Therapeutic and Preventive Effects of Griseofulvin in Guinea Pigs1

THERAPEUTIC AND PREVENTIVE EFFECTS OF GRISEOFULVIN IN GUINEA PIGS* STANLEY A. ROSENTHAL, PH.D., NORMAN GOLDFARB, M.D. AND RUDOLF L. BAER, M.D. With the technical assistance of REVA WISE, BA. AND ANTONIO OLIvER

Ever since the report of Gentles (1) that from the clinical, histologic and microbiologic viewpoints? What is the minimal preventive dose? Is there a lasting prophylactic action of

griseofulvin cured infections due to 3liero.sporom canis and Trichophyton mentagrophytes in guinea

pigs, this drug has aroused intense interest as a griseofulvin after the drug has been discontinued? therapeutic agent for superficial mycoses. While EXPERIMENTAL Gentles originally administered griseofulvin at a In order to ascertain the minimal effective dose dosage of 60 mg/kg/day, he subsequently found

(2) 15 mg/kg/day to be an effective dose in

and the relative speed of healing of fungous infec-

guinea pigs. Martin (3) successfully treated T. mentagrophytes infections in guinea pigs with griseofulvin at 25 mg/kg/day and to some extent with lower doses. Lauder and O'Sullivan (4) found 60 mg/kg/day to be effective in curing

griseofulvin, we treated 38 guinea pigs which had been experimentally infected with M. canis for this purpose. Wood's light positive hairs from another infected guinea pig had been applied to a slightly scarified area on the flank. Ten to fourteen days

tions on various doses of orally administered

later, when the fungous lesions were well de-

existing infections and in preventing experimental infections in cattle with T. verrucosum. Clinical experience in man by others (2, 5, 6)

veloped and fluoresced under Wood's light, treatment was started by force-feeding of water sus-

and by our group has amply demonstrated the

pensions of griseofulvin*. Groups of animals

efficacy of griseofulvin in many forms of mycotic infection. Blank and Roth (7) have demonstrated the high degree of sensitiveness of many dermato-

weight daily, 5 days a week for 4—5 weeks.

received 60, 30, 15, 10, 6 or 3 mg per kg of body

Guinea pigs were also infected but left un-

phytes to griseofulvin but noted that the deep fungi and Candida albicans are insensitive. We have been able to confirm these findings in as

treated to serve as a control for comparison with griseofulvin-fed animals. Animals were observed clinically at intervals of 1 to 3 days. Cultures and Wood's light examinations were made approximately weekly.

yet unpublished studies.

In man, the most commonly used dosage for the treatment of superficial fungous infections hitherto has been 1000 mg per day. Based on an average weight of 70 kg, this amounts to 14.3

RESULTS

In six animals, fed 60 mg/kg/day, slight clinical improvement was noted as early as the third day after the start of therapy; there was less erythema than in the untreated control animals, and bloody

mg/kg/day, a figure which is very close to the 15 mg/kg/day dosage used by several investigators crusts had not formed in the treated animals. in laboratory animals. In our own studies, we attempted to investigate After seven days (i.e. a total of 5 feedings) the a number of questions which, thus far, have not lesions were definitely in the process of cure, the been elucidated: What is the minimal effective inoculated sites showing only slight scaling with dose of griseofulvin per kg/day in infected ani- no erythema. Control animals at this time had

mals? Can a quicker healing of the lesions be anticipated with larger than the minimal effective doses? How does the speed of healing compare

bloody crusted lesions with surrounding crythema. After 9 days, hair had started to regrow

in all six animals and after 14 days they appeared clinically cured. However, it should be noted that, *From the Department of Dermatology and even after 14 days, the previously affected sites

Syphilology (Dr. Marion B. Sulzberger, Chair- continued to fluoresce and were still culturally man), New York University Post-Graduate Medical School and the Skin and Cancer Unit of positive for M. canis. Figures 1—5 show a typical University Hospital, New York, N. Y. *Griseofulvin was supplied by Dr. Gavin Presented at the Twentieth Annual Meeting of The Society for Investigative Dermatology, Inc., Hildick-Smith of Johnson & Johnson, New BrunsAtlantic City, N. J., June 7, 1959. wick, New Jersey. 419

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FIG. 1. GP 149. Infected with M. coals 13 days

prevlously. On day treatment with griseofulvin started.

r

FIG. 3. GP 149. Two weeks after start of feedings. Infected 27 days previously.

I FIG. 2. GP 149. One week after start of feedings. (60 mg/kg). Infected 20 days previously.

FIG. 4. GP 152. Infected with M. coals 13 days previously.

day, when a decrease in sealing was noted. By

therapeutic response in a guinea pig receiving 60 mg/kg, as well as an untreated control animal. In 5 animals, fed 30 mg/kg/day, the first signs of clinical improvement, noted on the fourth day

after starting treatment, were decreased ery-

the sixteenth day, hair had started to regrow. The animals appeared almost normal after 3 weeks,

while the infected sites in untreated animals at this time remained slightly inflammatory. In four animals fed 10 mg/kg/day, no clinical

thema and crusting as compared with the control improvement was noted during the first week guinea pigs. By the eleventh day, normal appear- after start of therapy. On the ninth day there was ing hair had started to regrow at the sites of a slight but definite decrease in erythema, crustinoculation, and the animals appeared clinically ing and edema. However, despite continued cured by day 16. treatment, no further improvement was noted durIn 7 animals, fed 15 mg/kg/day, a definite de- ing the subsequent observation period which crease in erythema was noted by the fourth day could not he attributed to the spontaneous cure after the start of treatment. However, no further process which normally occurs in untreated clinical improvement occurred until the eleventh infected guinea pigs.

THERAPEUTIC AND PREVENTIVE EFFECTS OF GRISEOFULVIN

In ten animals fed 6 mg/kg and in six animals fed 3 mg/kg, the course of the fungous infection appeared unaltered as compared with untreated control animals. In the course of these therapeutic assays, we carried out comparative histologic studies of skin biopsies from 16 guinea pigs infected with ill. canis. Ten to twelve days after infection, 8 of the

421

animals started to receive griseofulvin, 60 mg/kg,

5 times a week in the manner previously described, and the rest were left untreated as controls. Biopsy specimens were taken before the start of therapy and on days 4 and 8 of therapy.

The sections were stained by the HotehkissMcManus method and were read by Dr. Morris Rothstein. * Typical sections are presented in Figs. 6—8. RESULTS

Before treatment, the sections showed numerous hyphae in the horny layer and numerous infected hairs and follicle walls in the epidermis;

40—85% of the hairs in the upper eutis were involved. After four days of treatment, numerous

filaments were still present in the horny layer. The hairs in the epidermis were infected while the follicle walls were free of fungi. In the upper cutis, only 10% of the hairs were infected. By the

eighth day, the number of fungous elements in the horny layer was very much reduced and no FIG. 5. GP 152. Infected with M. canis 27 days

fungi could be seen in the hairs or follicle walls of the epidermis or upper cutis. The untreated con-

previously. No griseofulvin therapy. Compare *We wish to thank Dr. Morris Rothstein for his with Figure 3. help in evaluating the tissue sections.

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FIG. 6. GP 69. Before start of griseofulvin therapy. Infected 10 days previously. Hotchkiss-McManus stain.

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422

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Pie.?. OP 69. After 4 feedIngs with grlssofulvin 60 mg/kg. Infected 14 days previously. Hotehkia-

VaUnqs stain.

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60mg/kg (8 days after start of treatment). Infected

THERAPEUTIC AND PREVENTIVE EFFECTS OF GRISEOFULVIN

423

TABLE 1 They were examined at weekly intervals for Clinically apparent infection in guinea pigs clinical evidence of infection for three weeks. simultaneously infected with M. canis and treated with griseofulvin

Griseoful-

yin per

Feeding,

mg/kg

60 30 15 10 15 15 10 5 10 2 2

No. of Feedings Per Week

5 5 S

5 3 2 3 5 2 5 3

300 150 75

50 45 30 30 25 20 10

6 0

The results of this experiment are presented in Table 1. When a total of 50 mg per kg or more

No. of Animals Infected After:

Total Weekly Dose,

mg/kg

RESULTS

1 week 2 weeks 3 weeks

0/3 0/3 0/5 0/5 0/4

0/3 0/3 0/5 0/5

1/4

4/4 3/4

0/4 0/5 3/3 2/7

3/4

2/3 3/5

0/3 0/3 0/5 0/5 3/3 4/4 3/5

of griseofulvin was fed per week, clinically apparent disease was prevented during the three week period of observation. Lower doses either were essentially without effect, or, at best, delayed the appearance of clinical changes. How-

ever, it was noted that even in those animals receiving as little as 10 mg/kg/week the lesions which did develop were much smaller than in the control animals. EXPERIMENT ON DURATION OF PROPHYLACTIC

5/7 4/4

5/7

13/13

TABLE 2 Effects of preceding treatment with griseofulvin on subsequent infection with M. canis

EFFECT FROM PRIOR MEDICATION

In another series of guinea pigs, we attempted to ascertain whether the previous administration of griseofulvin has a prophylactic effect on subsequent experimental infection with M. canis.

Groups of guinea pigs were fed varying

amounts of griseofulvin for varying lengths of time. Three and six days after the last day of treatment, the animals were challenged with M. can.is and were then observed for 7—10 days for signs of fungous infection. RESULTS

The dosage schedules, time intervals and results are presented in Table 2. This shows that prefeeding griseofulvin for as long as three weeks,

at a level of 60 mg/kg, did not prevent the sub-

sequent development of fungous disease in animals which were deliberately infected with M.

canis three days after discontinuation of treattrol animals biopsied at the same intervals as ment. those receiving griseofulvin showed the full blown

features of fungous infection during the entire period of observation.

DISCUSSION

ill. canis infections in guinea pigs responded favorably to griseofulvin therapy as long as the EXPERIMENTS IN PROPHYLAXIS dose, given daily for five days during each week, In further tests, we attempted to ascertain the was at a level of 15 mg/kg/day or more. There minimum dose of orally administered griseofulvin was some indication that with the higher doses, which will prevent the development of experi- cure took place at a significantly faster rate than mental infection with M. canis. Guinea pigs were with the minimum effective dose of 15 mg/kg/ exposed to M. canis by placing Woods light day. A dose of 10 mg/kg/day had a very slight positive hairs on slightly abraded areas on their inhibitory but non-curative effect on M. canis flanks. Starting on the day of infection, groups infection, while doses of 6 and 3 mg/kg/day had of animals were fed varying amounts of griseo- no noticeable effects on established infections. fulvin according to the schedule given in Table 1. These findings appear to be in satisfactory agree-

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ment with those of Gentles (cited in 2) and of

possibility that pathogenic fungi may survive in

Swartz, et al. (8), who studied T. mentagrophytes infections in guinea pigs.

the patient's skin for some time after clinical

If the results of our investigations in guinea pigs are applicable to man, the presently used average dose of 1000 mg per day represents the

disappear from the hairs and hair follicles of

minimum effective dose for a person weighing 70 kg. It would appear that a correspondingly higher dose shou]d be administered in patients weighing more than 70 kg. Furthermore, in patients where,

feedings of 60 mg/kg showed a decrease in the

"cure". During griseofulvin therapy fungi rapidly

guinea pigs, but not as rapidly from their surface

horny layers. Sections obtained after 4 daily number of infected hairs and follicles in the upper cutis, no fungi in the lower cutis, but no marked

reduction in the number of fungi in the horny

for one reason or another, healing within the

layer. This is during the phase when reduction of shortest possible time appears desirable, doses of erythema was first noted. After eight days, when 30 to 60 mg/kg/day would be indicated, unless the animals were in the process of "cure", sections further experience in man suggests prohibitive showed no fungi in hairs or follicle walls but the side effects at the higher dosage levels. horny layer still showed some fungi, although in Although the minimal effective dose in man markedly reduced numbers. has not yet been ascertained, our clinical experiOur attempts to prevent infections showed

ence to date is in accord with our results in that, if treatment is started simultaneously with animals. In a few cases in children who received exposure to M. canis, active disease can b' such a small dose as 10 mg/kg/day for Ill. prevented with 50 mg/kg/week. This amount is audouini infections of the scalp, no beneficial equivalent to 9 the minimal therapeutic dose in effects were observed. guinea pigs. It appears, therefore, that perhaps We wish to call attention to still another find- once active disease has been "cured", it may be ing of ours which may be of clinical importance: possible to prevent recurrences by maintaining In guinea pigs treated with doses as high as 60 patients on 10 mg/kg/day. However, our findmg/kg/day, the previously infected but clinically ings in guinea pigs clearly demonstrate that as "cured" sites remained culturally positive at 14 early as three days after discontinuation of days and longer. In fact, cultures from these griseofulvin therapy in doses as high as 60 mg/ griseofulvin-fed animals did not start to become kg/day, the skin is unprotected against infection negative until the cultures in untreated, control with M. canis. Similar findings have been seen in animals began to become negative. We have noted the independent studies of Swartz, et at (8).

parallel findings in some of our patients, where positive cultures still could be obtained from

previously infected sites, even though these clinically appeared "cured." Indeed, some of these patients subsequently developed clinical recurrences of their disease after discontinuing therapy. It must therefore be anticipated, since griseofulvin is only a fungastatic (2), that, at least in some cases it perhaps makes healthy carriers out of patients who have been "cured" of their active disease, i.e., possibly it does not entirely eliminate all fungi from the skin even after prolonged therapy. Such an effect would not be unlike that seen in patients with furuncles who have been treated systemically with broad spectrum antibiotics; the medicament may help to cure the furunculosis but certainly does not eliminate all pathogenic staphylococci from the patient's skin and does not prevent recurrences once it has been discontinued.

Our histologic studies tend to support the

SUMMARY

1. Guinea pigs infected with M. canis responded favorably to force feedings of griseofulvin

at a level of 15 mg or more per kg five times a week as evidenced by accelerated clinical healing

of the lesions as compared with controls. Cultures, however, still were positive at that time. Biopsy studies showed rapid disappearance of fungi from infected hairs and hair follicles, but reduced numbers of fungi were still demonstrable

in the horny layer eight days after inception of treatment. The possible implications of these findings regarding therapeutically effective doses

in man and regarding recurrences of "cured" infections are discussed.

2. When griseofulvin therapy was started simultaneously with deliberate infection with M. canis, a dose of 50 mg/kg/week was found to be the minimal dose required to prevent infection. Thus, in guinea pigs, the minimal preventive dose

THERAPEUTIC AND PREVENTIVE EFFECTS OF GRISEOFULVIN

425

the minimal curative dose. The possible implications of these findings regarding prevention

3. MARTIN, A. R.: The systemic and local treatment of experimental dermatophytoses with

of recurrences in "resistant" fungous infections

528, 1959. 4. LAUDER, I. M. AND O'SULLIVAN, J. G.: Ring-

is

in man are discussed.

3. It was found that the protective effect of griseofulvin is lost within 3 days after the drug has been discontinued.

griseofulvin. J. Invest. Dermat., 32: 525—

worm in cattle. Prevention and treatment with griseofulvin. Vet. Rec., 70: 949—951, 1958.

5. WILLIAMS, D. I., MARTEN, R. H. AND

Editor's note: This paper was discussed jointly with that of Frank J. Roth, Jr., Bennett Saliman

and Harvey Blank, "In Vitro Studies of the Antifungal Antibiotic Griseofulvin" which immediately precedes the present paper. REFERENCES 1. GENTLES, J. C.: Experimental ringworm in guinea pigs: Oral treatment with griseoI ulvin. Nature, 182: 476—477, 1958.

2. BLANK, H. AND ROTH, F.: The treatment of

dermatomycoses with orally administered griseofulvin. Arch. Dermat. & Syph., 79:

259—266, 1959.

SARKANY, I.: Oral treatment of ringworm with griseofulvin. Lancet, 2: 1212—1213, 1958.

6. RIEHL, G.: Griseofulvin—Ein Peroral Wirken-

des Antimykoticum. Der Hautarzt, 10: 136, 1959.

7. ROTH, F. AND BLANK, H.: In Vitro studies of

the antifungal antibiotic griseofulvin. J. Invest. Dermat., In Press. 8. SWARTZ, H. E., GRUTTER, F. H., KAMP, L. F.

AND GAUGHRAN, E. R. L.: The effect of

griseofulvin on trichophyton mentagrophytes infected guinea pigs. Bact. Proc.,

Page 81, 1959. Presented before the Society of American Bacteriologists, May 10—14, 1959, St. Louis, Missouri.

DISCUSSION DR. JACOB H. SWARTZ (Boston, Mass.): I should like to ask Dr. Roth if tests for hemolysis were done with griseofulvin? I should also like to ask whether mutants developed following treatment with griseofulvin? If so, were these mutants more resistant to this antifungal antibiotic? DR. BERNARD GORDON (Bellevue Hospital, New York City): On behalf of Dr. Fred Reiss I

In T. tonsurans infections during therapy one sees transformation of the round endospores into rectangular anthrospores and eventually irregularly shaped, bizarrely outlined, distorted hyphae. DR. PAUL H. JACOBS (Washington, D. C.): I

enjoyed both papers and the beautiful pictures. I would like to ask two questions: 1, regarding the time lag period after the pellets were removed

would like to comment on the clinical and from the griseofulvin solution, did this vary in mycological effects of griseofulvin as seen on

treating 30 children (ages 2 to 11) with ringworm infections of the scalp at Bellevue hospital.

length depending upon how long they were in the solutions; and 2, was any resistance noted

Griseofulvin (250 mg. twice a day) was ad- in the organisms studied?

DR. LOUIS H. WINER (Beverly Hills, Calif.): ministered orally to 25 children with M. audouini I had the pleasure of reading and discussing a infections of the scalp. The average length of time paper by Dr. Harry Robinson et at. on Grifulvin*. to cure ranged from 21 to 74 days. The average Using cultures of Trichophyton rvbrum and was 44 days. Our criterion for cure was two consecutive Micros poron canis and transpiating the organisms to gradually increased strengths of Grifulvin in negative cultures. Five children with T. ton.surans infections of vitro. Has anyone been able to obtain positive

the scalp were cured in an average of 53 days. animal inoculation with this type of attenuated The range was from 36 to 75 days. organism. There were no side effects.

DR. LAWRENCE C. GOLDBERG (Cincinnati,

During therapy the morphological character- Ohio): We heard what the minimum amount of istics of M. audouini in the hair revealed transi- griseofulvin was necessary to cure the fungus tion from the typical microid mosaic pattern of infection in these animals. By any chance does the spores to frequent formation of hyphal anyone know how much griseofulvin would have elements. After the 3rd week of therapy, these to be given to kill a guinea pig. hyphae showed rectangular fragmentation and groups of bizarrely shaped spores.

*Griseofulvin of McNeil.

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THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

DR. A. J. ORFUSS (New York, N. Y.): I would dermatophytes. Our studies in this area, as relike to know, firstly whether there were any ported in our manuscript, have been restricted to reactions in the animals that were given this one species, Trichophyton rubrum. We have been

unable to demonstrate significant adaptation or know the dose that these animals received pre- produce resistant mutants even after prolonged cisely? Was this material given in the water these exposure of the fungus to the antibiotic. The work animals were drinking, and if so, what was the of Dr. Robinson on induced resistance was done way he determined the amount that they with other species of dermatophytes. received? I believe the remaining questions can best be DR. FRANK J. ROTH (in closing): In answer to answered by Dr. Rosenthal. the first question, we have not undertaken any Da. S. A. ROSENTHAL (in closing): The findings direct studies on the possible hemolytic activity in our laboratory concerning increased resistance of griseofulvin. No such action has been ob- of Trichophyton rubrum and other pathogenic served, however, in any of our clinical trials of fungi to griseofulvin are in agreement with Dr. the antibiotic. A number of other investigators Roth. We have been able to grow T. rubrurn on have evaluated the drug in this respect and have increasingly higher concentrations of griseofulvin found no indication of such an effect. by culturing the parent strain on subinhibitory We have not as yet observed any hereditary concentrations of this drug. Using a technic changes in the morphology or metabolism of different from that of Dr. Roth, certain of our sensitive fungi following their exposure to gris- strains of T. rubrum were made to grow in coneofulvin. Culture isolates obtained from patients centrations as high as 7 micrograms per milliliter. on therapy and dermatophytes introduced and However, these so-called resistant strains, when antibiotic, and secondly, how does Dr. Rosenthal

maintained in varying concentrations of the transferred back onto ordinary Sabouraud's antibiotic have been carefully studied. A pro- medium for one generation, lost all increased longed lag phase is observed after a dermato- resistance and became just as susceptible as the phyte has been removed from an inhibitory con-

parent cultures.

centration of the antibiotic. This lag period is Dr. Robinson has apparently anticipated us. especially evident after the fungus has resided We have experiments in progress now attempting for 10 days or more in a minimal inhibitory con- to induce resistance to griseofulvin in Microcentration of griseofulvin. We believe that there .sporum canis. If we develop resistance in this is a loose binding of the antibiotic to the my- fungus, we plan to inoculate the resistant strain celium or a small intracellular depot formed which must be dissociated or removed by elution before

a normal rate of growth can be resumed. With higher concentrations a state of enforced dormancy is produced in susceptible fungi—a state which must first be broken before growth will occur. Efforts to establish a persistent retardation of growth by prior treatment of the fungus with griseofulvin have been unsuccessful. Strains of Trichophyton rubrum which showed an increase

of the minimal inhibitory concentration to 1.8 mgm./ml. developed normally in media free of the antibiotic and upon reassay demonstrated .a drop back to the original sensitivity (0.20 mgm. /ml.). This observation was considered to represent transitory adaptation or "training"

into guinea pigs, treat them with griseofulvin and compare them with treated animals infected with a non-resistant strain.

I cannot tell you the maximum amount that can be fed to guinea pigs. We have fed some of our animals up to five weeks at a level of 60 mg. per kilogram five days a week with no apparent adverse effect. Perhaps Dr. Hildick-Smith may have more information on that. As much as 200 mg. per kg. given intravenously in rats does not

kill, although it causes inhibition of mitotic activity (Paget, G. E. and Walpole, A. L.: Some cytological effects of griseofulvin. Nature, 182: 1320—1321,

1958).

I think I have answered Dr. Orfuss' questions, except that the amount of griseofulvin to be fed

phenomenon on the part of the fungus. was determined by weighing the animals and I am familiar with Dr. Robinson's recent work force-feeding the calculated amount of a 30 mg/ on the induction of resistance to griseofulvin by ml water suspension of griseofulvin.

THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

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