ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, May 1990, p. 909-910
0066-4804/90/050909-02$02.00/0 Copyright ©D 1990, American Society for Microbiology
Vol. 34, No. 5
Fluconazole Levels in Plasma and Vaginal Secretions of Patients after a 150-Milligram Single Oral Dose and Rate of Eradication of Infection in Vaginal Candidiasis ELIZABETH T. HOUANG,1* OLIVER CHAPPATTE,1 DOMINIC BYRNE,' PAUL V. MACRAE,2 AND JOHN E. THORPE3 Queen Charlotte's and Chelsea Hospital for Women, London W6 OXG,1 and Departments of Drug Metabolism2 and Clinical Research,3 Pfizer Central Research, Sandwich, Kent, United Kingdom Received 16 August 1989/Accepted 9 February 1990 Mean peak concentrations of fluconazole in plasma and vaginal secretions of females after a 150-mg single oral dose were shown to be 2.82 ,g/ml and 2.43 ,g/g, respectively. Our results indicate that clinically efficacious concentrations of fluconazole in vaginal secretions are easily achieved after this single oral dose.
Fluconazole is a new bis-triazole antifungal agent which has shown excellent activity against candidiasis both in animal models and in patients with oropharyngeal or vaginal candidiasis (1, 3, 4). As well as having good absorption after oral administration (>90% bioavailability), a long plasma half-life (25 h), and low protein binding (12%), fluoconazole is rapidly distributed in body tissue fluids (2). The activity of fluoconazole against oropharyngeal candidiasis has been partially attributed to its presence in saliva, where levels of magnitude similar to those in plasma are attainable. Fluconazole would be expected to achieve levels similar to those in plasma in both vaginal secretions and tissues to account for its success in the treatment of vaginal candidiasis. The aim of the present study was to determine the simultaneous levels of fluconazole in plasma and vaginal secretions in patients receiving treatment for vaginal candidiasis and to measure the rate of eradication of fungal infection. We also report the first use of a capillary gas chromatographic assay combined with ion trap detection for measurement of fluconazole in samples of small volume. From a group of patients participating in a multicenter study to determine the efficacy, safety, and toleration of fluconazole in the treatment of vaginal candidiasis (results of which will be published separately), patients who worked locally were approached for additional informed consent to enter this investigation. Only patients aged 18 years or older, with a clinical diagnosis of vaginal candidiasis (including symptoms of either vaginal pruritus or burning), who were willing to refrain from sexual intercourse until 7 days after fluconazole treatment were allowed to enter. Excluded were those who were either pregnant or lactating; had any other clinically relevant disease or impairment of renal or hepatic function; had concomitant venereal disease, trichomoniasis, or nonspecific vaginitis; were undergoing any other antifungal treatment; were taking any drugs that are cleared predominantly by metabolism and that have a low therapeutic ratio; or had taken any investigational drug in the previous month. Thus the first nine females (mean age, 30 years; range, 23 to 41 years) who agreed to return were enrolled in the study. Just before a patient received an oral 150-mg dose (three 50-mg capsules) of fluconazole, samples of blood and of vaginal secretion were taken. Further samples were taken at *
2, 8, 24, 72, 120, and 168 h postdosing. Blood samples were collected in heparin tubes, and plasma was separated immediately by centrifugation and stored at -20°C until assayed. Vaginal secretion samples were collected by suction into thin plastic tubes. The bulk of the sample was stored at -20°C until assayed, but a portion in each case was used to determine fungal colony counts (in CFU per gram) at each time point. A weighed sample of secretion was first vortexed in 1 ml of peptone water broth (Oxoid Ltd., London, United Kingdom). Serial 10-fold dilutions were then made in peptone water broth, and 0.1 ml of each dilution was spread with a sterile glass spreader onto Sabouraud agar (Oxoid). The yeast colonies were enumerated after incubation for 48 h at 370C. The small size (range, 5 to 450 mg) of some of the vaginal secretion samples necessitated use of an assay more sensitive than the usual gas chromatographic method for the determination of fluconazole levels (5). A capillary gas chromatographic assay combined with ion trap detection was developed (P. V. Macrae, in E. Reid, ed., Methodological Surveys in Biochemistry and Analysis, vol. 20, in press). This assay was also used for determinations of fluconazole levels in plasma in this study. For plasma and vaginal secretions, calibration curve coefficients of correlation were 0.997 and 0.998, respectively; mean recoveries from quality assurance samples, with fluconazole concentration ranges of 0.07 to 3.6 ,ug/ml for plasma and 0.3 to 1.6 pug/g for vaginal secretions, were 97% (n = 6; standard deviation, 4%) and 105% (n = 4; standard deviation, 11%), respectively. The method had a limit of detection of 2 ng and was linear over the range of 2 to 200 ng, equivalent to concentration ranges of 0.04 to 4 ,ug/ml and 0.04 to 4 ,ug/g for plasma and vaginal
secretions, respectively. Mean fluconazole concentrations in plasma and vaginal secretions are shown in Fig. 1. For plasma, the peak concentrations ranged from 2.44 to 3.58 jig/ml (mean, 2.82 ,ug/ml; n = 8) 2 h after the dose was administered. At 24 h, levels ranged from 1.36 to 2.25 ±g/ml (mean, 1.78 ,ug/ml; n = 8). Fluconazole was still detectable in plasma at 168 h, although the levels were low (mean, 0.06 ,ug/ml; n = 8). For vaginal secretions, the peak concentrations of fluconazole occurred between 8 and 24 h after the dose was administered. The mean concentration at both of these time points was 2.42 ,ug/g (at 8 h, n = 9; at 24 h, n = 8). The ranges of
Corresponding author. 909
910
NOTES
ANTIMICROB. AGENTS CHEMOTHER. 4 0 z
pg/ml plasma ug/g vaginal secretions
A
2H 3 cc H z C.)
z 0 2 C-)
LU
-J
0 N z
0 1 -J UL
0
20
40
60 80 100 120 TIME (HOURS POST DOSE)
140
160
180
FIG. 1. Mean concentrations of fluconazole in plasma and vaginal secretions from female patients after a single oral dose (150 mg) of the drug.
fluconazole concentrations at 8 and 24 h postdose were 1.10 to 3.90 and 1.19 to 2.93 pLg/g, respectively. Candida albicans colony counts of vaginal-secretion samples from eight patients (one patient had no pretreatment growth) pre- and posttreatment are shown in Table 1. Counts ranged from 4.00 x 102 to 3.07 x 107 CFU/g pretreatment. No growth was seen in samples from two patients at 24 h and in samples from all eight patients by 72 h. All patients became free of symptoms at the end of the study. No side effects were reported by the patients. The pharmacokinetics of fluconazole in plasma measured in our study (based on data for mean concentration in plasma [n = 9]: half-life, 30.2 h, 90% confidence limits, 28.5 to 32.2 h; apparent clearance rate, 0.32 ml/min per kg; apparent volume of distribution, 0.843 liters/kg) were similar to those previously reported (2). In contrast to saliva, in which fluconazole pharmacokinetics are similar to those in plasma (2), the drug persisted longer in vaginal secretions than in plasma, with levels of 1 ,ug/g present for up to 72 h. This may TABLE 1. Yeast colony counts pre- and posttreatment from patients treated with 150 mg of oral fluconazole Colony count (CFU/g) at indicated time (h) Patient 0 (pretreatPosttreatment ment)
1 3.07 x 107 2 3.14 x 103 3 2.50 x 103 4 2.30 x 105 5 1.27 x 104 6 4 x 102 7 2.5x i04 8 2.7 x 104 a NG, No growth. b NS, No sample.
2
8.20 1.02 4.68 1.08 4.5 1.09 2.5 1.8
8
107 1.80 x 107 X 104 3.60 x 103 x 10o 4.11 x 104 x 10' 1.17 x 10' x 103 1.70 x 103 x 104 8.8 x 102 x 104 1.0x 1i x 104 1.7 x 104 x
24
72
reflect the fact that it is not actively lost except as part of the vaginal discharge. It is interesting to note that the pretreatment vaginal yeast concentrations ranged from approximately 102 to approximately 10i CFU/g, although all patients were symptomatic. A significant reduction (>10-fold) in yeast count was observed in four of the seven patients from whom samples were obtained 24 h after dosing. No yeast was isolated by 72 h in any of the patients. The MICs of fluconazole for most strains of C. albicans range from 0.4 to 0.8 p.g/ml (1). The persistence of fluconazole in vaginal secretions may contribute to the mycological and clinical efficacy seen in our study as weli as in the much larger study already reported (1). The results of our study show that a single oral dose of 150 mg of fluconazole gives rise to a peak concentration in vaginal secretions 8 to 24 h later. Fluconazole, being a stable compound, persists in the vagina in concentrations greater than the MICs for most strains of C. albicans for at least 3 days.
1. 2.
168
2.77 x 104 NGa NG NSb NG NG 2.84 x 103 NG NG 5.00 x 103 NG NG 8.30 x 103 NG NG NS NG NG NG NG NG 1.5 x 104 NG NG
3. 4.
5.
LITERATURE CITED Brammer, K. W., and J. M Feczko. 1988. Single-dose oral fluconazole in the treatment of vaginal candidosis. Ann. N.Y. Acad. Sci. 554:561-563. Brammer, K. W., and M. H. Tarbit. 1987. Fluconazole pharmacokinetics in animals and man, p. 141-149. In R. A. Fromtling (ed.), Recent trends in the discovery, development and evaluation of antifungal agents. J. R. Prous Science Publishers, Barcelona, Spain. Dupont, B., and E. Drouhet. 1988. Fluconazole in the management of oropharyngeal candidosis in a predominantly HIV antibody-positive group of patients. J. Med. Vet. Mycol. 26:67-71. Kutzer, E., R. Oittner, S. Leodolter, and K. W. Brammer. 1988. A comparison of fluconazole and ketoconazole in the oral treatment of vaginal candidiasis; report of a double-blind multicentre study. Eur. J. Obstet. Gynecol. Reprod. Biol. 29:305-313. Wood, P. R., and M. H. Tarbit. 1986. Gas chromatographic method for the determination of fluconazole, a novel antifungal agent, in human plasma and urine. J. Chromatogr. 383:179-186.
0066-4804/90/050909-02$02.00/0 Copyright ©D 1990, American Society for Microbiology
Vol. 34, No. 5
Fluconazole Levels in Plasma and Vaginal Secretions of Patients after a 150-Milligram Single Oral Dose and Rate of Eradication of Infection in Vaginal Candidiasis ELIZABETH T. HOUANG,1* OLIVER CHAPPATTE,1 DOMINIC BYRNE,' PAUL V. MACRAE,2 AND JOHN E. THORPE3 Queen Charlotte's and Chelsea Hospital for Women, London W6 OXG,1 and Departments of Drug Metabolism2 and Clinical Research,3 Pfizer Central Research, Sandwich, Kent, United Kingdom Received 16 August 1989/Accepted 9 February 1990 Mean peak concentrations of fluconazole in plasma and vaginal secretions of females after a 150-mg single oral dose were shown to be 2.82 ,g/ml and 2.43 ,g/g, respectively. Our results indicate that clinically efficacious concentrations of fluconazole in vaginal secretions are easily achieved after this single oral dose.
Fluconazole is a new bis-triazole antifungal agent which has shown excellent activity against candidiasis both in animal models and in patients with oropharyngeal or vaginal candidiasis (1, 3, 4). As well as having good absorption after oral administration (>90% bioavailability), a long plasma half-life (25 h), and low protein binding (12%), fluoconazole is rapidly distributed in body tissue fluids (2). The activity of fluoconazole against oropharyngeal candidiasis has been partially attributed to its presence in saliva, where levels of magnitude similar to those in plasma are attainable. Fluconazole would be expected to achieve levels similar to those in plasma in both vaginal secretions and tissues to account for its success in the treatment of vaginal candidiasis. The aim of the present study was to determine the simultaneous levels of fluconazole in plasma and vaginal secretions in patients receiving treatment for vaginal candidiasis and to measure the rate of eradication of fungal infection. We also report the first use of a capillary gas chromatographic assay combined with ion trap detection for measurement of fluconazole in samples of small volume. From a group of patients participating in a multicenter study to determine the efficacy, safety, and toleration of fluconazole in the treatment of vaginal candidiasis (results of which will be published separately), patients who worked locally were approached for additional informed consent to enter this investigation. Only patients aged 18 years or older, with a clinical diagnosis of vaginal candidiasis (including symptoms of either vaginal pruritus or burning), who were willing to refrain from sexual intercourse until 7 days after fluconazole treatment were allowed to enter. Excluded were those who were either pregnant or lactating; had any other clinically relevant disease or impairment of renal or hepatic function; had concomitant venereal disease, trichomoniasis, or nonspecific vaginitis; were undergoing any other antifungal treatment; were taking any drugs that are cleared predominantly by metabolism and that have a low therapeutic ratio; or had taken any investigational drug in the previous month. Thus the first nine females (mean age, 30 years; range, 23 to 41 years) who agreed to return were enrolled in the study. Just before a patient received an oral 150-mg dose (three 50-mg capsules) of fluconazole, samples of blood and of vaginal secretion were taken. Further samples were taken at *
2, 8, 24, 72, 120, and 168 h postdosing. Blood samples were collected in heparin tubes, and plasma was separated immediately by centrifugation and stored at -20°C until assayed. Vaginal secretion samples were collected by suction into thin plastic tubes. The bulk of the sample was stored at -20°C until assayed, but a portion in each case was used to determine fungal colony counts (in CFU per gram) at each time point. A weighed sample of secretion was first vortexed in 1 ml of peptone water broth (Oxoid Ltd., London, United Kingdom). Serial 10-fold dilutions were then made in peptone water broth, and 0.1 ml of each dilution was spread with a sterile glass spreader onto Sabouraud agar (Oxoid). The yeast colonies were enumerated after incubation for 48 h at 370C. The small size (range, 5 to 450 mg) of some of the vaginal secretion samples necessitated use of an assay more sensitive than the usual gas chromatographic method for the determination of fluconazole levels (5). A capillary gas chromatographic assay combined with ion trap detection was developed (P. V. Macrae, in E. Reid, ed., Methodological Surveys in Biochemistry and Analysis, vol. 20, in press). This assay was also used for determinations of fluconazole levels in plasma in this study. For plasma and vaginal secretions, calibration curve coefficients of correlation were 0.997 and 0.998, respectively; mean recoveries from quality assurance samples, with fluconazole concentration ranges of 0.07 to 3.6 ,ug/ml for plasma and 0.3 to 1.6 pug/g for vaginal secretions, were 97% (n = 6; standard deviation, 4%) and 105% (n = 4; standard deviation, 11%), respectively. The method had a limit of detection of 2 ng and was linear over the range of 2 to 200 ng, equivalent to concentration ranges of 0.04 to 4 ,ug/ml and 0.04 to 4 ,ug/g for plasma and vaginal
secretions, respectively. Mean fluconazole concentrations in plasma and vaginal secretions are shown in Fig. 1. For plasma, the peak concentrations ranged from 2.44 to 3.58 jig/ml (mean, 2.82 ,ug/ml; n = 8) 2 h after the dose was administered. At 24 h, levels ranged from 1.36 to 2.25 ±g/ml (mean, 1.78 ,ug/ml; n = 8). Fluconazole was still detectable in plasma at 168 h, although the levels were low (mean, 0.06 ,ug/ml; n = 8). For vaginal secretions, the peak concentrations of fluconazole occurred between 8 and 24 h after the dose was administered. The mean concentration at both of these time points was 2.42 ,ug/g (at 8 h, n = 9; at 24 h, n = 8). The ranges of
Corresponding author. 909
910
NOTES
ANTIMICROB. AGENTS CHEMOTHER. 4 0 z
pg/ml plasma ug/g vaginal secretions
A
2H 3 cc H z C.)
z 0 2 C-)
LU
-J
0 N z
0 1 -J UL
0
20
40
60 80 100 120 TIME (HOURS POST DOSE)
140
160
180
FIG. 1. Mean concentrations of fluconazole in plasma and vaginal secretions from female patients after a single oral dose (150 mg) of the drug.
fluconazole concentrations at 8 and 24 h postdose were 1.10 to 3.90 and 1.19 to 2.93 pLg/g, respectively. Candida albicans colony counts of vaginal-secretion samples from eight patients (one patient had no pretreatment growth) pre- and posttreatment are shown in Table 1. Counts ranged from 4.00 x 102 to 3.07 x 107 CFU/g pretreatment. No growth was seen in samples from two patients at 24 h and in samples from all eight patients by 72 h. All patients became free of symptoms at the end of the study. No side effects were reported by the patients. The pharmacokinetics of fluconazole in plasma measured in our study (based on data for mean concentration in plasma [n = 9]: half-life, 30.2 h, 90% confidence limits, 28.5 to 32.2 h; apparent clearance rate, 0.32 ml/min per kg; apparent volume of distribution, 0.843 liters/kg) were similar to those previously reported (2). In contrast to saliva, in which fluconazole pharmacokinetics are similar to those in plasma (2), the drug persisted longer in vaginal secretions than in plasma, with levels of 1 ,ug/g present for up to 72 h. This may TABLE 1. Yeast colony counts pre- and posttreatment from patients treated with 150 mg of oral fluconazole Colony count (CFU/g) at indicated time (h) Patient 0 (pretreatPosttreatment ment)
1 3.07 x 107 2 3.14 x 103 3 2.50 x 103 4 2.30 x 105 5 1.27 x 104 6 4 x 102 7 2.5x i04 8 2.7 x 104 a NG, No growth. b NS, No sample.
2
8.20 1.02 4.68 1.08 4.5 1.09 2.5 1.8
8
107 1.80 x 107 X 104 3.60 x 103 x 10o 4.11 x 104 x 10' 1.17 x 10' x 103 1.70 x 103 x 104 8.8 x 102 x 104 1.0x 1i x 104 1.7 x 104 x
24
72
reflect the fact that it is not actively lost except as part of the vaginal discharge. It is interesting to note that the pretreatment vaginal yeast concentrations ranged from approximately 102 to approximately 10i CFU/g, although all patients were symptomatic. A significant reduction (>10-fold) in yeast count was observed in four of the seven patients from whom samples were obtained 24 h after dosing. No yeast was isolated by 72 h in any of the patients. The MICs of fluconazole for most strains of C. albicans range from 0.4 to 0.8 p.g/ml (1). The persistence of fluconazole in vaginal secretions may contribute to the mycological and clinical efficacy seen in our study as weli as in the much larger study already reported (1). The results of our study show that a single oral dose of 150 mg of fluconazole gives rise to a peak concentration in vaginal secretions 8 to 24 h later. Fluconazole, being a stable compound, persists in the vagina in concentrations greater than the MICs for most strains of C. albicans for at least 3 days.
1. 2.
168
2.77 x 104 NGa NG NSb NG NG 2.84 x 103 NG NG 5.00 x 103 NG NG 8.30 x 103 NG NG NS NG NG NG NG NG 1.5 x 104 NG NG
3. 4.
5.
LITERATURE CITED Brammer, K. W., and J. M Feczko. 1988. Single-dose oral fluconazole in the treatment of vaginal candidosis. Ann. N.Y. Acad. Sci. 554:561-563. Brammer, K. W., and M. H. Tarbit. 1987. Fluconazole pharmacokinetics in animals and man, p. 141-149. In R. A. Fromtling (ed.), Recent trends in the discovery, development and evaluation of antifungal agents. J. R. Prous Science Publishers, Barcelona, Spain. Dupont, B., and E. Drouhet. 1988. Fluconazole in the management of oropharyngeal candidosis in a predominantly HIV antibody-positive group of patients. J. Med. Vet. Mycol. 26:67-71. Kutzer, E., R. Oittner, S. Leodolter, and K. W. Brammer. 1988. A comparison of fluconazole and ketoconazole in the oral treatment of vaginal candidiasis; report of a double-blind multicentre study. Eur. J. Obstet. Gynecol. Reprod. Biol. 29:305-313. Wood, P. R., and M. H. Tarbit. 1986. Gas chromatographic method for the determination of fluconazole, a novel antifungal agent, in human plasma and urine. J. Chromatogr. 383:179-186.
