Comparative Evaluation of Dehydroepiandrosterone Sulfate Potential to Predict Hepatic Organic Anion Transporting Polypeptide Transporter-Based Drug-Drug Interactions.

Pharmacokinetic drug-drug interactions (DDIs) on hepatic organic anion transporting polypeptides (OATPs) are important clinical issues. Previously, we reported that plasma dehydroepiandrosterone sulfate (DHEAS) could serve as an endogenous probe to predict OATP-based DDIs in monkeys using rifampicin as an OATP inhibitor. Since the contribution of hepatic OATPs to the changes in plasma DHEAS by rifampicin remains unclear, however, we performed an in vivo pharmacokinetic study to explore this issue. Since plasma DHEAS concentrations were low in our rat model, the disposition of externally administered DHEAS was evaluated. Intravenously administered DHEAS was recovered mainly in bile (29.1%) and less in urine (2.95%). The liver tissue-to-plasma concentration ratio (Kpliver) decreased from 41.8 to 5.07 by rifampicin, and this decrement was consistent with the decrease in distribution volume from 247 to 59 ml/rat. Comparison of the in vitro IC50 of rifampicin for DHEAS uptake by isolated rat hepatocytes and in vivo plasma rifampicin concentration suggested that the effect of rifampicin on the plasma DHEAS concentration was explained mostly by the inhibition of hepatic OATPs, demonstrating that DHEAS could be a biomarker of hepatic OATP activity. Next, previously reported rifampicin-induced changes in plasma concentrations evaluated as an AUC ratio (AUCR) of possible probe compounds were compared on the basis of rifampicin dose/body surface area. The AUCR values of endogenous compounds and i.v. administered statins, for which possible DDIs in the intestinal absorption process can be excluded, increased proportionally to the rifampicin dose. Simultaneous measurement of these endogenous compounds could be effective biomarkers for the prediction of OATP-based DDIs.

Dehydroepiandrosterone sulfate, a useful endogenous probe for evaluation of drug-drug interaction on hepatic organic anion transporting polypeptide (OATP) in cynomolgus monkeys.

Since drug-drug interaction (DDI) can affect organic anion-transporting polypeptide (OATP) and cause clinical events, prediction of such DDI is important in early clinical development. Although statins are useful probes for OATP-mediated DDI, endogenous probes would be more practical for predicting such DDI. In this study, we investigate the possible use of dehydroepiandrosterone sulfate (DHEAS), an endogenous OATP substrate, in predicting OATP-mediated DDI in cynomolgus monkeys as a first step toward in human assessment. In in vitro experiments, both human and cynomolgus monkey hepatocytes showed a time- and temperature-dependent DHEAS uptake. Rifampicin (RIF), a typical OATP inhibitor, inhibited this uptake, indicating the involvement of OATP in DHEAS uptake. In in vivo experiments, the area under the plasma concentration-time curve (AUC) and maximum plasma concentration (Cmax) of DHEAS were significantly increased following administration of RIF 10 mg/kg, although the extent of this increase was lower than that observed with the test-statins used in this study. However, based on the results of in vitro hepatic DHEAS uptake, changes in DHEAS concentration are expected to be more prominent in human than in monkey. This shows for the first time that DHEAS may be used as endogenous probe for predicting OATP-mediated DDI.

The pharmacokinetics and pharmacodynamics of dehydroepiandrosterone during use of an ethinylestradiol- and drospirenone-containing oral contraceptive.

BACKGROUND: Combined oral contraceptives (COCs) reduce the levels of ovarian and adrenal androgens. Co-administration of dehydroepiandrosterone (DHEA) may normalise androgen levels during COC use. OBJECTIVE: To investigate the effect of the addition of DHEA to a COC on the pharmacokinetics (PK) and pharmacodynamics (PD) of DHEA and its sulphate (DHEA-S), and on levels of total and free testosterone (T). METHODS: In a prospective, randomised, double-blind, placebo-controlled, cross-over study involving 21 female volunteers, the PK and PD of DHEA and DHEA-S were investigated during the use of one cycle of a COC containing 30 µg ethinylestradiol (EE) and 3 mg drospirenone (DRSP) with and without daily co-administration of 50 mg DHEA. RESULTS: Treatment during one cycle with a COC containing EE and DRSP reduces the exposure to DHEA and DHEA-S by at least 20%. This loss of adrenal androgens can be fully compensated by daily oral co-administration of 50 mg DHEA. With DHEA co-administration total T levels rise significantly (1.44 nmol/L with DHEA vs. 0.82 nmol/L with placebo; p < 0.001). Free T levels decrease significantly with both DHEA and placebo treatment, but significantly less during co-administration of DHEA (6.34 pmol/L with DHEA vs. 3.96 pmol/L with placebo; p < 0.001). CONCLUSION: By adding DHEA to a COC the loss of adrenal and ovarian androgens can be restored.

Organic anion transporter 3 mediates the efflux transport of an amphipathic organic anion, dehydroepiandrosterone sulfate, across the blood-brain barrier in mice.

The present study investigated the efflux transport systems of organic anions across the blood-brain barrier (BBB) using dehydroepiandrosterone sulfate (DHEAS) as a probe. The elimination of DHEAS from the brain after microinjection into the cerebral cortex was characterized in wild-type mice and mice with deficiency of well characterized organic anion transporters, organic anion-transporting polypeptide 1a4 (Oatp1a4)/Slco1a4 and organic anion transporter 3 (Oat3)/Slc22a8, at the BBB. The saturable efflux of DHEAS from the brain was completely inhibited by probenecid, benzylpenicillin, and estrone-3-sulfate and moderately inhibited by taurocholate and p-aminohippurate (50-57%). Uptake of DHEAS and estrone-3-sulfate was greater in murine Oat3 cRNA-injected oocytes than that in water-injected oocytes. Efflux of these compounds from the brain was significantly delayed in Oat3(-/-) mice compared with that in wild-type mice, indicating that indeed Oat3 is functionally important in vivo. Furthermore, probenecid and taurocholate inhibited DHEAS efflux completely in Oat3(-/-) mice. Contrary to the past report in rats that suggested involvement of Oatp1a4, specific uptake of DHEAS and estrone-3-sulfate by murine Oatp1a4 was not detected in vitro, and efflux of both compounds from the brain was not altered in Oatp1a4(-/-) mice. There was no significant difference in the uptake of DHEAS by brain slices prepared from wild-type, Oatp1a4(-/-), and Oat3(-/-) mice. Taken together, these results suggest that Oat3 plays a significant role in the efflux of steroid conjugates across the BBB in mice and that the BBB also expresses other unknown organic anion transporters for the efflux of DHEAS. Transport mechanisms of organic anions at the BBB are far more diverse than they were assumed to be.

Functional differences in steroid sulfate uptake of organic anion transporter 4 (OAT4) and organic anion transporting polypeptide 2B1 (OATP2B1) in human placenta.

Human trophoblasts depend on the supply of external precursors such as dehydroepiandrosterone-3-sulfate (DHEA-S) and 16alpha-OH-DHEA-S for synthesis of estrogens. Recently, we have characterized the uptake of DHEA-S by isolated mononucleated trophoblasts and identified different transporter polypeptides involved in this process. Immunohistochemistry of 1st and 3rd trimester placenta detected organic anion transporter 4 (OAT4) and organic anion transporting polypeptide 2B1 (OATP2B1, former name OATP-B) in cytotrophoblast membranes and at the basal surface of the syncytiotrophoblast, indicating that both transporter polypeptides are involved in placental uptake of foetal derived steroid sulfates. In the present study we have characterized and compared the kinetics of DHEA-S and estrone sulfate (E(1)S) uptake by these transporters stably expressed in FlpIn -HEK293 cells using the Flp recombinase-mediated site-specific recombination. Uptake of E(1)S by OAT4- and OATP2B1-transfected cells was highly increased compared to the non-transfected cells. In contrast, DHEA-S uptake was only highly increased in OAT4 (40 times), but only weakly enhanced in OATP2B1 cells. The uptake of DHEA-S and E(1)S by OAT4 was partly Na(+)-dependent (about 50%), whereas uptake of DHEA-S by OATP2B1 was Na(+)-independent. Kinetic analysis of the initial uptake rates of E(1)S by OAT4 and OATP2B1 gave very similar values for K(m) (about 20microM) and V(max) (about 600pmol/(minxmg protein)). In contrast, the affinity of DHEA-S towards OATP2B1 was about 10 times lower (K(m)>200microM) then for OAT4 (K(m)=29microM). Our results suggest different physiological roles of the two transporter polypeptides in placental uptake of foetal derived steroid sulfates. OATP2B1 seems not to be involved in de novo synthesis of placental estrogens but may contribute to the clearance of estrogen sulfates from foetal circulation.

DHEAS repeated treatment improves cognitive and behavioral deficits after mild traumatic brain injury.

Mild traumatic brain injury (mTBI) is characterized by diffused symptoms, which when combined are called "post-concussion syndrome". Dehydroepiandrosterone sulfate (DHEAS) is a neuroactive neurosteroid. Previously, we have reported that closed head mTBI causes long lasting cognitive deficits and depressive-like behavior. In the present study we describe the effects of DHEAS on the behavior of mice that suffered closed head mTBI. Following the induction of mTBI, mice were treated once a week with DHEAS (s.c. 20 mg/kg) and their performance in the passive avoidance test and the forced swimming test (FST) were evaluated 7, 30, 60 and 90 days post-injury. The most important interactions were between injury and injection (passive avoidance; p<0.001 and FST; p=0.001), meaning that DHEAS has beneficial effects only when given to injured animals. Our results demonstrate that the long-term cognitive and behavioral effects induced by mTBI may be improved by a repeated weekly treatment with DHEAS.

The steroid sulfatase inhibitor COUMATE attenuates rather than enhances access of dehydroepiandrosterone sulfate to the brain in the mouse.

Intraperitoneal injection of adult male mice with the neuroactive steroid dehydroepiandrosterone sulfate (DHEAS) at 1 and 40 mg/kg caused dose-dependent increases in the concentration of both this compound and its corresponding free steroid DHEA in brain within 1 h of injection. Pretreatment of these animals for 24 h with the steroid sulfatase inhibitor COUMATE at a dose (10 mg/kg, p.o.) shown previously to cause almost complete inhibition of this enzyme in liver and brain was expected to increase the amount of the DHEAS dose reaching the brain. Surprisingly however, the increases in brain concentrations of DHEAS and DHEA after injection of DHEAS i.p. were attenuated by pretreatment with COUMATE. The results suggest that the arylsulfamate based steroid sulfatase inhibitors such as COUMATE interfere with the influx of the DHEAS anion into the brain.

Terapéutica en AP. El tratamiento con dehidroepiandrosterona de la depresión resistente a los antidepresivos / Therapy in primary health care. DHEA treatment of anti-depressantresistant depression

La dehidroepiandrosterona (DHEA) es una hormona involucrada en la ansiedad y la depresión. Sus niveles correlacionan positivamente con la resistencia a estas enfermedades. Sin embargo, en España no suele prestarse mucha atención a los niveles de DHEA, ni se complementa la carencia que se manifiesta paulatinamente desde la mediana edad. Los suplementos de DHEA no están autorizados por el Ministerio de Sanidad, con lo cual la importación ilegal con sus riesgos en cuanto a la calidad del producto se está abriendo camino. La evidencia no justifica esta prohibición y los estudios estadounidenses más recientes confirman su efectividad en depresión, tanto menor como mayor, especialmente en la depresión resistente a los antidepresivos, sobre todo en los adultos mayores. La determinación de niveles hormonales en saliva tiene claras ventajas clínicas pero en España sólo se practica en contados centros o laboratorios

Dehydroepiandrosterone (DHEA) is a hormone that is involved in stress and in depression. Its levels correlate positively with resistance to these disorders. However, in Spain not much attention is paid to DHEA levels nor is its deficiency that gradually appears in middle age compensated. DHEA supplements are not authorized for sale by the Ministry of Health. This induces illegal importation with the inherent risks as to the quality of the product. The available evidence does not justify this prohibition and recent U.S. studies have confirmed its efficacy in minor and major depression, especially in midlife-onset antidepressant resistant depression. The analysis of hormones in saliva has clear clinical advantages. However, only a few clinical centers or laboratories offer this in Spain

Modification of OATP2B1-mediated transport by steroid hormones.

The family of the organic anion transporting polypeptides forms an increasing group of uptake transport proteins with a wide substrate spectrum. Although the expression of some members of this group, such as organic anion transporting polypeptide (OATP)-A or C, is limited to special tissues (such as liver or brain), the organic anion transporting polypeptide 2B1 (OATPB/SLCO2B1) is expressed in many organs, including liver, placenta, mammary gland, brain, and intestine. However, little is known about its function in those tissues because only a limited number of compounds, such as dehydroepiandrosterone-sulfate (DHEAS) and estrone-3-sulfate (E3S), have been characterized as OATP2B1 substrates. To further elucidate the role of OATP2B1 on steroid transport, we examined the influence of steroid hormones on OATP2B1-mediated E3S and DHEAS uptake using OATP2B1-overexpressing Madin-Darby canine kidney II cells. We identified unconjugated androgens (e.g., testosterone) as potent inhibitors for OATP2B1. In contrast, gestagenes such as progesterone enhanced E3S uptake in a concentration-dependent manner to up to 300% of the control, accompanied by a significant decrease in the OATP2B1 K(m) value for E3S (control, K(m) = 14 microM; in the presence of 31.6 muM progesterone, K(m) = 3.6 microM). Moreover, we demonstrated that testosterone and progesterone are not substrates of OATP2B1, indicating an allosteric mechanism for the observed effects. Furthermore, we showed that progesterone enhances the OATP2B1-dependent pregnenolone sulfate transport. Taken together, the results indicate functional modification of OATP2B1-mediated E3S and DHEAS as well as pregnenolone sulfate transport through steroid hormones such as progesterone. These effects can have physiological consequences for the organ-specific uptake of steroids.

Localization of the sulfate/anion exchanger in the rat liver.

Although the sulfate/anion transporter (sat-1; SLC26A1) was isolated from a rat liver cDNA library by expression cloning, localization of sat-1 within the liver and its contribution to the transport of sulfate and organo sulfates have remained unresolved. In situ hybridization and immunohistochemical studies were undertaken to demonstrate the localization of sat-1 in liver tissue. RT-PCR studies on isolated hepatocytes and liver endothelial and stellate cells in culture were performed to test for the presence of sat-1 in these cells. In sulfate uptake and efflux experiments, the substrate specificity of sat-1 was evaluated. Sat-1 mRNA was found in hepatocytes and endothelial cells. Sat-1 protein was localized in sinusoidal membranes and along the borders of hepatocytes. The canalicular region and bile capillaries were not stained. Sulfate uptake was only slightly affected by sulfamoyl diuretics or organo sulfates. Sulfate efflux from sat-1-expressing oocytes was enhanced in the presence of bicarbonate, indicating sulfate/bicarbonate exchange. Estrone sulfate was not transported by sat-1. Sat-1 may be responsible for the uptake of inorganic sulfate from the blood into hepatocytes to enable sulfation reactions. In hepatocytes and endothelial cells, sat-1 may also supply sulfate for proteoglycan synthesis.

Investigation of efflux transport of dehydroepiandrosterone sulfate and mitoxantrone at the mouse blood-brain barrier: a minor role of breast cancer resistance protein.

Breast cancer resistance protein (Bcrp/Abcg2) is a new efflux transporter found at the blood-brain barrier (BBB) of humans and pigs. Since it has been hypothesized that Bcrp may act as a new type of efflux transporter at the BBB, we investigated the involvement of Bcrp in the efflux transport of typical substrates, dehydroepiandrosterone sulfate (DHEAS) and mitoxantrone, across the mouse BBB. The expression of Bcrp in mouse brain capillaries was confirmed by quantitative polymerase chain reaction, Western blot, and immunohistochemical analysis. The role of Bcrp as an efflux transporter was evaluated using the in situ brain perfusion method in wild-type and P-glycoprotein (P-gp) knockout mice with or without treatment with GF120918 (Elacridar), an inhibitor of both Bcrp and P-gp. The increased brain uptake of [(3)H]DHEAS and [(3)H]mitoxantrone by GF120918 in wild-type and P-gp knockout mice suggested the existence of a GF120918-sensitive and P-gp-independent efflux transporter for DHEAS and mitoxantrone across the BBB. However, the brain uptake of [(3)H]DHEAS in Bcrp knockout mice was comparable with that in wild-type mice, and the effect of GF120918 was still observed in Bcrp knockout mice. In addition, the brain uptake of [(3)H]mitoxantrone was also similar in wild-type and Bcrp knockout mice. These results suggest that although BCRP is expressed at the BBB it plays a minor role in active efflux transport of DHEAS and mitoxantrone out of brain and that one or more GF120918-sensitive efflux transporters distinct from BCRP or P-gp contributes to the brain efflux of DHEAS and mitoxantrone.

Searching for new markers of endogenous steroid administration in athletes: "looking outside the metabolic box".

A simple means of detecting the abuse of steroids that also occur naturally is a problem facing doping control laboratories. Specific markers are required to allow the detection of the administration of these steroids. These markers are commonly measured using a set of data obtained from the screening of samples by gas chromatography-mass spectrometry (GC-MS). Doping control laboratories further need to confirm identified abuse using techniques such as gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS). An interesting urinary species was found while following the pharmacokinetics and changes to the steroid profile from single and multiple oral doses of the International Olympic Committee/World Anti Doping Agency (IOC/WADA) prohibited substance, dehydroepiandrosterone (DHEA). The urine samples collected from the administration studies were subject to GC-MS and GC-C-IRMS steroid analysis following cleanup by solid phase extraction techniques. A useful urinary product of DHEA administration was detected in the urine samples from each of the administration studies and was identified by GC-MS experiments to be 3alpha,5-cyclo-5alpha-androstan-6beta-ol-17-one (3alpha,5-cyclo). This compound occurs naturally but the concentrations of 3alpha,5-cyclo were elevated following both the single DHEA administration (up to 385 ng/mL) and multiple DHEA administrations (up to 1240 ng/mL), in relation to those observed prior to these administrations (70 and 80 ng/mL, respectively). A reference distribution of urine samples collected from elite athletes (n = 632) enabled the natural concentration range of 3alpha,5-cyclo to be established (0-280 ng/mL), with a mean concentration of 22 ng/mL. Based on this an upper 3alpha,5-cyclo concentration limit of 140 ng/mL is proposed as a GC-MS screening marker of DHEA abuse in athletes. GC-C-IRMS analysis revealed significant 13C depletion of 3alpha,5-cyclo following DHEA administration. In the single administration study, the delta13C value of 3alpha,5-cyclo changed from -24.3 per thousand to a minimum value of -31.1 per thousand at 9 h post-administration, before returning to its original value after 48 h. The multiple administration study had a minimum delta13C 3alpha,5-cyclo of -33.9 per thousand during the administration phase in contrast to the initial value of -24.2 per thousand. Preliminary studies have shown 3alpha,5-cyclo to most likely be produced from DHEA sulfate found at high levels in urine. The complementary use of GC-MS and GC-C-IRMS to identify new markers of steroid abuse and the application of screening criteria incorporating such markers could also be adapted by doping control laboratories to detect metabolites of androstenedione, testosterone and dihydrotestosterone abuse.

Enhancement of dehydroepiandrosterone solubility and bioavailability by ternary complexation with alpha-cyclodextrin and glycine.

In the present work we investigated the possibility of improving dehydroepiandrosterone (DHEA) solubility and bioavailability by high-energy cogrinding with alpha-cyclodextrin (alpha-Cd) in the presence or absence of different auxiliary substances (glycine, biomaltodextrin, poly(vinyl pyrrolidone), poly(ethylene glycol) 400). In all cases, ternary products exhibited higher drug solubilizing properties than the binary DHEA-alpha-Cd coground system. Glycine was the most effective component. The best combinations, corresponding to 1:1:2 and 1:2:3 drug-alpha-Cd-glycine molar ratios, were characterized by differential scanning calorimetry and X-ray powder diffractometry and evaluated for dissolution rate. The presence of glycine favored destruction of DHEA crystalline structure during cogrinding, as evidenced by the strong reduction in both time and vibration frequency of milling necessary to obtain total drug amorphization. Both ternary products showed better dissolution properties than the drug alone, affording, respectively, a 40 and 60% increase of dissolution efficiency. The 1:2:3 coground product was then selected for in vivo bioavailability studies in women suffering from adrenocortical insufficiency. DHEA and DHEA sulfate blood levels were significantly higher (p < 0.001) after oral administration of the coground product than after oral administration of untreated drug, with a 100% increase in the area under the curve (AUC) of concentration versus time. Moreover, the time to reach maximum concentration (t(max)) decreased from 2.2 with the untreated drug to 0.5 h with the coground product, and the mean permanence time of DHEA within physiological levels was four times longer for the coground product compared with the untreated drug. These results indicate that the developed product is particularly suitable for oral DHEA formulations in hormone replacement therapies.

ABCG2 transports sulfated conjugates of steroids and xenobiotics.

The mechanism for the cellular extrusion of sulfated conjugates is still unknown. In the present study, we investigated whether human wild type ABCG2 transports estrone 3-sulfate (E1S) using membrane vesicles from cDNA-transfected mouse lymphoma cell line (P388 cells). The uptake of [3H]E1S into ABCG2-expressing membrane vesicles was stimulated by ATP, and the Km value for [3H]E1S was determined to be 16.6 microm. The ABCG2-mediated transport of [3H]E1S was potently inhibited by SN-38 and many sulfate conjugates but not by glucuronide and glutathione conjugates or other anionic compounds. Other sulfate conjugates such as [3H]dehydroepiandrosterone sulfate (DHEAS) and [35S]4-methylumbelliferone sulfate (Km = 12.9 microm) and [35S]6-hydroxy-5,7-dimethyl-2-methylamino-4-(3-pyridylmethyl)benzothiazole (E3040) sulfate (Km = 26.9 microm) were also transported by ABCG2. Although [3H]methotrexate, [3H]17beta-estradiol-17beta-D-glucuronide, [3H]2,4-dinitrophenyl-S-glutathione, and [14C]4-methylumbelliferone glucuronide were transported by ABCG2, this took place to a much lesser extent compared with [3H]E1S. It was suggested that ABCG2 preferentially transports sulfate conjugates and that E1S and DHEAS are the potential physiological substrates for this transporter.

Characterization and identification of steroid sulfate transporters of human placenta.

Human trophoblasts depend on the supply of external precursors, such as dehydroepiandrosterone-3-sulfate (DHEA-S) and 16 alpha-OH-DHEA-S, for synthesis of estrogens. The aim of the present study was to characterize the uptake of DHEA-S by isolated mononucleated trophoblasts (MT) and to identify the involved transporter polypeptides. The kinetic analysis of DHEA-(35)S uptake by MT revealed a saturable uptake mechanism (K(m) = 26 microM, V(max) = 428 pmol x mg protein(-1) x min(-1)), which was superimposed by a nonsaturable uptake mechanism (diffusion constant = 1.2 microl x mg protein(-1) x min(-1)). Uptake of [(3)H]DHEA-S by MT was Na(+) dependent and inhibited by sulfobromophthalein (BSP), steroid sulfates, and probenecid, but not by steroid glucuronides, unconjugated steroids, conjugated bile acids, ouabain, p-aminohippurate (PAH), and bumetanide. MT took up [(35)S]BSP, [(3)H]estrone-sulfate, but not (3)H-labeled ouabain, estradiol-17beta-glucuronide, taurocholate, and PAH. RT-PCR revealed that the organic anion-transporting polypeptides OATP-B, -D, -E, and the organic anion transporter OAT-4 are highly expressed, and that OATP-A, -C, -8, OAT-3, and Na(+)-taurocholate cotransporting polypeptide (NTCP) are not or are only lowly expressed in term placental tissue and freshly isolated and cultured trophoblasts. Immunohistochemistry of first- and third-trimester placenta detected OAT-4 on cytotrophoblast membranes and at the basal surface of the syncytiotrophoblast. Our results indicate that uptake of steroid sulfates by isolated MT is mediated by OATP-B and OAT-4 and suggest a physiological role of both carrier proteins in placental uptake of fetal-derived steroid sulfates.

Polarized glucose transporters and mRNA expression properties in newly developed rat syncytiotrophoblast cell lines, TR-TBTs.

In this study, we have established new syncytiotrophoblast cell lines (TR-TBTs) from the recently developed transgenic rat harboring temperature-sensitive simian virus 40 large T-antigen gene (Tg-rat). Four conditionally immortalized syncytiotrophoblast cell lines (TR-TBT 18d-1 approximately 4) were obtained from pregnant Tg-rats at gestational day 18. These cell lines had a syncytium-like morphology, could be prepared as monolayers, expressed cytokeratins and rat syncytiotrophoblast markers, and exhibited apical or basal GLUT1 localizations and apical GLUT3 localizations. TR-TBTs express large T-antigen and grow well at 33 degrees C with a doubling time of about 30 h. TR-TBTs have processes for the uptake of dehydroepiandrosteron-3-sulfate (DHEAS) and these are predominantly located on the basal side, and this is the first report of an in vitro model of blood placental barrier (BPB) able to incorporate DHEAS. Therefore, TR-TBTs are an appropriate in vitro model for investigating carrier-mediated transport functions at the BPB. Moreover, TR-TBTs express betaine/GABA transporter (GAT-2/BGT-1), concentrative nucleoside transporter 2 (CNT2), equilibrative nucleoside transporter 1 (ENT1), and ENT2 and the expression of these transporters has been reported in blood-brain barrier (BBB). Thus, the expression patterns of nucleoside and neurotransmitter transporters examined are quite similar in both the BPB and BBB.

Effects of oral prasterone (dehydroepiandrosterone) on single-dose pharmacokinetics of oral prednisone and cortisol suppression in normal women.

This study sought to determine effects of multiple dosing of prasterone (DHEA, dehydroepiandrosterone) on the pharmacokinetics of prednisolone and endogenous cortisol secretion. These drugs are likely to be coadministered to patients with systemic lupus erythematosus. Fourteen normal women (ages 30.1 +/- 5.4 years) received single-dose oral prednisone (20 mg) before and after 200 mg/day of oral prasterone for one menstrual cycle (approximately 28 days). Identical assessments, timed to onset of menses, were conducted pretreatment (baseline) and at days 28 and 29 of prasterone treatment and included serum total and free prednisolone, prednisone, DHEA, DHEA-S (dehydroepiandrosterone sulfate), ACTH-stimulated cortisol, and sex hormones and 24-hour urine free cortisol. Pharmacokinetic parameters of prednisolone as assessed by Cmax, t 1/2, AUC, or serum protein binding were not affected by prasterone. The ACTH-stimulated plasma cortisol concentrations were mildly reduced, but 24-hour urinefree cortisol excretion was unchanged during prasterone administration. Serum androstenedione and testosterone increased, while no changes in serum estradiol or estrone occurred. The administration of 200 mg oral prasterone produced serum concentrations of DHEA and DHEA-S significantly greater than endogenous levels. Chronic dosing with 200 mg/day of prasterone did not alter either prednisolone pharmacokinetics or inhibition of cortisol secretion by prednisolone.

Neurosteroids stimulate G protein-coupled sigma receptors in mouse brain synaptic membrane.

Dehydroepiandrosterone, its sulfate (DHEAS) and pregnenolone sulfate, representative neurosteroids as well as (+)-pentazocine concentration-dependently stimulated the [35S]GTPgammaS binding in synaptic membranes of mouse prefrontal cortex. These stimulations were blocked by NE-100, a sigma-receptor antagonist, and by progesterone, another type of neurosteroid. The DHEAS-induced stimulation was blocked by the pertussis toxin (PTX)-treatment, and completely recovered by reconstitution of PTX-treated membranes with recombinant G(i1), but not with G(oA). DHEAS also stimulated the [35S]GTPgammaS binding in the coronal sections of mouse brain in NE-100- or progesterone-reversible manner. These findings suggest that some neurosteroids may act on metabotropic sigma receptors, and this study may be the first to show the coupling of neurosteroid binding site and G(i).

Functional analysis and androgen-regulated expression of mouse organic anion transporting polypeptide 1 (Oatp1) in the kidney.

Mouse Oatp1 was recently identified as a new murine member of the organic anion transporting polypeptide (Oatp) family and suggested to represent the counterpart of rat Oatp1. Northern blot analysis detected expression of several mouse Oatp-transcripts predominantly in liver and kidney. In the present study we describe the strict androgen-dependent expression of mouse Oatp1 mRNA in kidney and obtained further information about its substrate specificity using Xenopus oocytes. In addition to the previously reported estrone-3-sulfate, we demonstrate that mouse Oatp1 mediates sodium-independent uptake of the anionic steroid conjugates dehydroepiandrosterone sulfate (K(m) approximately 8 microM) and estradiol-17-glucuronide (K(m) approximately 5 microM) and also of the prostaglandin PGE(2).

Steroid sulfatase in the human hair follicle concentrates in the dermal papilla.

5 alpha-dihydrotestosterone is known to play a crucial part in the regulation of hair growth and in the development of androgenetic alopecia. 5 alpha-dihydrotestosterone is formed locally within the hair follicle from the systemic precursor testosterone by cutaneous steroid 5 alpha-reductase. Moreover, adrenal steroids such as dehydroepiandrosterone are converted to 5 alpha-dihydrotestosterone by isolated hair follicles, which may provide an additional source of intrafollicular 5 alpha-dihydrotestosterone levels. Elevated urinary dehydroepiandrosterone and serum dehydroepiandrosterone sulfate have been reported to be present in balding young men. These reports suggest that dehydroepiandrosterone sulfate may act as an important endocrine factor in the development of androgenetic alopecia. Hence the question arises whether the dehydroepiandrosterone sulfate can be metabolized within the hair follicles to yield dehydroepiandrosterone by the microsomal enzyme steroid sulfatase, and where steroid sulfatase might be localized. We therefore performed immunostaining for steroid sulfatase on human scalp biopsies as well as analysis of steroid sulfatase enzyme activity in defined compartments of human beard and occipital hair follicles ex vivo. Using both methods steroid sulfatase was primarily detected in the dermal papilla. Steroid sulfatase activity was inhibited by estrone-3-O-sulfamate, a specific inhibitor of steroid sulfatase, in a concentration-dependent way. Furthermore, we show that dermal papillae are able to utilize dehydroepiandrosterone sulfate to produce 5 alpha-dihydrotestosterone, which lends further support to the hypothesis that dehydroepiandrosterone sulfate contributes to androgenetic alopecia and that steroid sulfatase inhibitors could be novel drugs to treat androgen-dependent disorders of the hair follicle such as androgenetic alopecia or hirsutism.