IN VITRO THIOCARBAMATE RESISTANCE OF TRICHOPHYTON MENTAGROPHYTES

IN VITRO THIOCARBAMATE RESISTANCE OF TRICHOPHYTON MENTAGROPHYTES Hongo, Bunkyo-ku, Tokyo 113, Japan (Received November 17, 1989' Accepted February 1, 1990) vitro Trichophyn mentagrophytes piritetrate (M-732), a new thiocarbamate antidermaphytic compound, studied comparison with a reference thiocarbamate, lnaftate. It found that this fungus not readily able develop eir se compounds. After a long period serial subculturg six stras Sabouraud dextrose broths on Sabouraud dextrose agar slants contag creasg concentrations compound, only one stra each eventually became highly resistant lnaftate eir media. None stras acquired piritetrate. two lnaftate-resistant stras thus obtaed manifested mphological, biochemical pathological changes showed partial cross- piritetrate. Key wds: Piritetrate (M-732), Tolnaftate, Thiocarbamate, Trichophyn mentagrophytes INTRODUCTION Piritetrate (M-732) is a new thiocarbamate compound [methyl (6-methoxy-2-pyridyl) carbamothioic acid 0-5, 6, 7, 8-tetrahydro-2- naphthalenyl ester; C18 H20 N2O2 S] which had been synsized by Chemical Research Labary Tosoh Cpation (fmerly, Toyo Soda Manufacturg Co., Ltd.), SHINNANYO- SHI, YAMAGUCHI-KEN, J APAN. structure piritetrate is shown Fig. 1, ger with that a reference thiocarbamate, lnaftate [methyl (3-methylphenyl) carbamothioic acid O-2 -naphthalenyl ester; C19H17N0S]1). Piritetrate is a white crystalle powder with a meltg pot 98.5-99.5t. It is soluble water, slightly soluble n -hexane, methanol ethanol, readily soluble benzene, er, acene, chlm, dimethylsulfoxide N, N-dimethyl- Piritetrate Tolnaftate (M-732) Fig. 1. Structures piritetrate (M-732) lnaftate. fmamide. potent vitro vivo antidermaphytic activity piritetrate had been itially

CHEMOTHERAPY MAY 1990 identified our screeng program on a number members thiocarbamate series which had been synsized as agricultural herbicides by Chemical Research Labary. Later2), we described detail results comparison vitro antifungal activity piritetrate, lnaftate, lciclate (a recently developed thiocarbamate compound; [O-(1,2,3,4-tetrahydro-1,4- methanonaphthalen-6- yl m-n- dimethylthiocarbanilatei3) clotrimazole (a classic azole compound; [1-{(2- chlophenyl) diphenylmethyl) imidazolej4'5), found that piritetrate possessed strongest antidermaphytic activity among se antifungals. In same paper, we also demonstrated me potent vivo activity piritetrate than lnaftate dermal model fections guea pigs with T. mentagrophytes. Me recently6), we repted action mechanism piritetrate compared with that lnaftate fmer a much me potent hibir fungal squalene epoxidation. This paper deals with results comparative vitro studies conducted on piritetrate lnaftate agast Trichophyn mentagrophytes. (A prelimary rept this wk presented at 24 th Interscience Conference on Antimicrobial Agents Chemapy, Ocber 8 `10, 1984, WASHINGTON, D. C.) MATERIALS AND METHODS Anhfungals. Piritetrate lnaftate supplied by Tosoh Cpation. se compounds dissolved dimethyl sulfoxide (DMS0), sck solutions 20 mg/ml kept at 4 Ž dark. Twold dilutions test media prepared from se sck }olutions. Trichophyn mentagrophytes stras. Six stras, namely, MTU numbers 19021, 19023, 19024, 19025, 19027 VUT 74023, used. se had been mataed our labaries by subculturg on Sabouraud dextrose agar (SDA; 2% dextrose, ph 6.5 }) slants. None m known have been exposed drugs, cludg piritetrate lnaftate, vitro vivo. Stra VUT 74023 had been isolated from a naturally-fected guea pig identified as Arthroderma vanbreuseghemii by Dr. H ASEGAWA A, Animal House, Faculty Agriculture, University Tokyo. Pri present study, we reconfirmed that this stra highly virulent f guea pigs upon dermal fection produced ascomyceus fm. Later, it proved be only stra which developed lnaftate. Stra MTU 19025, which had been isolated by us from a patient with tea pedis, representatively selected f a comparative study primary with stra VUT 74023. Stras MTU 19025 VUT 74023 highly susceptible both piritetrate lnaftate, with each showg MICs 0.01 gg/m1 0.01 ug/ml Sabouraud dextrose broth (SDB; 2% dextrose, ph 6.5 }) 0.02ƒÊg/ml 0.1ƒÊg/ml on SDA, respec. tively. Media. SDA SDB used f various tests. se will hereafter be simply called solid medium liquid medium, respectively, unless wise specified. 'Ordary Sabouraud dextrose agar' (hereafter referred as dary SDA; 4% dextrose, ph 6.5 }) employed obta a sufficient amount conidia f oculation tests on mphological biochemical properties as well as on virulence resistant. F demonstration perfect stage stra VUT 74023 its subcultures, Takashio's medium') used its gredients are neopepne (Difco) 0.1 g, dextrose 0.2 g, KI-121)04 0.1g, MgSO4 E7 H20 0.1 g, Bacagar 2.0g, made up 1,000 ml with distilled water. Animals. Male guea pigs, weighg ca. 300g, purchased from Shizuoka Labary Animals Cpation, Ltd., S HIZUOKA, JApAN used f determation virulence subcultures comparison with ir sensitive stras. Preparation oculum. An appropriate volume sale contag 0.1% Tween 80 poured on culture T. mentagrophytes grown on dary SDA slants at 27t f 4 weeks, surface culture scratched with a platum loop free conidia. conidium suspension mixed with a small amount hphae transferred a flask allowed

VOL. 38 NO.5 Thiocarbamate st f an appropriate period until small pieces precipitated. After filterg suspension, excludg precipitates through sterile folded gauze, supernatant, contag almost pure conidia, suspended same sale adjusted ca. 10 CFU/ml conidia on basis an optical density 0.35 at 540 mp. Immediately pri oculation, conidium suspension shaken gently ensure a homogeneous state. Determation MICs. MICs piritetrate lnaftate determed by broth agar dilution methods. Conidia oculated 5 ml broth L- fm tubes on 20 ml agar plates contag twold dilutions antifungal. oculum sizes ca. 103 CFU/ml ca. 103 CFU, respectively. After cubation at 27 Ž f one week liquid medium with shakg, f 3 weeks on solid medium, MICs read. Testg f development. F development liquid medium, th culture, which had grown fairly well at a subhibiry concentration closest MIC, gently crushed a mtar obta a homogeneous oculum suspension adjusted ca. 107 CFU/ml. conidium suspension n oculated a series same a little higher twold dilutions antifungal. Subculturg at 27t with shakg perfmed every week f 27 weeks. At time transfer solid medium cultures, conidia harvested from colonies grown on plates contag antifungal at a subhibiry concentration closest MIC. Subculturg carried out every 3 weeks, 27 times tal. oculum sizes liquid medium on solid medium same as those dicated determation MICs. Testg f demonstration primary. existence number cells resistant 1ƒÊg/ml compounds. number visible colonies fmed after cubation f one, week at same temperature counted. Demonstration back mutation. two lnaftate-resistant which eventually obtaed passed 10 times liquid medium absence antifungal. level checked after 5 10 passes determe if back mutation occurred. Demonstration cross-. Two resistant ir stra (VUT 74023) examed f cross- piritetrate lnaftate terms MIC on plates contag appropriate concentrations antifungal. Testg f cultural mphological changes resistant. To exame f possible cultural mphological changes, cubated at 27t liquid medium with shakg on dary SDA plates, ir growth rates colony fms observed over a period 4 6 weeks, respectively, comparison with ir stra. Cells grown dary slide culture usg SDA at same temperature also compared with those ir stra by means light microscopy. Possible loss ascomycemaus fm examed cultures resistant grown on Takashio's medium at 27t f 2 months. Testg f changes virulence resistant. resistant compared with ir stra f ir virulence each ten guea pigs. An area (ca. 8 by 10 cm) on dsal trunk each animal shaved, two circular areas 3 cm diameter separated by an appropriate distance gently abraded with spaper. About 106 CFU conidia se stras grown on dary SDA at 27 Ž f 5 weeks n oculated on se treated sites. both compounds population stras VUT 74023 MTU 19025 determed. About 109 CFU conidia harvested from each stra after growth on dary SDA at 27t f 4 weeks plated on plates contag 0, 0.01, 0.1 RESULTS Development. Five T. mentagrophytes stras, i.e., 19021, 19023, 19024, 19025 19027, did not develop- eir compound

CHEMOTHERAPY MAY 1990 Fig. 2. Development stras Trichophyn mentagrophytes piritetrate lnaftate liquid medium. Initial susceptibility stra VUT 74023 piritetrate lnaftate 0.01 0.1 geml, while fal susceptibility 0.04 >80ƒÊg/ml. respective figures f stra MTU 19025 0.01 0.1ƒÊg/ml 0.08 0.2ƒÊg/ml. Fig. 4. Comparison distribution piritetrate- lnaftate-resistant mutant cells existg sensitive stras Trichophyn mentagrophytes VUT 74023 MTU 19025. Ca. 109 CFU conidia stras grown on SDA slants by cubatg at 27 Ž f 4 weeks plated on SDA plates contag drug at 0.01 80 gem' visible colonies fmed after cubatg f one week at same temperature counted. Symbols: A, piritetrate; B, lnaftate; VUT 74023 stra,;------, MTU 19025 stra. drug late stage serial subculturg on solid medium. se resistant progenies designated L-1 mutant mutant, respectively. We consider that both progenies reached highest-level, over an 800-fold crease, because y grew at 80mg/ml, highest tested concentration lnaftate its maximum solubility Fig. 3. Development stras Trichophyn mentagrophytes piritetrate lnaftate solid medium. Initial susceptibility stra VUT 74023 piritetrate lnaftate 0.02 0.1 jig/ml, while fal susceptibility 0.08 >80 jig/ml. respective figures f stra MTU 19025 0.02 0.1ƒÊg/ml 0.04 0.04ƒÊg/ml. eir medium. ir MIC values creased only 2-4-fold. Stras 19023 19025 showed a transient 8-fold crease,piritetrate lnaftate, respectively, liquid medium. In contrast, only stra MT 74023 developed high-level olnaftate both media. This stra developed gradual lnaftate midway through series subculturg liquid medium progressive media used (piritetrate also). F simplicity, Figs. 2 3 show results f stra MTU 19025, selected as representative five stras which did not develop, stra VUT 74023. Distribution resistant mutant cells existg sensitive stras. Usg about 109 CFU conidium stras VUT 74023 MTU 19025, all cells stra MTU 19025 sensitive 0.1ƒÊg/ml less both compounds, whereas relatively many cells stra VUT 74023 a little less sensitive lnaftate, with MICs between 0.1 1ƒÊg/ml (Fig. 4). This difference susceptibility piritetrate lnaftate, although not very conspicuous, may evidence fact that stra VUT 74023 eventually developed a high level lnaftate durg repeated subculturg

VOL. 38 NO.5 Thiocarbamate Table 1. Degree back mutation 439 L1 S 1 lnaftate-resistant Trichophyn rnentagrophytes VUT 74023 Resistant obtaed by serial transfers stra sensitive piritetrate lnaftate liquid on solid medium contag creasg concentrations L-I: Tolnaftate-resistant drug (see Fig. 1.). mutant obtaed a SDB; tested f back mutation by serial transfers drug-free SDB. : Tolnaftate-resistant mutant b obtaed on SDA; tested f back mutation by 10 serial transfers Table 2. Piritetrate See liquid medium five failed mutant its Cross 2. re between grow at a even after 10 mutant, its itial on solid piritetrate slight results medium but are defite lnaftate L-1 80ƒÊg/ml whereas after did stra susceptibility,. 1, not susceptibility not MIC after changed 0.08 test 5 revert even had 74023 which however, igal ir transfers, its contrast Table concentration VUT compound. level lnaftate-resistant 1. 19025, eir shown highest much transfers. from completely As MTU SDA. mentagrophytes Table medium, lost transfers. 10 lnaftate mutant solid sensitivity Trichophyn footnote cludg. retaed able on stras, develop Stability on drug-free ttg/ml. f presented cross Table cross- both L-1 Fig. 5. Colonies mutant (resistant lnaftat; left) its stra (VUT 74023; right). Note pigment fmation by mutant.

CHEMOTHERAPY 440 Fig. 6. Light stra. cell with medium f different, brown did As seen streakg a 2 pm shown conidia whereas 6 globose. that ospes spiral with fmed stra. reverse deeper hyphae 0.3 size stra. resistant also 0.7 shape. pm Few no aleuri- its shape. than macroconidia by on antifungal agents duction resistant is much me difficult than with bacteria. has thus received little attention, contrast critical imptance bacterial. Dermaphytes versus antifungals are no exception this generalization; re have been only a few repts on ir, ambrutic fmation conspictus 1 segmen- variants Fungal number Meover, ovoid, hyphae me as stra conspicuous a 1.5 produced shape globose caused hyphae by size human pathogenic fungi dicate that naturally -occurrg is very rare, with notable exception flucyset1. rangg stra, Most repts with width i.e., compared various characterized conidia, mutant. easily (/400) stras positive tissue mycological response at 19 20 fected foci under same test conditions. oculated much disrted tation f f stra. ten became diameter, conidia entirely 2 pm longer left) two DISCUSSION diameter Fig. 5, Fig. size 5 pm reddish- colonies pigs given a dermal oculation ca. 106CFU conidia two at shaved sites on back showed no recognizable lesions at any 20 fected foci each tal both groups, while group stra gave clearly colonies a plate greater 2.5 (s); solid lnaftate; obverse. Microscopically, from ir deeper liquid on between Genetic property resistant. Both resistant cubated on Takashio's medium found have completely lost ability produce a perfect stage durg 3month observation period. Virulence resistant. Ten guea significantly from seen suspension on than not fmation conidial fms 27 Ž week differ pigment ne. where one change period, col by (resistant differences resistant colony not soluble col mutant at f prcipal cubation weeks, mutant rates shakg by Note cubated six stra. area mphologies when medium cells right)( ~400). growth, 74023;. Cultural microscopy (VUT MAY 1990 such as griseulv9-16) (cyclopropylpyran acid) 17.18) However, view widespread frequent use antifungal agents recent years, studies have become newly exploited imptant, agents. particularly with

VOL. 38 NO.5 Thiocarbamate In this study we found that only one six test stras T. mentagrophytes became resistant lnaftate after relatively many transfers, while none acquired piritetrate. se results suggest that this fungal species, probably dermaphytes as well, do not readily develop se thiocarbamate compounds. However, stra VUT 74023 might not necessarily be such an exception its acquisition lnaftate view repts on primary / secondary antifungals such as azoles pathogenic fungi, cludg dermaphytes4.539-21). possible existence a few stras which are able acquire such thiocarbamates durg longterm rapy might be responsible f occasional cases unsuccessful treatment dermaphyses. It is notewthy that acquired lnaftate-resistant three ders magnitude greater than stra. test stras, cludg stras MTU 19021 VUT 74023, transiently developed 8-fold less liquid medium on solid medium. However, such a transient rise MIC values cannot be regarded as acquisition true sense, sce evaluated exceed upper limit MIC range. MIC values do not It seems likely that se multi-step obtaed by means serial subculturg presence creasg concentrations lnaftate obviously differ from one-step resistant flucyse ir mode acquirg 8,22). That acquisition me progressive on solid than liquid medium might be due various facrs, f stance, physiological conditions both media f growth, preparation cell suspensions f oculation, etc. Overall, VUT 74023 MTU 19025 showed similar cell population structural patterns terms susceptibility piritetrate lnaftate befe transfer. However, a culture VUT 74023 cluded me cells less sensitive lnaftate than piritetrate. This difference considered small but not negligible sce VUT 74023 eventually became resistant lnaftate but not piritetrate by repeated subculturg eir medium. reasons why mutant became resistant earlier than L-1 mutant, yet L-1 mutant me stable retention than S- 1 mutant, are unclear. Possible facrs are slight differences techniques f subculturg f development f back mutation, stra variation. fdg that cross- lnaftate-resistant piritetrate partial may derive from differences chemical structures se thiocarbamates hence ir vitro vivo antidermaphytic activity : piritetrate has a pyride nucleus with a methoxy group tetrahydronaphthalene moiety, while lnaftate has a benzene nucleus with a methyl group naphthalene moiety. It is particular terest that acquisition stra VUT 74023 lnaftate accompanied by a characteristic pigment production matured colonies its mutant. pigment might be a carotenoid (s) consideration its col solubility solid medium. Changes cellular mphology two resistant also characteristic, f stance, enlargement conidia, strikg segmentation hyphae that produced a great many aleuriospes, accelerated fmation spiral hyphae, etc. se features might be due qualitative quantitative alterations imptant cellular constituents such as nucleic acids prote, as repted f fungal cells treated with flucyses). loss perfect stage resistant ir concomitant loss reduction virulence f guea pigs upon dermal fection with conidia might be due a lowerg ir gemral metabolic activity. This 'loss mutation' found be irreversible both. Thus it ic clear that se changes mphological, r hysioi ogic a 1 pathological properties lnaftate-resistant closely related each. In summary, a new thiocarbamate compound,

CHEMOTHERAPY MAY 1990 piritetrate, a reference thiocarbamate, lnaftate, did not readily duce T. mentagrophytes stras vitro by serial transfer both liquid on solid medium contag creasg drug concentrations. However, one six test stras became resistant lnaftate after many transfers. se results suggest possible failure long-term rapy with such thiocarbamates if resistant cells develop. characteristic mphological changes, concomitant loss perfect stage virulence, cross stability acquired two resultant lnaftate-resistant are described. ACKNOWLEDGMENTS We thank Dr. HASEGAWA A, Animal House, Faculty Agriculture, University Tokyo, TOKYO Tosoh Cpation f providg stra VUT 74023 T. mentagrophytes two thiocarbamate compounds, respectively. We also thank Tomoko Kamoshida f her technical assistance. REFERENCES 1) NOGUCHI T, Krul A, IGARASHI Y, SHIGEMATSU A, TANIGUCHI K. Antitrichophyn activity naphthiomates. Antimicrob Agents Chem 1962: 259 267, 1963 2) IWATA K, YAMASHITA T, UEHARA H: In vitro vivo activity piritetrate (M-732), a new antidermaphytic thiocarbamate. Antimicrob Agents Chem 33:2118 2125, 1989 3) de CARNERI I, Mown G, B IANCHI S, CASTELLINO S, MEINARDI G, MANDELLI V: Tolciclate agast dermaphytes. Arzneim-Fsch (Drug Res) 26: 769 `772, 1976 4) PLEMPEL M, BARTMANN K: Experimental studies on antimycotic action clotrimazole (Canesten) vitro after local application vivo. Drugs Germ 15 : 103 120, 1972 5) SMITH K J, WARNOCK D W, K ENNEDY C T, J OHNSON E M, H OPWOOD V, VAN C UTSEM J, VANDENBOSSCHE H: Azole Cida albicans. J Med Vet Mycol 24: 133-144, 1986 6) M ORITA T, IWATA K, NOZAWA Y: Inhibiry effect a new mycotic agent, piritetrate on ergosterol biosynsis pathogenic fungi. J 7) TAKASHIO M: Sexual reproduction some Arthroderma Nannizzia on diluted Sabouraud agar with without salts, Mykosen 15: 11 17, 1972 8) S CHOLER H J: Flucyse, pp. 35 106. Antifungal Chem., Speller D C E, 1980 9) ARTIS W M, BONNIE M, Jos H E Griseulvresistant dermaphysis crelates with vitro. Arch Dermal 117: 16 19, 1981 10) A YTOUN R S C, CAMPBELL A H, NAPIER E J, SEILER D A L: Mycological aspects action griseulv agast dermaphytes. Arch Dermal 81: 650 656, 1960 11) DAVIES R R, EVERALL J D, HAMILTON E: Mycological clical evaluation griseulv f chronic onychomycosis. Br med J iii: 464 468, 1967 ` 12) GREENBERG J H: Griseulv. Int J Dermal 18: 249 `260, 1979 13) GRIN E I, NADAZDIN M, OzwGOVIC L: Investigations dermaphyte sensitivity griseulv. Acta Derm Venerol 45: 283 `287, 1965 14) H ANTSCHK D, GoETZ H: Griseulv Resistanz. Z Hautkr 56 : 1326 `1333, 1981 15) LENHART K: Griseulv-resistant dermaphytes: II Physiological genetic studies. Mykosen 13: 139 `144, 1970 16) YOUNG C N: Sensitivity patterns griseulv Trichophyn rubrum rgwm fungi. Trans Rep St John's Hosp Derm Soc 58: 226 `234, 1972 17) RINGEL S M, GREENOUGH R C, RoaMEx S, Coo D, Gurr A L, B LAIR B, KANTER G, STRANDTMANN M: Ambrutic (W7783), a new antifungal antibiotic. J Antibiol 30: 371 375, 1977 18) S HADOMY S, DIXON D M, ESPINEL-INGRcFF A, WAGNER G E, Yu H P, SHADOMY H J: In vitt v studies with ambrutic, a new antifungal antibiotic. Antimicrob Agents Chem 14: 99 104, 1978 19)HOLT R J, Amu A: Miconazole-resistant cida. Lancet i:50, 1978 20) JOHNSON E M, RICHARDSON M D, WARNOCK D W: In-vitro imidazole antifungals Cida albicans. J Antimicrob Chem 13: 31 43, 1984 21) RYLEY J F, WILSON R G, BARRETT-BEE K J: Azole Cida albicans. Sabouraudia 22:53 63, 1984 22) HAMILTON -MILLER J M T: Physiological properties mutagen-duced variants Cida albicans resistant polyene antibiotics. J Med Microbiol 5: 425 429, 1972 Med Vet Mycol 27: 17 25, 1989

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