Gas chromatography/mass spectrometry characterization of urinary metabolites of danazol after oral administration in human

Danazol (17alpha-pregna-2,4-dien-20-yno [2,3-d]-isoxazol-17beta-ol), is a synthetic derivative of ethisterone, structurally related to stanozolol. For this reason its use as doping agent has been investigated. Danazol (Runch) (200 mg) were orally administered to two healthy male volunteers. Urine samples were collected up to 1-week post-dose. Four new metabolites have been identified in addition to the five previously reported. We propose the monitorization of 6beta-hydroxy-2-hydroxymethyl-1,2-dehydroethisterone and 6beta,16epsilon-dihydroxy-2epsilon-hydroxymethyl-ethisterone by free fraction analysis. In a same way, we proposed to detect the principal isomer of a mono-hydroxylated metabolite of 6beta-hydroxy-2epsilon-hydroxymethylethisterone in the conjugated fraction. We conclude that new metabolites can be included for the detection of danazol abuse since the main metabolite ethisterone is excreted relatively fast in urine.     

Identification of Ginkgo biloba flavonol metabolites after oral administration to humans

An extract of Ginkgo biloba leaves (EGb) was given to healthy volunteers. Urine samples were collected for 3 days, and blood samples were withdrawn every 30 min for 5 h. The samples were purified through SPE C₁₈ cartridges and analyzed by reversed-phase LC–diode array detection for the presence of EGb metabolites. Only urine samples contained detectable amounts of substituted benzoic acids, i.e., 4-hydroxybenzoic acid conjugate, 4-hydroxyhippuric acid, 3-methoxy-4-hydroxyhippuric acid, 3,4-dihydroxybenzoic acid, 4-hydroxybenzoic acid, hippuric acid and 3-methoxy-4-hydroxybenzoic acid (vanillic acid). In contrast to rats no phenylacetic acid or phenylpropionic acid derivatives were found in urine, thus indicating that in humans a more extensive metabolism takes place. As for rats the metabolites found in human urines accounted for less than 30% of the flavonoids given. The same procedure was applied to blood samples, and no metabolites could be detected.     

Effect of progesterone (norethindrone) only oral contraceptive on mouse uterus--a SEM study

Impact of progesterone (norethindrone) only oral contraceptive (MICRONOR) on mouse uterus was investigated by SEM. The three dimensional SEM photographs revealed that, flat wave like mucous folds of the uterus (control) become more thick and highly convoluted after 50 days of MICRONOR feeding. The uteri became highly secretory as indicated by increased thickness of nonciliated secretory cell population with their increased microvilli. Low doses of norethindrone in combination with circulating estrogen, instead of causing regression, maintained a steady and highly secretory state which may interfere implantation/gamete transport.     

Distribution and percentages of non-protein bound contraceptive steroids in human serum

It has been shown that albumin bound steroids are taken up by the rat brain in addition to nonprotein bound steroids and it has also been suggested that cortisol binding globulin (CBG) may facilitate progesterone uptake by the rat uterus but not the brain. Recently serum sex-hormone binding globulin (SHBG) has been identified in the cytoplasm of sex steroid target cells. Thus the distribution of synthetic steroids between various protein bound and nonprotein bound components in serum may influence their bioavailability at different target tissues. The authors employed a newly developed technique, centrifugal ultrafiltration-dialysis. The results showed that there are no differences in percentages of nonprotein bound ethinyl estradiol (EE2), and cyproterone acetate (CA) with respect to sex or serum SHBG and CBG binding capacities. However serum percentages of nonprotein bound norethisterone (NET) (p0.05) are significantly lower in women than in men. Also the percentages of nonprotein bound NET and D-norgestrel are both very much lower (p0.001) in serum from pregnant women when compared to nonpregnant women. These differences appear to be inversely related to serum SHBG binding capacity. The percentages of nonprotein bound NET and D-norgestrel in heat treated serum from men and nonpregnant women are identical and largely represent the contribution of albumin binding alone. In addition heat labile binding proteins do not appear to influence the percentages of nonprotein bound EE2 and CA and it can be inferred that EE2 and CA are almost exclusively bound by albumin in native serum; 98.5% of EE2 and 93% of CA are bound to albumin. In contrast the percentages of nonprotein bound NET and D-norgestrel in native serum are inversely related to SHBG binding capacity. This data indicate that the nonprotein bound and albumin bound factors of NET and D-norgestrel may vary by as much as 2-3 fold between women who are known to have subnormal or supranormal levels of serum SHBG binding capacity and it is suggested that measurements of serum SHBG binding capacity may provide a method of assessing the lowest effective dose of these 2 progestins in individual subjects to help reduce side effects associated with their use. Future studies should address the effect of serum steroid concentrations on the actual nonprotein bound serum concentrations and distribution of these progestins.     

The pharmacokinetic profile of crocetin in healthy adult human volunteers after a single oral administration

Crocetin, a unique carotenoid with a short carbon chain length, is an active compound of saffron and Gardenia jasminoides Ellis used as traditional herbal medicine. The present study was undertaken to investigate the pharmacokinetic profiles of crocetin in healthy adult subjects. The study was conducted as an open-label, single dose escalation with 10 Filipino volunteers (5 men and 5 women). The subjects received a single dose of crocetin at three doses (7.5, 15 and 22.5 mg) in one week interval. Blood samples were collected from the brachial vein before and at 1, 2, 4, 6, 8, 10 and 24 h after administration. Plasma concentrations of crocetin were determined by high-performance liquid chromatography (HPLC). Crocetin was rapidly absorbed and detected within an hour of administration with a mean time to reach maximum concentration (T_max) of crocetin ranging from 4.0 to 4.8 h. The mean values of C_max and AUC_0-24 h ranged from 100.9 to 279.7 ng/ml and 556.5 to 1720.8 ng^.h/ml respectively. C_max and AUC values increased with dose proportional manner. Crocetin was eliminated from human plasma with a mean elimination half life (T_1/2) of 6.1 to 7.5 h.In summary, there were no serious adverse events up to 22.5 mg dose of crocetin while crocetin was found to be absorbed more quickly than the other carotenoids such as β-carotene, lutein and lycopene.     

Influence of the oral administration of micronized progesterone on plasma and tissue levels of steroids in human pregnancy

A single dose of micronized progesterone, orally administered to women at different stages of pregnancy, induced an immediate increase in the plasma levels of progesterone. Progesterone was administered to another group of women just before elective cesarean section. Levels of progesterone, oestradiol-17 beta and oestrone were determined in plasma, placenta and at different sites of the myometrium obtained during the surgical procedure. Results were compared to those observed in a control group of women who did not receive progesterone. Progesterone levels demonstrated a marked increase in plasma and in the whole myometrium 150 min after administration; the levels then decreased rapidly to control values within one hour. The concentrations of progesterone in the placenta did not show any change. Evident modifications of the oestrogen levels appeared in the myometrium and in the placenta after the administration of progesterone whereas no change occurred in the plasma. No difference was observed in estradiol-17 beta in the myometrium whereas an increase was seen in the placenta. Estrone levels decreased in the myometrium and in the placenta. This study demonstrates a prompt delivery of an orally administered natural progesterone to the myometrial target tissue and subsequent modifications in the estrogen pattern. This provides a theoretical mechanism for the clinical use of progesterone in the prevention of premature labor.     

The presence of cellular metabolites in milk

Cellular metabolites are present in goats' milk. The concentrations of UDPgalactose and some nucleotides were higher in milk than in mammary tissue. Other metabolites were present in milk at similar concentrations to those found in the mammary gland. It is proposed that the concentrations of these metabolites in milk reflect their Golgi-vesicular and cytosolic concentrations.     

Relative bioavailability of ethinyl estradiol from two different oral contraceptive formulations after single oral administration to 18 women in an intraindividual cross-over design

Two low-dose oral contraceptives, both containing the same dose of ethinyl estradiol (EE2) but different progestins (gestodene and desogestrel, respectively), were compared with respect to the relative bioavailability of EE2. After single-dose administration of both formulations to 18 women in an intraindividual cross-over design, there was no difference in the target variables for EE2 (Cmax, tmax and AUC). With respect to EE2, both formulations were bioequivalent. The observation of others, reporting higher EE2 levels in the serum of women taking the gestodene-containing formulation as compared to those taking the desogestrel-containing formulation, was not confirmed.     

Bioequivalence of two fexofenadine formulations in healthy human volunteers after single oral administration

AIM: A randomized, two-way, crossover, bioequivalence study was conducted in 25 fasting, healthy, male volunteers to compare two brands of fexofenadine 180 mg tablets, FEXOFENADINE 180 mg Film Tablet (Drogsan A.S., Ankara, Turkey) as test and Telfast 180 mg Tablet (Aventis Pharma, Frankfurt am Main, Germany) as a reference product. METHOD: One tablet of either formulation was administered after 10 h of overnight fasting. After dosing, serial blood samples were collected during a period of 48 hours. Plasma samples were analysed for fexofenadine by a validated HPLC method. The pharmacokinetic parameters AUC(0-48), AUC(0-alpha), C(max), T(max), K(el), T(1/2), and CL were determined from plasma concentration-time profiles for both formulations and were compared statistically. RESULTS AND CONCLUSIONS: The analysis of variance (ANOVA) did not show any significant difference between the two formulations and 90% confidence intervals (CI) fell within the acceptable range, satisfying the bioequivalence criteria of the FDA. Based on these statistical inferences it was concluded that the two brands exhibited comparable pharmacokinetics profiles and that Drogsan's Fexofenadine is equivalent to Telfast of Aventis Pharma, Frankfurt am Main, Germany.     

Detection and quantification of glucuro- and sulfoconjugated metabolites in human urine following oral administration of xenobiotic 19-norsteroids

Recently, the endogenous origin of nandrolone (19-nortestosterone) and other 19-norsteroids has been a focus of research in the field of drug testing in sport. In the present study, we investigated metabolites conjugated to a glucuronic acid and to a sulfuric acid in urine following administration of four xenobiotic 19-norsteroids. Adult male volunteers administered a single oral dose (10 mg) of each of four 19-norsteroids. Urinary samples collected from 0 to 120 h were subjected to methanolysis and beta-glucuronidase hydrolysis and were derivatized by N-methyl-N-trimethylsilyltrifluoroacetamide (MSTFA) before gas chromatography-mass spectrometry analysis. We confirmed that 19-norandrosterone (19-NA) and 19-noretiocholanolone (19-NE) were present in both glucuronide (g) and sulfate (s) conjugates and 19-norepiandrosterone (19-NEA) was excreted exclusively as a sulfate fraction in urine of all 19-norsteroids tested. The overall levels of the three metabolites can be ranked as follows: 19-NA(g+s)>19-NE(g+s)>19-NEA(s). The concentration profiles of these three metabolites in urine peaked between 2 to 12h post-administration and declined thereafter until approximately 72-96 h. 19-NA was most prominent throughout the first 24 h post-administration, except for a case in which an inverse relationship was found after 6h post-administration of nandrolone. Furthermore, we found that sulfate conjugates were present in both 19-NA and 19-NE metabolites in urine of all 19-norsteroids tested. The averaged total amounts of metabolites (i.e. 19-NA(s+g)+19-NE(s+g)+19-NEA(s)) excreted in urine were 38.6, 42.9, 48.3 and 21.6% for nandrolone, 19-nor-4-androsten-3,17-dione, 19-nor-4-androsten-3beta,17beta-diol and 19-nor-5-androstene-3beta,17beta-diol, respectively. Results from the excretion studies demonstrate significance of sulfate-conjugated metabolites on interpretation of misuse of the 19-norsteroids.     

Stereoselective pharmacokinetics of clausenamide enantiomers and their major metabolites after single intravenous and oral administration to rats

The pharmacokinetics of clausenamide (CLA) enantiomers and their metabolites were investigated in Wistar rat. After intravenous and oral administration at a dose of 80 and 160 mg/kg each enantiomer, plasma concentrations of (-)- or (+)-CLA and its major metabolites were simultaneously determined by reverse-phase HPLC with UV detection. Notably, stereoselective differences in pharmacokinetics were found. The mean plasma levels of (+)-CLA were higher at almost all time points than those of (-)-CLA. (+)-CLA also exhibited greater t(max), C(max), t(1/2beta), AUC(0-12h), and AUC(0--> infinity) and smaller CL (or CL/F) and V(d) (or V(d)/F), than its antipode. The (+)/(-) isomer ratios for t(1/2beta), t(max), AUC(0-12 h), and AUC(0--> infinity), which ranged from 1.26 to 2.08. The ratio for CL (or CL/F) was about 0.5, and there were significant differences in these values between CLA enantiomers (P < 0.05), implying that the absorption, distribution, and elimination of (-)-CLA were more rapid than those of (+)-CLA. Similar findings for (-)-7-OH-CLA, the major metabolite of (-)-CLA, and (+)-4-OH-CLA, the major metabolite of (+)-CLA, can be also seen in rat plasma. The contributing factors for the differences in stereoselective pharmacokinetics of CLA enantiomers appeared to be involved in their different plasma protein binding, first-pass metabolism and interaction with CYP enzymes, especially with their metabolizing enzyme CYP 3A isoforms.     

The effect of oral contraceptive steroid therapy on the urinary metabolites of radioactive estrone and estradiol-17beta

Eight urinary metabolites of radioactive estrone and estradiol-17β (estrone, estradiol-17β, 2-hydroxyestrone, 2-methoxyestrone, 2-hydroxyestrone 3-methyl ether, 16α-hydroxyestrone, 2-hydroxyestradiol and estriol) have been measured by reverse isotope dilution from young women on oral contraceptive therapy. There was a decrease in the sum of the 16α-hydroxy1ated metabolites in the Ketodase liberated fraction from the subjects taking ethynylestradiol containing preparations as compared to those taking preparations containing mestranol and those subjects who were taking no oral contraceptives. This report is also the first to document and measure 2-hydroxyestradiol as a urinary metabolite of radioactive estrone and estradiol.     

Gas chromatographic–mass spectrometric identification of main metabolites of stanozolol in cattle after oral and subcutaneous administration

An analytical method has been developed in order to control the illegal use of stanozolol as growth promoter in livestock. The procedure was based on enzymatic hydrolysis, purification on a Clean Screen DAU column and derivatization with heptafluorobutyric anhydride prior to GC–MS analysis. This method allowed us to study the metabolism of stanozolol in cattle after oral and subcutaneous administrations. Urinary metabolites were identified by mass spectrometry. Stanozolol and 16-hydroxystanozolol were detected after oral administration, while 16-hydroxystanozolol and 4,16-dihydroxystanozolol were found after subcutaneous administration.