Source: Health Products and Food Branch (CA) Revision Year: 2020
Clotrimazole acts primarily by damaging the permeability barrier in the cell membrane of fungi. Clotrimazole brings about inhibition of ergosterol biosynthesis, an essential constituent of fungal cell membranes. If ergosterol synthesis is completely or partially inhibited, the cell is no longer able to construct an intact cell membrane. This leads to death of the fungus.
Exposure of Candida albicans to clotrimazole causes leakage of intracellular phosphorus compounds into the ambient medium with a concomitant breakdown of cellular nucleic acids and potassium eflux. The onset of these events is rapid and extensive after exposure of the organism to the drug, and causes a time-dependent and concentration-dependent inhibition of fungal growth.
Pharmacokinetic investigations after vaginal application have shown that only a small amount of clotrimazole (3-10%) is absorbed. Due to the rapid hepatic metabolization of absorbed clotrimazole into pharmacologically active metabolites, the resulting peak plasma concentrations of clotrimazole after vaginal application of a 500 mg dose were less than 10 ng/mL, suggesting that clotrimazole applied intravaginally is unlikely to lead to measurable systemic effects or side effects.
Metabolism studies performed after oral or intravenous administration have shown that in most species studies, levels of clotrimazole in tissue and serum are low. The majority of the drug is excreted as metabolites in the feces, with small amounts excreted in the urine. Human studies indicate slow excretion following oral administration of 14C-labelled clotrimazole (greater than 6 days). After intraperitoneal and subcutaneous administration, very low levels have been observed in the urine. The absorption and organ distribution of the drug is very poor when administered parenterally.
The pharmacokinetics of topically applied clotrimazole in human subjects have been evaluated by Duhm et al. who reported on the penetration of radioactive clotrimazole 1% cream and 1% solution into intact and acutely inflamed skin. Six hours after application of the drug, the concentration of clotrimazole found in skin layers varied from 100 µg/cm 3 in the stratum corneum to 0.5 to 1.0 µg/cm³ in the stratum reticulare and <0.1 µg/cm 3 in the subcutis. No measurable amount of radioactivity (0.001 µg/mL) was found in the serum within 48 hours after application of 0.5 mL of the solution or 0.8 g of the cream.
Intravaginal application of 14C-labelled clotrimazole tablets containing 100 mg of active substance in human subjects has shown that the amount absorbed is less than 1/200 of that absorbed after the oral administration of 1.5 gof clotrimazole. Themaximum serum concentration values were between 0.016 and 0.05 µg/mL from one to three days after intravaginal application. Intravaginal application in human subjects of 5 mL 14C-labelled clotrimazole vaginal cream containing 50 mg of active substance has shown that the systemic absorption of clotrimazole from the vaginal cream is quantitatively proportional to that from the vaginal tablets.
In animal experiments, clotrimazole exerts an in vitro and in vivo, dose-dependent, stimulating effect on certain microsomal enzyme systems which is approximately equal to that of phenobarbital in its inductive potential. However, thisstimulating effectsubsides rapidly when treatment is discontinued. The enzyme-inductive effect of clotrimazole has been found to be intact in adrenalectomized animals.
Clotrimazole is an antifungal agent with a broad spectrum of activity. In general, the in vitro activity of clotrimazole corresponds to that of tolnaftate, griseofulvin, and pyrrolnitrin against dermatophytes (Trichophyton, Microsporum and Epidermophyton species) and to that of the polyenes, amphotericin B and nystatin, against budding fungi (Candida and Histoplasma species).
In vitro, clotrimazole is fungistatic for most isolates of pathogenic fungi at concentrations of 0.02 to 10 µg/mL. The drug is fungicidal for many isolates of Trichophyton, Microsporum, Epidermophyton and Candida species at concentration of 0.1 to 2 µg/mL.
No one-step or multiple-step secondary resistance to clotrimazole has developed during successive passages of C. albicans, C. krusei, C. pseudotropicalis, T. mentagrophytes, T. rubrum, Cryptococcus neoformans, Aspergillus niger, and A. nidulans. Only a few isolates have been designated as having primary resistance to clotrimazole: a single isolate of C. guillermondii,six isolates of C. neoformans, three isolates of Paracoccidioides brasiliensis and two isolates of Blakeslea trispora.
Topical application of clotrimazole has been effective in the treatment of skin infections experimentally induced in the guinea pig with T. mentagrophytes and T. quinckeanum.
Clinical studies conducted as double-blind trials with mycological control have shown that clotrimazole is effective in the treatment of tinea cruris, tinea corporis, tinea pedis, tinea versicolor and cutaneous candidiasis. Mycological examinations have proven its efficacy against Trichophyton rubrum, T. mentagrophytes, Malassezia furfur and Candida albicans. Griseofulvin- resistant dermatophytes show no cross resistance to clotrimazole. Itmay be assumed, therefore, that the site of action of this drug is different from that of other antimycotics. Consequently, there is no cross resistance between these agents.
Minimum inhibitory concentrations (MICs) of clotrimazole were determined in serial dilution in broth or agar and in agar diffusion tests using the punched hole procedure. Conventional culture substrates, incubation times, and incubation temperatures were used. At concentrations less than 2 µg/mL, clotrimazole wasfungicidalfor many isolates of C. albicans, Trichophyton sp., Microsporum sp., and Epidermophyton sp., tested, and at concentrations less than 5 µg/mL, clotrimazole was fungistatic for other isolates of these species. Addition of bovine serum to the culture media at a final concentration of 30% resulted in somewhat higher MICs of clotrimazole.
The in vitro antifungal activity of clotrimazole was comparable to that of pyrrolnitrin; either compound at 0.78 µg/mL was fungicidal for most strains of Trichophyton sp., Microsporum sp. and Epidermophyton sp., tested.
The type of action of clotrimazole was determined in the Warburg apparatus by measuring the oxygen consumption of proliferating organisms exposed to varying concentrations of the drug. Additionalstudies were performed using a classicalsubculture technique with organism counts made after 16, 24 and 48 hours of exposure to the drug. These experiments showed that the primary action of clotrimazole at concentrations up to 20 µg/mL is fungistatic and affects only proliferating organisms. At concentrations greater than 20 µg/mL, clotrimazole was fungicidal for some organisms.
The determinations of MICs of clotrimazole for budding fungi and for biphasic fungi in the yeast phase have been shown to be dependent on the size of the inoculum and the length of incubation time. MICs for several isolates of Candida albicans and Torulopsis glabrata were higher when the inoculum size or incubation time or both were increased.
The effects of inoculum size has been attributed to binding of clotrimazole to the surface of the fungal cells. This was established in a study of turntable cultures of C. albicans. After 24 hours, the amount of clotrimazole in a nutrient substrate was reduced from 1 µg/mL to 0.7 µg/mL by an inoculum of 1 to 5 × 105 cells/mL.
A larger inoculum, 1 × 108 cells/mL, reduced the drug concentration from 1 µg/mL to 0.3 µg/mL. When the cultures were centrifuged and the cell sediment was washed with physiological saline solution, the wash solutions contained clotrimazole in concentrations of 0.2 µg/mL to 0.4 µg/mL.
The effect of incubation time on the determination of MIC values is thought to be related to the mechanism of action of clotrimazole. Initial studies indicated that clotrimazole acted as an antimetabolite upon the amino acid and protein metabolism of the fungi, causing a gradual inhibition of fungal growth.
However, recent studies using C. albicans as the test organism have shown that the primary mode of action of clotrimazole is damage to the permeability of the cell membrane. Exposure of C. albicans to clotrimazole caused leakage of intracellular phosphorus compounds into the ambient medium with a concomitant breakdown of cellular nucleic acids. The onset of these events wasrapid and extensive after exposure of C. albicans to the drug and caused a timedependent and concentration-dependent inhibition of fungal growth.
Only a few isolates have been designated as having primary resistance to clotrimazole; a single isolate of Candida guillermondii, six isolates of Cryptococcus neoformans, three isolates of Paracoccidioides brasiliensis, and two isolates of Blakeslea trispora. The potential for development ofsecondary resistance to clotrimazole was determined forseveral organisms by successive passages in a liquid medium, successive passages on a solid medium, or the Warburg proliferation test. Growth of dermatophytes and yeasts on Szybalski plates was also used as a method for determining the development of secondary resistance.
No change in sensitivity was detected for C. albicans in any of the tests forsecondary resistance, and no change in sensitivity was detected for Trichophyton mentagrophytes, T. rubrum, C. krusei, C. pseudotropicalis, C. neoformans, Aspergillus niger, or A. nidulans after successive passages on liquid and solid media. Possible resistance development was noted in successive passages of Torulopsis glabrata and other Torulopsis species. Data obtained from Szybalski plate growth and from other tests indicated that dermatophytes and yeasts do not develop onestep or oligo-step secondary resistance.
Non-clinical data reveal no special hazards for humans based on conventional studies of safety pharmacology, genotoxicity and carcinogenic potential. Effects in nonclinical studies, such as the effects on the liver (elevation of transaminases and alkaline phosphatase, liver cell hypertrophy) in the repeat-dose toxicity studies, the effects on the survival of the neonate in a rat fertility study, the species-specific indirect effects on the growth/survival of the fetus in a rat teratology study were observed with oral administration but only at exposures in excess of the maximum human exposure indicating little relevance to clinical use. Given the limited absorption of clotrimazole following a topic application, the potential for toxicity with the occasional use of Clotrimaderm Vaginal 6 is further limited.
Carcinogenicity of clotrimazole was evaluated in a 78-week oral dosing study in rats and the results did not show any carcinogenic effect of clotrimazole.
Clotrimazole has been extensively studied in in vitro and in vivo mutagenicity assays, and no evidence of genotoxic potential was found. In an Ames test, an in vitro biological assay to detect the mutagenicity of chemical compounds, clotrimazole showed no evidence of mutagenic activity. Clotrimaozle was found to be non-mutagenic in two additional in vitro studies, a gene mutation test in V79 cell lines and an Unscheduled DNA Synthesis (UDS) in primary rat hepatocytes. Studies evaluating the mutagenicity of clotrimazole in germ cells did not demonstrate mutagenic effects in a spermatogonia test in male hamsters, or in a dominant lethal test in male mice. Additionally, in mice, clotrimazole was not clastogenic in a micronucleus test.
| Species | LD50 mg/kg |
|---|---|
| Mouse | 761-923 |
| Rat | 708-718 |
| Rabbit | >1000 |
| Cat | >1000; vomiting from 100 mg/kg |
| Dog | >2000; vomiting from 100 mg/kg |
Multidose Local Tolerance:
In 453 cases under treatment which were evaluated with respect to photosensitivity and phototoxicity, no reactions were encountered.
Twenty normal subjects were tested in a controlled study for sensitivity to ultraviolet radiation. Areas of skin treated with clotrimazole were irradiated for 30 seconds on the first day and for onehalf minute longer each time on every second day thereafter. One of the 20 subjects was irradiated once only; 9 subjects three times, and 10 subjects four times. One subject developed papule formation after the first exposure to ultraviolet radiation.
There were undesirable effects in three (0.5%) of 653 patients treated with clotrimazole vaginal cream which were possibly related to treatment. Discontinuation of treatment was necessary in a patient with a sensation of vaginal burning and in another patient with a possible allergic reaction, manifested by vaginal burning, local irritation and erythema. Treatment was, however, continued in a patient with intercurrent cystitis.
At dosages up to 100 mg/kg (oral), clotrimazole was well tolerated by pregnant mice, rats and rabbits, and it had no embryotoxic or teratogenic effect.
When given to pregnant rats at oral doses of 100 mg/kg from day 6 through day 15 of
gestation, the number of resorptions was higher and the fetal weights were lower than the controls, but the number of fetal malformations did not differ significantly from that of the control group.
Rats treated with clotrimazole for 10 weeks at dosage up to 50 mg/kg/day did not show any difference from the control group in the duration of estrus, fertility, duration of pregnancy, or in the number of implantations and resorptions. The dose of 50 mg/kg/day impaired the development of the young, and dams receiving this dose level raised fewer offspring. The intravaginal administration of 100 mg/kg clotrimazole from the sixth to the fifteenth day of gestation was well tolerated by pregnant rats, and there were no harmful effects on the fertilization rate, the resorption rate, the mean fetal weight, and the frequency of stunted forms and of fetuses with slight bone alterations. No malformations were produced by this dose.
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