zoles into the central nervous system has not been well human (HUTUBBM), C. elegans (CEBEN1, CETUB 1, studied. However, in hums with neurocysticercosis, and O oral dose of 15 mg/kg resulted in albendazole levels in and CEMEC7), C. albicans (CABT), and S. cerevisiae CSF of about 0.4 pg/ml, which exceeds the IC50 for (SCBT). Tubulins are labelled S if they are sensitive to most of the Cineoformans strains tested. any benzimidazole, R if they are resistant to all benz Since clinical usefulness of a drug may be limited by imidazoles (CABT appears sensitive when expressed in the frequency at which spontaneous resistance occurs, S. cerevisiae, but this could be artifactual). 15 this frequency was estimated in vitro. Mutants resistant The alignment of 6-tubulin residues (Table 4) permits to 5 ug/ml albendazole or mebendazole occured at the calculation of the similarity (% identity) of the two frequencies of <3X106; resistance to 1 g/ml fenben C. neoformans (3-tubulins (CNBTB and CNBTA) to dazole occured at a frequency of only 5X109. other previously published 6-tubulins, several of which Our current investigations of the mechanism of action are shown. These calculations confirm that C. neofor 20 of these drugs against C. neoformans are focused on the mans CNBTB (8-tubulin is indeed very similar to other characterisation of tubulin genes from wild type C. fungal 6-tubulins, such as Aspergillus nidulans benA. neoformans and benzinidazole resistant mutants. Frag This alignment also permits us to determine if specific ments of two 6-tubulin genes from C. neoformans have amino acids (positions 167, 198, 200, and 241) that have been cloned, and sequencing has identified amino acid been previously implicated in sensitivity to the antifun 25 residues previously implicated in benzimidazole ac gal benzimidazole benomyl are present in C. neoformans tively. g-tubulin. These are indeed present in CNBTB; in par What is claimed is: ticular, amino acid 200 (F) is highly correlated with 1. A method of treatment of Cryptococcus neoformans benzimidazole sensitivity in fungi (such as ANBENA), infection which comprises administering to a patient in helminths (such as CEBEN 1 ), and protozoa. One 30 need thereof an effective amount of a benzimidazole additional amino acid (position 165) has been previously compound selected from the group consisting offen implicated in sensitivity to the anthelminthic/antifungal bendazole, albendazole, mebendazole, oxfendazole, par benzimidazole thiabendazole. Position 165 is variable bendazole, cambendazole, flubendazole, ricobendazole among many (3-tubulins examined. There is currently and luxabendazole, or a prodrug thereof. no data that identifies amino acid positions specifically 35 2. A method according to claim 1 in which the benz involved in sensitivity to anthelminthic benzimidazoles imidazole compound is selected from the group consist such as albendazole and fenbendazole. However, we ing offenbendazole, albendazole and mebendazole. predict that the presence of cat position 165 in CNBT 3. A method of treatment of meningitis caused by is partly responsible for the unexpected sensitivity of C. Cryptococcus neoformans infection in AIDS patients 40 which comprises administering to a patient in need neoformans to these anthelminthic benzimidazoles. thereof an effective amount of a benzimidazole con Conclusions pound selected from the group consisting of fenben dazole, albendazole, mebendazole, oxfendazole, parben Benzimidazoles fall into four groups on the basis of dazole, cambendazole, flubendazole, ricobendazole and activity versus C. neoformans (Table 1). Fenbendazole 45 luxabendazole, or a prodrug thereof. is highly active (IC50=0.02 g/ml), the carbendazim 4. A method according to claim 3 in which the benz group is weakly active (IC50=2 ug/ml) and thiabenda imidazole compound is selected from the group consist zole is inactive at the highest concentration tested. In ing offenbendazole, albendazole and mebendazole. comparison to amphotericin B, the currently recom 5. A method according to claim 2 in which the benz mended anticryptoccocal agent, the albendazole group 50 imidazole compound is albendazole. was generally 5-10 less inhibitory and 2-4 less cidal, but 6. A method according to claim 4 in which the benz fenbendazole was 2-3 fold more inhibitory and 8-9 fold imidazole compound is albendazole. more cidal. k k k i k
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