Enzymatic preparation of high-specific-activity beta-D-[6,6'-3H]fructose-2,6-bisphosphate: Application to a sensitive assay for fructose-2,6-bisphosphatase.

beta-D-Fructose-2,6-bisphosphate (Fru-2,6-P(2)) is an important regulator of eukaryotic glucose homeostasis, functioning as a potent activator of 6-phosphofructo-1-kinase and inhibitor of fructose-1,6-bisphosphatase. Pharmaceutical manipulation of intracellular Fru-2,6-P(2) levels, therefore, is of interest for the treatment of certain diseases, including diabetes and cancer. [2-(32)P]Fru-2,6-P(2) has been the reagent of choice for studying the metabolism of this effector molecule; however, its short half-life necessitates frequent preparation. Here we describe a convenient, economical, one-pot enzymatic preparation of high-specific-activity tritium-labeled Fru-2,6-P(2). The preparation involves conversion of readily available, carrier-free d-[6,6'-(3)H]glucose to [6,6'-(3)H]Fru-2,6-P(2) using hexokinase, glucose-6-phosphate isomerase, and 6-phosphofructo-2-kinase. The key reagent in this preparation, bifunctional 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase from human liver, was produced recombinantly in Escherichia coli and purified in a single step using an appendant C-terminal hexa-His affinity tag. Following purification by anion exchange chromatography using triethylammonium bicarbonate as eluant, radiochemically pure [6,6'-(3)H]Fru-2,6-P(2) having a specific activity of 50 Ci/mmol was obtained in yields averaging 35%. [6,6'-(3)H]Fru-2,6-P(2) serves as a stable, high-specific-activity substrate in a facile assay capable of detecting fructose-2,6-bisphosphatase in the range of 10(-14) to 10(-15) mol, and it should prove to be useful in many studies of the metabolism of this important biofactor.

Steady-state pharmacokinetics following application of a novel transdermal estradiol spray in healthy postmenopausal women.

This study was designed to evaluate the steady-state pharmacokinetics (PK) of estradiol and its metabolites, estrone and estrone sulfate, following application of a novel estradiol transdermal spray to healthy postmenopausal women. Participants were randomly assigned in parallel to receive 1-, 2-, or 3-spray doses (24 participants/dose level) of a 1.7% estradiol metered-dose transdermal spray (1.53 mg/spray) once daily for 14 days. Blood was collected predose on days 1 to 14 and over 7 days after the last dose. Serum concentrations for all 3 analytes reached steady state by day 7 or 8 and were still slightly above baseline on day 21. Estradiol, estrone, and estrone sulfate serum concentrations generally increased with increasing dose. Mean estradiol and estrone maximum serum concentration (C(max)) following 1, 2, or 3 sprays for 14 days were 36 and 50, 57 and 60, and 54 and 71 pg/mL, respectively. Estradiol time when maximum concentration occurred (t(max)) was 18 to 20 hours. The area under the serum concentration-time curve over 24 hours following the last dose of study drug (AUC(0-24 h)) on day 14 for the 1-, 2-, and 3-spray groups, respectively, was 471, 736, and 742 pg.h/mL for estradiol; 886, 1208, and 1367 pg x h/mL for estrone; and 16,501, 26,515, and 27,971 pg x h/mL for estrone sulfate. The metered-dose estradiol transdermal spray delivers estradiol at therapeutic levels and produces low serum estrone concentrations.

The effects of skin-to-skin contact, application site washing, and sunscreen use on the pharmacokinetics of estradiol from a metered-dose transdermal spray.

OBJECTIVE: This study evaluated the transfer of estradiol by skin-to-skin contact and the influence of washing and sunscreen use on the absorption of estradiol from a transdermal spray. DESIGN: Studies were conducted in the same group of 20 healthy postmenopausal women over a period of 18 days. The women were dosed with three sprays of study medication once daily (a total daily dose of 4.59 mg). To evaluate skin-to-skin transfer, estradiol levels in 20 untreated men were evaluated before and after direct skin-to-skin contact with the application sites of 20 treated women after application of study medication on study days 1 to 3. To examine the effect of washing the application site, estradiol absorption was evaluated when the application site was washed 1 hour after application compared with the unwashed site on study days 10 to 12. To examine the effects of sunscreen use, estradiol pharmacokinetic profiles were evaluated when sunscreen was applied before and after study drug application on study days 14 to 17. RESULTS: The 90% CI of the ratios of the areas under the serum estradiol-time curves (AUC0-24) in untreated men before and after contact with treated women was 1.00 to 1.07, which was within the prespecified equivalence range (0.8-1.25). The 90% CI of the AUC0-24 ratios with and without application site washing was 0.92 to 1.15. Application of sunscreen 1 hour after study drug resulted in a 90% CI of AUC0-24 ratios of 0.76 to 1.08. Application of sunscreen 1 hour before study drug resulted in a 90% CI of AUC0-24 ratios of 0.86 to 1.23. CONCLUSIONS: The use of a transdermal estradiol spray did not result in a significant transfer of estradiol by skin-to-skin contact. Washing the application site did not significantly affect absorption of estradiol. Estradiol absorption was slightly decreased due to the application of sunscreen after study drug application, but was unaffected when sunscreen was applied before study drug.

The effects of intravaginal clindamycin and metronidazole therapy on vaginal lactobacilli in patients with bacterial vaginosis.

OBJECTIVE: The purpose of this study was to determine the effects of 3 intravaginal antibacterial treatments on vaginal lactobacilli in patients with bacterial vaginosis (BV). STUDY DESIGN: Retrospective analyses were performed on Lactobacillus scores from 3 similar studies evaluating 2 2% clindamycin vaginal creams and 0.75% metronidazole gel at baseline and at 21 to 30 days in 408 patients with BV. Scores were compared using a 1-way global F test and McNemar's test. RESULTS: All groups had similar mean Lactobacillus scores at baseline (P = 0.37) and at 21 to 30 days (P = .71). The 3 groups were also comparable at both visits with respect to the distributions of scores within each group. In all groups, there was significant improvement in the percentages of patients with no lactobacilli present at 21 to 30 days compared with baseline (P < .0001 for all comparisons). CONCLUSION: Clindamycin and metronidazole promoted similar levels of restoration of vaginal lactobacilli at 21 to 30 days.

An open-label, two-period, crossover study of the systemic bioavailability in healthy women of clindamycin phosphate from two vaginal cream formulations.

BACKGROUND: A clindamycin phosphate 2% single-dose vaginal cream (CSDVC) formulation has been designed to provide release of clindamycin equivalent to 7 daily doses of a conventional clindamycin phosphate 2% vaginal cream (CVC). OBJECTIVE: The purpose of this study was to compare the systemic bioavailability of clindamycin from 1 dose of CSDVC with that from 1 dose from a 7-day regimen of CVC in healthy women. METHODS: This was a single-center, open-label, randomized, 2-period, 2-sequence crossover study that enrolled healthy, nonpregnant, adult women. Subjects were randomly assigned to receive a single 5-g intravaginal dose of CSDVC or CVC. Blood samples were then collected for 96 hours after study medication administration. Subjects were crossed over after a 14-day washout period, and received a single dose of the other medication. Blood samples were then collected for 96 hours after administration of the second drug. The plasma clindamycin pharmacokinetic profiles were determined, using a validated assay with a lower limit of detection of 0.2 ng/mL, and compared between treatments. RESULTS: The median age of women was 43.5 years(range, 18-66 years), the median weight was 65.0 kg (range, 47.7-91.8 kg), and the median body mass index was 25.4 kg/m2 (range, 19.2-34.7 kg/m(2)). AUC from time 0 to the last detectable concentration (AUCO(0-t)) and from time 0 to infinity (AUCO(0-infinity)) and C(max) were significantly lower with CSDVC than with CVC (geometric means of 98.61 vs 794.21 ng . h/mL for AUCO(0-t), 100.33 vs 809.14 ng . h/mL for AUC(0-infinity), and 3.18 vs 42.27 ng/mL for C(max); all comparisons, P < 0.001 between formulations). Overall bioavailability of clindamycin from CSDVC was approximately 12% of that from CVC, as measured by AUC. The arithmetic mean T(max) was significantly longer with CSDVC (26.4 vs 9.8 hours; P < 0.007). There were 18 adverse events reported during this study. The most common adverse event with each formulation was headache (CSDVC, 10%; CVC, 25%). CONCLUSIONS: Systemic bioavailability of clindamycin was significantly lower and systemic absorption was significantly slower with the CSDVC formulation than with the single dose of 7-day CVC formulation in these healthy volunteers.