Components characterisation of Berchemia lineata (L.) DC. by UPLC-QTOF-MS/MS and its metabolism with human liver microsomes.

Investigations were performed on the determination of the main components in Berchemia lineata (L.) DC. (BL) and its metabolism with human liver microsomes (HLM). A total of 35 compounds were detected in BL extracts and 25 of them including 6 naphthopyrones, 10 flavonoids, 2 phenolic acids, 2 phenols, 4 fatty acids and 1 quinone were unambiguously or tentatively identified by UPLC-QTOF-MS/MS. Among them, naphthopyrones were first identified in BL extracts and labelled in chromatography. In addition, the weak inhibitory effects of BL extracts (IC50＝149.25 µg/mL) and rubrofusarin-6-O-α-L-rhamnosyl-(1-6)-O-ß-D-glu-copyranside (the main component of BL extracts, M0; IC50＝82.14 µM) on CYP3A4 were also proved using testosterone as specific probe drug. The main metabolic pathway of M0 by HLM was hydroxylation in its aglycone, the metabolite was tentatively identified as 10-hydroxy-rubrofusarin-6-O-α-L-rhamnosyl-(1-6)-O-ß-D-glucopyranside. Components characterisation and the metabolism with HLM could help the further development and application of BL.

Modulation of the Lipid Profile of Reconstructed Skin Substitutes after Essential Fatty Acid Supplementation Affects Testosterone Permeability.

Skin models with efficient skin barrier function are required for percutaneous absorption studies. The contribution of media supplementation with n-3 and n-6 polyunsaturated fatty acids (PUFAs) to the development of the skin barrier function of in vitro skin models remains incompletely understood. To investigate whether PUFAs, alpha-linolenic acid (ALA, n-3 PUFA) and linoleic acid (LA, n-6 PUFA), could enhance the impermeability of a three-dimensional reconstructed human skin model, skin substitutes were produced according to the self-assembly method using culture media supplemented with either 10 µM ALA or 10 µM LA. The impact of PUFAs on skin permeability was studied by using a Franz cell diffusion system to assess the percutaneous absorption of testosterone and benzoic acid. Our findings showed that ALA supplementation induced a decrease in the absorption of testosterone, while LA supplementation did not significantly influence the penetration of testosterone and benzoic acid under present experimental conditions. Both ALA and LA were incorporated into phospholipids of the skin substitutes, resulting in an increase in n-3 total PUFAs or n-6 total PUFAs. Collectively, these results revealed the under-estimated impact of n-3 PUFA supplementation as well as the importance of the n-6 to n-3 ratio on the formation of the skin barrier of in vitro reconstructed human skin models.

Effect of testosterone therapy on the urinary bladder in experimental hypogonadism of rats.

Testosterone (T) deficiency is prevalent particularly in elderly men and lead to physical and sexual morbidities. Although low levels of T are associated with low urinary tract symptoms, the correlation between T deficiency and bladder dysfunction is not clearly identified. The aim of this study was to investigate the effect of high dose testosterone replacement therapy (TRT) on the histological structure of the UB in castrated rats. Twenty-five adult male rats were divided into three groups: control, castrated and castrated + TRT. T was administrated in high dose (100 mg/kg) two intramuscular injections/week for 60 days. UB sections were prepared and stained with H&E, Masson's trichrome and immunohistochemical detection of Cytokeratin 20 (Ck20). All data were morphometrically and statistically analyzed. In castrated group, significant atrophy of the urothelium (P < 0.001) accompanied with widening of the corium were observed. The smooth muscle appeared thin with marked increase in the collagen fibers. On treating the castrated group with TRT, atypical Ck20 expression as well as significant increase in urothelial thickness (P < 0.05) and smooth muscle/collagen ratio (P < 0.001) were detected. In castrated rat model, high dose TRT has a positive effect on the UB smooth muscle rather than the urothelium which acquired atypical patterns.

Pinelliae rhizoma, a toxic chinese herb, can significantly inhibit CYP3A activity in rats.

Raw Pinelliae Rhizoma (RPR) is a representative toxic herb that is widely used for eliminating phlegm or treating cough and vomiting. Given its irritant toxicity, its processed products, including Pinelliae Rhizoma Praeparatum (PRP) and Pinelliae Rhizoma Praeparatum cum Zingibere et Alumine (PRPZA), are more commonly applied and administered concomitantly with other chemical drugs, such as cough medications. This study aimed to investigate the effects of RPR, PRP, and PRPZA on CYP3A activity. Testosterone (Tes) and buspirone (BP) were used as specific probe substrates ex vivo and in vivo, respectively. CYP3A activity was determined by the metabolite formation ratios from the substrates. Ex vivo results show that the metabolite formation ratios from Tes significantly decreased, indicating that RPR, PRP, and PRPZA could inhibit CYP3A activity in rats. CYP3A protein and mRNA levels were determined to explore the underlying mechanism. These levels showed marked and consistent down-regulation with CYP3A activity. A significant decrease in metabolite formation ratios from BP was also found in PRPZA group in vivo, implying that PRPZA could inhibit CYP3A activity. Conclusively, co-administration of PR with other CYP3A-metabolizing drugs may cause drug-drug interactions. Clinical use of PR-related formulae should be monitored carefully to avoid adverse interactions.

In vitro percutaneous absorption of ketoprofen and testosterone: comparison of pluronic lecithin organogel vs. pentravan cream.

An in vitro human percutaneous absorption study was conducted to assess the delivery of ketoprofen and testosterone from two base formulations, a Pluronic lecithin organogel and Pentravan Cream. Each formulation was applied to ex vivo human trunk skin (from three skin donors) on triplicate sections mounted onto Franz Diffusion Cells. Following a 5-mcL/cm2 applied dose, serial dermal receptor solutions were collected over 48 hours. For both compounds, a greater rate and extent of absorption was found from the Pentravan formulation than from the Pluronic lecithin organogel formulation: 3.8-fold greater for ketoprofen, 1.7-fold greater for testosterone, for amount absorbed.

Testosterone supplementation in male obese Zucker rats reduces body weight and improves insulin sensitivity but increases blood pressure.

Androgen levels are lower in obese men as compared with normal weight individuals. However, there are no safety data regarding the chronic use of androgen supplements in middle-aged men. The present study was undertaken to determine the cardiovascular and metabolic effects of chronic (10 weeks) testosterone treatment in male obese Zucker rats, starting at 22 weeks of age, when testosterone levels were significantly decreased. Testosterone supplements increased plasma levels, 10-fold in both obese Zucker rats and lean Zucker rats. In obese Zucker rats, testosterone supplements reduced body weight, plasma insulin, and cholesterol levels and improved the oral glucose tolerance test. None of these parameters were affected in lean Zucker rats. Mean arterial pressure was significantly increased in obese Zucker rats but not lean Zucker rats. Testosterone supplements increased proteinuria and accelerated renal injury in lean Zucker rats only. Thus, treatment of obese men with chronic testosterone supplements should be done with careful monitoring of blood pressure.

[Interaction between four herb compounds and a western drug by CYP3A4 enzyme metabolism in vitro].

OBJECTIVE: To explore the interaction between herbal medicines and western drugs based on CYP3A4 enzyme metabolism by using testotesrone as a probe in liver microsome metabolism system in vitro. METHOD: The mixed liver microsome enzymatic system consisting of rat liver microsomes by ultra-high-speed centrifuge was established. The substrate testosterone was added into the system and enzyme CYP3A4 metabolic activity was expressed by the output of 6beta-hydroxy-testosterone which was measured by HPLC method. The proper conditions for testotesrone metabolism in liver microsome system included substrate concentration, incubation time, pH and incubation temperature. When the conditions in vitro were determined, three kinds of Chinese herbal medicinal ingredients (Tetrahydropalmatine, neferine, panax notoginseng saponins) were diluted into different concentrations and incubated with testotesrone in the liver microsomes incubation system, respectively. The results were measured through metabolite production with or without the presence of Chinese medicines. We assessed the Chinese herbal medicinal ingredients effect on the metabolism of CYP3A4 enzyme through 6beta-hydroxy metabolite of testosterone production. RESULT: Liver microsomes were incubated in the system, the testosterone metabolited into 6beta-hydroxy testosterone. The metabolism conditions were proper at the concentration of testosterone 200 micromol x L(-1) which was incubated for 3.5 hours at 37 degrees C in pH 7.0, PBS 0.1 mol x L(-1). The inhibition of tetrahydropalmatine and panax notoginseng saponins on testotesrone were weak with IC50 > 100 micromol x L(-1). The neferine had a little inhibition on testotesrone metabolism, IC50 < 100 micromol L(-1). CONCLUSION: Tetrahydropalmatine and panax notoginseng saponins had no obvious effect on testotesrone metabolism. Neferine had a little effect on testotesrone metabolism. It prompted that drug-interaction could not be apparent between two kinds of Chinese medicines and the CYP3A4 enzyme substrate, Neferine could bring about drug-interaction.

Biotransformation of sildenafil in the male rat: evaluation of drug interactions with testosterone and carbamazepine.

The biotransformation of sildenafil to its major circulating metabolite, UK-103,320, was studied in male rat liver microsomes. The conversion of sildenafil to UK-103,320 by rat microsomes followed Michaelis-Menten kinetics, for which the parameters were V(max) = 1.96 microM/minand K(m) = 27.31 microM. Using substrates of CYP3A4 of testosterone and carbamazepine, the active sites on CYP3A4 responsible for metabolizing sildenafil were also evaluated. Sildenafil biotransformation was inhibited in the individual presence of testosterone and carbamazepine. The results showed drug interaction was observed in the sildenafil-testosterone and sildenafil-carbamazepine. Although testosterone and carbamazepine can inhibit sildenafil demethylation in concentration- and incubation time-dependent manners, sildenafil did not inhibit testosterone hydroxylation or carbamazepine epoxidation. These results may be explained by a model in which multiple substrates or ligands can concurrently bind to the active site of a single CYP3A4 molecule. However, the contribution of separate allosteric sites and conformational heterogeneity to the atypical kinetics of CYP3A4 cannot be ruled out in this study.

Improvement of the experimental setup for skin absorption screening studies with reconstructed skin EPISKIN.

Percutaneous penetration studies are usually performed in human skin samples set up in a Franz cell device. The ability to perform these studies may depend on the availability of skin samples. Reconstructed skin models are an interesting alternative to overcome such limitations but are less easily mounted in diffusion cell devices. Previous data showed that EPISKIN was a highly performing model to carry out such studies. However, the setup in a PermeGear cell device is time consuming and therefore unsuitable for screening purposes. Another approach could be using EPISKIN in its cell culture insert. The aim of this study was to compare cutaneous penetration of chemicals applied to EPISKIN samples in a PermeGear cell versus in their own insert. Eight chemicals having widely different chemical structures and penetration potentials were studied. Six test chemicals showed a similar penetration level in both devices. Using the PermeGear cell device, the penetration level was overestimated for the other 2 tested chemicals. The results demonstrated that percutaneous studies with EPISKIN samples could be easily performed using the insert setup. The EPISKIN model has been greatly improved in the recent years and it is now possible to develop screening tests for the evaluation of skin penetration with a higher reliability.

Enhancing the drug metabolism activities of C3A--a human hepatocyte cell line--by tissue engineering within alginate scaffolds.

In this study, we investigated the applicability of C3A--a human hepatocyte cell line--as a predicting tool for drug metabolism by applying tissue-engineering methods. Cultivation of C3A cells within alginate scaffolds induced the formation of spheroids with enhanced drug metabolism activities compared to that of two-dimensional (2-D) monolayer cultures. The spheroid formation process was demonstrated via histology, immunohistochemistry, and transmission electron microscope (TEM) analyses. The C3A spheroids displayed multilayer cell morphology, characterized by a large number of tight junctions, polar cells, and bile canaliculi, similar to spheroids of primary hepatocytes. Spheroid formation was accompanied by a reduction in P-glycoprotein (Pgp) gene expression and C3A cell proliferation was limited mainly to cells on the spheroid outskirt. The 3-D constructs maintained a nearly constant cell number according to MTT assay. Drug metabolism by the two most important cytochrome p-450 (CYP) enzymes in human liver, CYP1A2 and CYP3A4, was tested using preferred drugs. With CYP1A2, 3-fold enhancement in activity per cell was seen for converting ethoxyresorufin to resorufin compared to C3A cell monolayers. The spheroids responded to the inducer beta-naphthoflavone and to the inhibitor furafylline of CYP1A2. Enhanced metabolizing activity of CYP3A4, measured by the amount 6beta-testosterone formed from testosterone, and that of the phase II enzyme glucuronosyltransferases (UGT) further indicated that the tissue-engineered C3A spheroids may provide an efficient experimental tool for predicting drug activities by these CYPs. Moreover, the maintenance of constant cell number, as well as the elevated hepatocellular functions and drug metabolism activities, suggest that the tissue-engineered C3A may be applicable in replacement therapies.

Plasticity in anterior hypothalamic vasopressin correlates with aggression during anabolic-androgenic steroid withdrawal in hamsters.

In hamsters, adolescent anabolic-androgenic steroid (AAS) exposure facilitates offensive aggression, in part by altering the development and activity of anterior hypothalamic arginine vasopressin (AH-AVP). This study assessed whether these effects were lasting by examining aggression and AH-AVP during AAS withdrawal. Adolescent hamsters administered AAS were tested as adults for aggression at 1, 4, 11, 18, or 25 days of withdrawal, sacrificed the following day, and examined for AH-AVP afferent innervation using immunohistochemistry. Through Day 12 of withdrawal, aggression and AVP were significantly higher in AAS-treated hamsters than in controls. These differences were no longer observable by Day 19 of withdrawal, at which point the behavior and neurobiology of AAS-treated hamsters reverted to that observed in controls. These data indicate that adolescent AAS exposure has short-term, reversible effects on both aggression and AH-AVP, correlating AH-AVP with the aggressive/nonaggressive behavioral phenotype during AAS withdrawal.

Establishment of a yeast system that stably expresses human cytochrome P450 reductase: application for the study of drug metabolism of cytochrome P450s in vitro.

Cytochrome P450s (CYPs) hold a balance in studying pharmacokinetics, toxico-kinetics, drug metabolism, and drug-drug interactions, which require association with cytochrome P450 reductase (CPR) to achieve optimal activity. A novel system of Saccharomyces cerevisiae useful for expression studies of mammalian microsomal CYPs was established. Human CPR (hCPR) was co-expressed with human CYP3A4 (hCYP3A4) in this system, and two expression plasmids pTpLC and pYeplac195-3A4 containing the cDNA of hCPR and hCYP3A4 were constructed, respectively. The two plasmids were applied first and controlled by phosphoglycerate kinase (PGK) promoter. S. cerevisiae BWG1-7alpha transformed with the expression plasmids produced the respective proteins in the expected molecular sizes reactive with both anti-hCYP3A4 immunoglobulin (Ig) and anti-hCPR Ig. The activity of hCPR in yeast BWG-CPR was 443.2 nmol reduced cytochrome c/min/mg, which was about three times the CPR activity of the microsome prepared from the parental yeast. The protein amount of hCYP3A4 in BWG-CPR/3A4 was 35.53 pmol/mg, and the 6beta-hydroxylation testosterone formation activity of hCYP3A4 expressed was 7.5 nmol/min/nmol CYP, 30 times higher than the activity of hCYP3A4 expressed in the parental yeast, and almost two times the activity of hCYP3A4 from homologous human liver microsome. Meanwhile, BWG-CPR/3A4 retained 100 generations under nonselective culture conditions, indicating this yeast was a mitotically stable transformant. BWG-CPR was further tested daily by the PCR amplification of hCPR of yeast genome, Western blot analysis, and the activity assay of hCPR of yeast microsome. This special expression host for CYPs was validated to be stable and efficient for the expression of CYPs, applying as an effective selection model for the drug metabolism in vitro.

Pharmacokinetics and pharmacodynamics of injectable testosterone undecanoate in castrated cynomolgus monkeys (Macaca fascicularis) are independent of different oil vehicles.

Testosterone undecanoate (TU) dissolved in soybean oil was developed in China to improve the pharmacokinetics of this testosterone ester in comparison with TU in castor or tea seed oil. As a pre-clinical primate model, three groups of five castrated cynomolgus macaques received either a single intramuscular injection of 10 mg/kg body weight TU in soybean oil, in tea seed oil, or in castor oil (equals 6.3 mg pure T/kg body weight for all preparations). Testosterone, estradiol, luteinizing hormone, and follicle-stimulating hormone as well as prostate volume, body weight and ejaculate weight were evaluated. After injection supraphysiological testosterone levels were induced. There were no significant differences in the pharmacokinetics of the three TU preparations for testosterone and estradiol. The gonadotropin levels showed a high individual variation. Prostate volumes increased equally in all groups after administration and declined to castrate level afterwards. The results suggest that TU in soybean oil produces similar effects as TU in the other vehicles. This study in non-human primates provides no objection to testing of this new preparation in humans.

The role of aromatization in testosterone supplementation: effects on cognition in older men.

OBJECTIVE: To determine the contribution of conversion of testosterone (T) to estradiol on cognitive processing in a population of healthy older men who received T supplementation. METHODS: Sixty healthy, community-dwelling volunteers aged 50 to 90 years completed a randomized, double-blind, placebo-controlled study. Participants were randomized to receive weekly IM injections of 100 mg T enanthate plus daily oral placebo pill (T group, n = 20), 100 mg testosterone enanthate plus 1 mg daily of anastrozole, an aromatase inhibitor (oral pill), to block the conversion of T to estradiol (AT group, n = 19), or saline injection and placebo pill (placebo group, n = 21) for 6 weeks. Cognitive evaluations using a battery of neuropsychological tests were conducted at baseline, week 3 and week 6 of treatment, and after 6 weeks of washout. RESULTS: Circulating total T was increased from baseline an average of 238% in the T and AT treatment groups. Estradiol increased an average of 81% in the T group and decreased 50% in the AT group during treatment. Significant improvements in spatial memory were evident in the AT and T treatment groups. However, only the group with elevated estradiol levels (T group) demonstrated significant verbal memory improvement. CONCLUSION: In healthy older men, improvement in verbal memory induced by testosterone administration depends on aromatization of testosterone to estradiol, whereas improvement in spatial memory occurs in the absence of increases in estradiol.

Testimony of the correlation between DHEA and bioavailable testosterone using a biochromatographic concept: effect of two salts.

In a previous paper (C. André et al., submitted to J. Chromatogr. B) a mathematical model based on the Langmuir theory was developed to visualize the competition effect between testosterone and deshydroepiandrosterone (DHEA) for their identical human serum albumin (HSA) binding cavity. In this work, the thermodynamic mechanisms of (i) the binding of two hormones, DHEA and testosterone to HSA and (ii) the testosterone displacement of its HSA binding cavity by DHEA was studied by biochromatography. The Na+ cation effect used as physico-chemical marker of these binding processes was clearly described. The Gibbs free energy value (DeltaGo ) of the displacement equilibrium was always negative demonstrating that DHEA well displaced testosterone of its HSA binding cavity. The thermodynamic data also showed that this displacement equilibrium was enthalpically controlled. Moreover, the effect of (Mg2+) concentration (x') on the two binding mechanisms was analyzed. It appeared that for old men with a deficit of testosterone, Mg(2+) supplementation during treatment with DHEA can increased the free testosterone concentration and its biological effect. All these results must be confirmed by in vivo test.

Azadirachta indica adversely affects sperm parameters and fructose levels in vas deferens fluid of albino rats.

Azadirachta indica treatment for 24 days in albino rats resulted in a decrease in the total sperm count, sperm motility, and forward velocity in vas deferens fluid. The percentage of abnormal sperm increased and the fructose content decreased. As diminished levels of fructose parallel androgen deficiency, we conclude that reduced androgen levels resulting from the anti-androgenic property of A. indica leaves probably influences the physiological maturation of sperm.

Ex vivo bioadhesion and in vivo testosterone bioavailability study of different bioadhesive formulations based on starch-g-poly(acrylic acid) copolymers and starch/poly(acrylic acid) mixtures.

Starch-g-poly(acrylic acid) copolymers or grafted starches synthesized by 60Co irradiation or chemical modification and co-freeze-dried starch/poly(acrylic acid) mixtures were evaluated on their ex vivo bioadhesion capacity. The buccal absorption of testosterone from a bioadhesive tablet formulated with the grafted starches or starch/poly(acrylic acid) mixtures was investigated. The results were compared to a reference formulation (physical mixture of 5% Carbopol 974P and 95% Drum Dried Waxy Maize). Rice starch-based irradiated grafted starches showed the best bioadhesion results. Partial neutralization of the acrylic acid with Ca(2+) ions resulted in significantly higher bioadhesion values compared to the reference. Ca(2+) and Mg(2+) partially neutralized maltodextrin-based irradiated grafted starches showed significantly higher bioadhesion values compared to the reference formulation. The chemically modified grafted starches showed significantly higher adhesion force values than for the reference tablet. None of the co-freeze-dried starch/poly(acrylic acid) mixtures showed significantly higher bioadhesion results than the reference (Bonferroni test, P<0.05). A chemically modified grafted starch could sustain the 3 ng/ml plasma testosterone target concentration during +/- 8 h (T(>3 ng/ml)). By lyophilization of a partially neutralized irradiated grafted starch, the in vivo adhesion time (22.0 +/- 7.2 h) and the T(>3 ng/ml) (13.5 +/- 1.3 h) could be increased. The absolute bioavailability of the lyophilized formulation approached the reference formulation. Some of the grafted starches showed to be promising buccal bioadhesive drug carriers for systemic delivery.

Alterations of prostate biomarker expression and testosterone utilization in human LNCaP prostatic carcinoma cells by garlic-derived S-allylmercaptocysteine.

BACKGROUND: This study determined the effects of S-allylmercaptocysteine (SAMC), a phytoconstituent from garlic, on the expression of androgen-responsive biomarkers, prostate specific antigen (PSA), and prostate specific membrane antigen (PSMA), in human prostatic carcinoma cells (LNCaP). METHODS: Secretion of PSA was determined as well as the activity of PSMA measured as a function of its ability to hydrolyze poly-gamma-glutamated folate and N-acetylaspartylglutamate (NAAG). Folate hydrolase capacity was also determined in SAMC-treated cells grown in charcoal stripped fetal calf serum (CS-FCS). In addition, testosterone disappearance was measured from culture media of SAMC-treated LNCaP and PC-3 cells as well as from cell free lysates. RESULTS: PSA secretions were significantly decreased compared to control values at 1 day (8.4 +/- 2.6 vs. 18.9 +/- 1.7, P < 0.01), 4 days (18.9 +/- 5.3 vs. 73.8 +/- 4. 4, P < 0.001), and 6 days (35.6 +/- 2.1 vs. 96.5 +/- 17.9 ng/10(5) cells, P < 0.01; mean +/- SD). By contrast, PSMA activity measured as either folate hydrolase or NAAG dipeptidase (NAALADase) activity increased in cells treated with SAMC. PSMA-folate hydrolase activity in SAMC-treated cells grown in CS-FCS increased beyond that observed in cells grown in CS-FCS alone. Pre-exposure of LNCaP cells to SAMC resulted in enhanced rate of testosterone disappearance from culture media at 6 hr (P < 0.01) and at 48 hr (P < 0.001) compared to media from cells not previously exposed to SAMC. Results similar to these were also observed in androgen-independent PC-3 cells treated with SAMC. In lysates of SAMC-treated LNCaP cells, the rate of testosterone catabolism was twice that from phosphate buffered saline (PBS)-treated cells. SAMC-treated LNCaP cells grown in media supplemented with testosterone temporarily exhibited enhanced growth over a 2 day period but cell numbers declined later to levels similar to those of SAMC treatment. CONCLUSIONS: These results show that SAMC exhibits differential effects on recognized biomarkers for LNCaP cells similar to those produced by androgen deprivation and strongly suggests that this effect may be mediated, in part, by diminishing the trophic effects of testosterone, likely by converting it to metabolites less reactive toward androgen receptors.

Development of a time-resolved fluoroimmunoassay (TR-FIA) for testosterone: measurement of serum testosterone concentrations after testosterone treatment in the rainbow trout (Oncorhynchus mykiss).

A sensitive time-resolved fluoroimmunoassay (TR-FIA) for testosterone was developed, and the assay system was used for measuring serum testosterone concentrations in rainbow trout. Testosterone-3-(O-carboxymethyl)oxime-bovine serum albumin (T-3-CMO-BSA) was immobilized by physical adsorption to the wells of microtiter plates. A competitive assay using two antibodies was performed among T-3-CMO-BSA in the solid-phase, unknown amounts of testosterone, testosterone antibodies, and europium labeled secondary antibodies, followed by measurements using a time-resolved fluorometer (DELFIA system). The TR-FIA had a sensitivity of 0.075 pg/50 microliters sample (1.5 pg/ml), and the range of the assay system was between 1.5 pg/ml and 25 ng/ml. The intra- and interassay coefficients of variation for the testosterone TR-FIA were satisfactorily low, and were between 1.62 and 6.38% and 2.96 and 8.29%, respectively. The assay system was applied to measure the serum testosterone concentrations after an injection of testosterone dissolved in saline, propyleneglycol, or coconut oil. Among the three solvents, the coconut oil group showed continuously high serum testosterone level. In contrast, the saline and propyleneglycol groups had maximum concentrations 24 hr after the injection, but their levels were significantly lower than that of the coconut oil group. The testosterone TR-FIA method is sensitive, repeatable, and is as accurate as conventional RIAs. It is very good for measuring serum testosterone concentrations.

Contributions of drug solubilization, partitioning, barrier disruption, and solvent permeation to the enhancement of skin permeation of various compounds with fatty acids and amines.

The contributions of several proposed mechanisms by which fatty acids and amines might increase skin permeation rates were assessed. Permeation rates of model diffusants with diverse physicochemical properties (naloxone, testosterone, benzoic acid, indomethacin, fluorouracil, and methotrexate) through human skin were measured in vitro. The enhancers evaluated were capric acid, lauric acid, neodecanoic acid, and dodecylamine. Increased drug solubility in the vehicle, propylene glycol (PG), in some cases accounted for the increases in flux in the presence of adjuvants, since permeability coefficients were unchanged. Partition coefficients of some drugs into isopropyl myristate or toluene were increased by the adjuvants, but this did not occur for combinations of an acid with a base (adjuvant-drug or drug-adjuvant). Increases in flux not accounted for by increases in drug solubility or partitioning were assumed to involve disruption of the barrier function of skin (increased skin diffusivity). All fatty acids increased skin diffusivity of naloxone, testosterone, indomethacin, and fluorouracil but not of methotrexate or benzoic acid. Dodecylamine increased skin diffusivity only for fluorouracil. Capric acid and dodecylamine, but not lauric acid or neodecanoic acid, increased the skin permeation rate of PG, suggesting that enhanced solvent penetration could also be involved as a mechanism for increased skin permeation of the drug. However, the increase in PG flux due to dodecylamine was nullified when methotrexate was added to the vehicle, possibly because of a dodecylamine/methotrexate interaction. These studies demonstrate that drug solubilization in the vehicle, increased partitioning, increased solvent penetration, and barrier disruption each can contribute to increased skin permeation rates in the presence of fatty acids and amines.(ABSTRACT TRUNCATED AT 250 WORDS)