Absence of pharmacokinetic interaction between orally co-administered naproxen sodium and diphenhydramine hydrochloride.

The potential for a pharmacokinetic interaction between naproxen and diphenhydramine was examined in a randomized three-way crossover design with a 1-week washout between dosing. Single oral doses of 220 mg of naproxen sodium and 50 mg of diphenhydramine hydrochloride were given separately and together to 30 healthy male and female subjects. Heparinized blood samples obtained for 48 h postdose were assayed for plasma naproxen and diphenhydramine concentrations using validated high-performance liquid chromatography (HPLC) and gas chromatography (GC) assay methods, respectively. The area under the plasma concentration-time curve (AUC), maximum plasma concentrations (C(max)), time of C(max) (T(max)) and terminal exponential half-life (t(1/2,z)), were analysed for significant treatment differences by analysis of variance (ANOVA). Based on absence of significant treatment effects on AUC and C(max), single-dose oral co-administration of 220 mg of naproxen sodium with 50 mg of diphenhydramine hydrochloride does not alter the pharmacokinetics of either naproxen or diphenhydramine. Significant treatment differences seen for naproxen T(max) (0.3 h, males only) and diphenhydramine t(1/2,z) (0.8 h, females only) were minor and are unlikely to have therapeutic consequences. Thus, efficacy and safety of concomitant naproxen and diphenhydramine should not be altered due to a pharmacokinetic interaction.

Development and validation of a sensitive method for tetracycline in gingival crevicular fluid by HPLC using fluorescence detection.

Increased interest in the clinical use of antibiotics for periodontal therapy required the development of a sensitive assay for the quantitation of tetracycline in gingival crevicular fluid (GCF). An HPLC method was developed and validated for tetracycline which separates and identifies the degradation component epi-tetracycline. The HPLC assay employs a C18 reversed-phase Hypersil column with a mobile phase composed of methanol and sodium acetate buffer containing CaCl2 and EDTA disodium salt. The chromatographic separation was monitored by a fluorescent detector with an excitation wavelength of 375 nm and an emission wavelength of 512 nm. Tetracycline was extracted from GCF collected on Periopapers by addition of a methanol solution containing the internal standard, doxycycline, and the mobile phase buffer (25:75, v/v). The mean percent recovery for the extraction method was 107.8% with all the % R.S.D. below 7.5%. The mean inter- and intra-batch accuracy was 104.1 and 105.3%, respectively with a coefficient of variation of less than 9.5%. The lower limit of detection was 2.5 ng on the Periopapers. The typical GCF volumes collected were 0.1-1 microliter. The method was validated for the linear concentration range 2.5-1000 ng of tetracycline on the Periopaper. This assay for tetracycline was shown to be an accurate, precise and rugged method.

A stability-indicating assay and the preformulation characteristics of the radiosensitizer, 1,2,4-benzotriazin-3-amine 1,4-dioxide.

A stability-indicating LC assay was developed for the analysis of 1,2,4-benzotriazin-3-amine 1,4-dioxide and applied to the preformulation characterization of the drug. The dissociation constants of the drug were determined using UV-vis spectrophotometry. The LC method was used to determine the aqueous stability of the drug under a variety of accelerated conditions, its solubility in a variety of pharmaceutical solvents and its octan-1-ol-water partition coefficient. The preformulation data were used to develop three prototype aqueous formulations of the drug at a concentration of 0.5 mg ml-1 in 5% Dextrose Injection USP, phosphate buffer (pH 7.4) and phosphate buffered mannitol. The 3-month stability of those formulations at room temperature was demonstrated.

Stereoselective pharmacokinetics and metabolism of the enantiomers of cyclophosphamide. Preliminary results in humans and rabbits.

[R(+),S(-)]-Cyclophosphamide [(R,S)-CP] is an anticancer drug, containing a chiral phosphorous atom, which is prepared and used clinically as the racemic mixture. A new high-performance liquid chromatographic assay suitable for pharmacokinetic studies of CP enantiomers in plasma has been reported recently by this laboratory (Reid et al., Anal Chem 61: 441-446, 1989). Briefly, the assay involves ethyl acetate extraction of CP enantiomers from plasma followed by derivatization to diastereomers in a two-step process utilizing chloral and (+)-naproxen acid chloride. Chromatographic analysis was performed on a reversed phase (ODS) column with detection at 232 nm. In the present study, preliminary results on the applicability of this assay to pharmacokinetic studies are presented. Several rabbits were used to compare the influence of i.p., i.v., and oral routes of administration on the stereoselective disposition of (R,S)-CP. Following i.p. administration, S-CP was cleared faster than R-CP. Following oral administration, only R-CP was detectable in plasma, while i.v. administration resulted in minor or no stereoselective disposition. These results indicated that there was a marked stereoselective metabolism of the S-CP enantiomer, with the i.p. and oral routes producing the greatest differences due to first-pass metabolism. Incubation of rabbit-liver microsomes with (R,S)-CP demonstrated that the monooxygenase system can exhibit marked stereoselectivity in its metabolism of CP. The ratio of R-CP to S-CP in the incubation medium increased during the incubation period from 1:1 initially to 4.5:1 after 60 min. The results from the experiments with rabbits indicate that the first-pass metabolism of this drug is highly stereoselective; in contrast, cancer patients who had received (R,S)-CP as an i.v. infusion showed no stereoselectivity in the elimination of the enantiomers. Pharmacokinetic studies with cancer patients, receiving (R,S)-CP as an oral dose, are in progress in order to determine if stereoselective first-pass metabolism of this drug also occurs in humans.

Analysis of 5-fluorouracil in plasma by precolumn derivatization with 4-bromomethyl-7-methoxycoumarin, followed by multi-dimensional high-performance liquid chromatography.

An assay for 5-fluorouracil (5-FU) has been developed that utilizes a double extraction with ethyl acetate, followed by precolumn derivatization with 4-bromo-methyl-7-methoxycoumarin. The reaction mixture was quenched with 5% acetic acid, extracted with hexane, and analyzed by multi-dimensional high-performance liquid chromatography. Derivatized 5-FU was injected into a cyanopropyl column and a heart cut containing the analyte was then switched to an octadecyl column and quantitated by fluorescence detection. The assay had a limit of detection of 0.5 ng 5-FU/ml plasma and was linear to 20 micrograms/ml. It was shown to be free of interferences from the other anticancer agents commonly used in combination with 5-FU. This assay should have the sensitivity needed to measure the low levels that occur after low-dose, continuous infusion of 5-FU.

Vitamin K1 and K2 in infant human liver.

It is not known to what extent humans store vitamin K in liver. We measured hepatic concentrations of vitamin K1 (phylloquinone) and K2 (menaquinones) in 11 human livers (eight infants and three adults). Relatively small amounts of vitamin K were found in the liver at any age compared to other fat soluble vitamins. Vitamin K1 was the predominant form with much smaller concentrations of vitamin K2. Long-chain menaquinones (vitamin K2) were readily identified in most liver specimens. Hepatic vitamin K2 concentrations also increased with increasing age. These observations have implications for vitamin K supplementation in infants.

Effect of various intakes of phylloquinone on signs of vitamin K deficiency and serum and liver phylloquinone concentrations in the rat.

The relationship between dietary phylloquinone, serum and liver concentrations of phylloquinone, and various indices of vitamin K adequacy have been studied in male rats fed a purified diet containing various levels of phylloquinone. In excess of 500 micrograms phylloquinone/kg diet was needed to prevent the most sensitive signs of vitamin K deficiency. Liver phylloquinone concentrations were shown to be correlated with dietary phylloquinone intake. Serum phylloquinone was not correlated with either diet or liver concentration of phylloquinone and did not increase with increased dietary intake until the liver contained sufficient vitamin to maintain optimal synthesis of vitamin K-dependent proteins. Because of the rapid loss of vitamin from the liver, prior ingestion of a high level of vitamin K had little influence on liver vitamin K concentrations beyond the first 2 d of a deficient period. When rats consumed a diet containing 500 micrograms phylloquinone/kg diet in 3 h, liver and serum phylloquinone concentrations fluctuated drastically following this feeding period. During the subsequent 24-h period, liver phylloquinone concentrations decreased to a level that would not support maximal activity of the hepatic vitamin K-dependent carboxylase.

High-performance liquid chromatography of 5-fluorouracil after derivatization with 4-bromomethyl-7-methoxycoumarin. Characterization of the derivative and the use of column switching for the improvement of resolution and the enhancement of sensitivity.

The derivatization of 5-fluorouracil with 4-bromomethyl-7-methoxycoumarin has been reported previously; however, the structure of the derivative was not confirmed. The synthesis and purification of the 5-FU derivative is described along with the spectroscopic (MS and NMR) determination that it is labelled at both heterocyclic nitrogens as expected. A column switching HPLC system is also presented which consists of primary separation on a cyanopropyl column followed by a final separation on an ODS column with fluorescence detection. This system removes all interferences from the derivatization system and has a limit of detection for the pure derivative of less than 50 fmol (injection volume = 100 microliters).

Effects of a vitamin K-deficient diet and antibiotics in normal human volunteers.

Decreased concentrations of vitamin K-dependent plasma clotting factors are a well-documented response of vitamin K-deprived patients administered broad-spectrum antibiotics. It has recently been claimed that antibiotics containing a N-methylthiotetrazole (NMTT) side chain cause this response through a direct effect of NMTT on the vitamin K-dependent posttranslational carboxylation of these clotting factors. To further study these relationships, 11 groups of three volunteers were fed a synthetic vitamin K-free diet for 2 weeks. During the last 10 days of vitamin K restriction, seven of the volunteer groups received a therapeutic dose of antibiotics not containing NMTT: ampicillin, sulfamethoxazole-trimethoprim (Bactrim), cefoxitin, cefotaxime, ceftazidime, clindamycin, and piperacillin, and three groups received NMTT-containing antibiotics: moxalactam, cefamandole, and cefoperazone. Serum phylloquinone (vitamin K1) concentrations reflected dietary intake and fell from 1.4 +/- 0.9 ng/ml after 3 days of hospital diet to 0.4 +/- 0.3 ng/ml after 13 days of vitamin K-free diet. Median stool excretion of phylloquinone was 19 micrograms/day while subjects consumed the hospital diet, and fell to 3 micrograms/day by day 6 on vitamin K-free diet. Prothrombin times remained within the normal range throughout the study. Suppression of vitamin K-dependent clotting factor biosynthesis was evident by decreased factor VII levels in seven of the volunteers and by an increased concentration of des-gamma-carboxy (abnormal) prothrombin in 21 of the volunteers. The changes occurred in the control subjects and in subjects receiving all nine of the 10 antibiotics with no consistent pattern.(ABSTRACT TRUNCATED AT 250 WORDS)

Menaquinone production and utilization in germ-free rats after inoculation with specific organisms.

Although menaquinones are biologically active forms of vitamin K, factors that influence their production by bacteria or their absorption from the gut are not well understood. Germ-free rats were inoculated with four different strains of organisms and fecal and tissue menaquinone concentrations were determined. No menaquinones were detected in the tissues or feces of rats colonized with Bifidobacterium longum or Clostridium ramosum, two organisms that have not been reported to produce menaquinones when grown in pure cultures. Rats colonized with Bacteroides vulgatus had high levels of fecal MK-10 with significant amounts of MK-9 and MK-11, whereas rats colonized with Escherichia coli had high levels of fecal MK-8 and small amounts of MK-7. The same menaquinones are produced in pure cultures of these organisms. The predominant fecal menaquinones were also detected in liver and were present in higher concentrations in the liver of those rats not maintained in coprophagy-preventing cages.