Topoisomerase inhibitors unsilence the dormant allele of Ube3a in neurons.

Angelman syndrome is a severe neurodevelopmental disorder caused by deletion or mutation of the maternal allele of the ubiquitin protein ligase E3A (UBE3A). In neurons, the paternal allele of UBE3A is intact but epigenetically silenced, raising the possibility that Angelman syndrome could be treated by activating this silenced allele to restore functional UBE3A protein. Using an unbiased, high-content screen in primary cortical neurons from mice, we identify twelve topoisomerase I inhibitors and four topoisomerase II inhibitors that unsilence the paternal Ube3a allele. These drugs included topotecan, irinotecan, etoposide and dexrazoxane (ICRF-187). At nanomolar concentrations, topotecan upregulated catalytically active UBE3A in neurons from maternal Ube3a-null mice. Topotecan concomitantly downregulated expression of the Ube3a antisense transcript that overlaps the paternal copy of Ube3a. These results indicate that topotecan unsilences Ube3a in cis by reducing transcription of an imprinted antisense RNA. When administered in vivo, topotecan unsilenced the paternal Ube3a allele in several regions of the nervous system, including neurons in the hippocampus, neocortex, striatum, cerebellum and spinal cord. Paternal expression of Ube3a remained elevated in a subset of spinal cord neurons for at least 12 weeks after cessation of topotecan treatment, indicating that transient topoisomerase inhibition can have enduring effects on gene expression. Although potential off-target effects remain to be investigated, our findings suggest a therapeutic strategy for reactivating the functional but dormant allele of Ube3a in patients with Angelman syndrome.

Pharmacokinetic Modeling of Lamivudine and Zidovudine Triphosphates Predicts Differential Pharmacokinetics in Seminal Mononuclear Cells and Peripheral Blood Mononuclear Cells.

The male genital tract is a potential site of viral persistence. Therefore, adequate concentrations of antiretrovirals are required to eliminate HIV replication in the genital tract. Despite higher zidovudine (ZDV) and lamivudine (3TC) concentrations in seminal plasma (SP) than in blood plasma (BP) (SP/BP drug concentration ratios of 2.3 and 6.7, respectively), we have previously reported lower relative intracellular concentrations of their active metabolites, zidovudine triphosphate (ZDV-TP) and lamivudine triphosphate (3TC-TP), in seminal mononuclear cells (SMCs) than in peripheral blood mononuclear cells (PBMCs) (SMC/PBMC drug concentration ratios of 0.36 and 1.0, respectively). Here, we use population pharmacokinetic (PK) modeling-based methods to simultaneously describe parent and intracellular metabolite PK in blood, semen, and PBMCs and SMCs. From this model, the time to steady state in each matrix was estimated, and the results indicate that the PK of 3TC-TP and ZDV-TP in PBMCs are different from the PK of the two in SMCs and different for the two triphosphates. We found that steady-state conditions in PBMCs were achieved within 2 days for ZDV-TP and 3 days for 3TC-TP. However, steady-state conditions in SMCs were achieved within 2 days for ZDV-TP and 2 weeks for 3TC-TP. Despite this, or perhaps because of it, ZDV-TP in SMCs does not achieve the surrogate 50% inhibitory concentration (IC50) (as established for PBMCs, assuming SMC IC50 = PBMC IC50) at the standard 300-mg twice-daily dosing. Mechanistic studies are needed to understand these differences and to explore intracellular metabolite behavior in SMCs for other nucleoside analogues used in HIV prevention, treatment, and cure.

Role of multidrug resistance-associated protein 4 in the basolateral efflux of hepatically derived enalaprilat.

Hepatic uptake and efflux transporters govern the systemic and hepatic exposure of many drugs and metabolites. Enalapril is a pharmacologically inactive prodrug of enalaprilat. Following oral administration, enalapril is converted to enalaprilat in hepatocytes and undergoes translocation into the systemic circulation to exert its pharmacologic effect by inhibiting angiotensin-converting enzyme. Although the transport proteins governing hepatic uptake of enalapril and the biliary excretion of enalapril and enalaprilat are well established, it remains unknown how hepatically derived enalaprilat translocates across the basolateral membrane into the systemic circulation. In this study, the role of ATP-binding cassette transporters in the hepatic basolateral efflux of enalaprilat was investigated using membrane vesicles. ATP-dependent uptake of enalaprilat into vesicles expressing multidrug resistance-associated protein (MRP) 4 was significantly greater (∼3.8-fold) than in control vesicles. In contrast, enalaprilat was not transported to a significant extent by MRP3, and enalapril was not transported by either MRP3 or MRP4. The functional importance of MRP4 in the basolateral excretion of derived enalaprilat was evaluated using a novel basolateral efflux protocol developed in human sandwich-cultured hepatocytes. Under normal culture conditions, the mean intrinsic basolateral efflux clearance (CLint ,basolateral) of enalaprilat was 0.026 ± 0.012 µl/min; enalaprilat CLint,basolateral was significantly reduced to 0.009 ± 0.009 µl/min by pretreatment with the pan-MRP inhibitor MK-571. Results suggest that hepatically derived enalaprilat is excreted across the hepatic basolateral membrane by MRP4. Changes in MRP4-mediated basolateral efflux may alter the systemic concentrations of this active metabolite, and potentially the efficacy of enalapril.

Hepatic basolateral efflux contributes significantly to rosuvastatin disposition II: characterization of hepatic elimination by basolateral, biliary, and metabolic clearance pathways in rat isolated perfused liver.

Basolateral efflux clearance (CLBL) contributes significantly to rosuvastatin (RSV) elimination in sandwich-cultured hepatocytes (SCH). The contribution of CLBL to RSV hepatic elimination was determined in single-pass isolated perfused livers (IPLs) from wild-type (WT) and multidrug resistance-associated protein 2 (Mrp2)-deficient (TR(-)) rats in the absence and presence of the P-glycoprotein and breast cancer resistance protein (Bcrp) inhibitor, elacridar (GF120918); clearance values were compared with SCH. RSV biliary clearance (CLBile) was ablated almost completely by GF120918 in TR(-) IPLs, confirming that Mrp2 and Bcrp primarily are responsible for RSV CLBile. RSV appearance in outflow perfusate was attributed primarily to CLBL, which was impaired in TR(-) IPLs. CLBL was ≈ 6-fold greater than CLBile in the linear range in WT IPLs in the absence of GF120918. Recovery of unchanged RSV in liver tissue increased in TR(-) compared with WT (≈ 25 versus 6% of the administered dose) due to impaired CLBL and CLBile. RSV pentanoic acid, identified by high-resolution liquid chromatography-tandem mass spectroscopy, comprised ≈ 40% of total liver content and ≈ 16% of the administered dose in TR(-) livers at the end of perfusion, compared with ≈ 30 and 3% in WT livers, consistent with impaired RSV excretion and "shunting" to the metabolic pathway. In vitro-ex vivo extrapolation between WT SCH and IPLs (without GF120918) revealed that uptake clearance and CLBL were 4.2- and 6.4-fold lower, respectively, in rat SCH compared with IPLs; CLBile translated almost directly (1.1-fold). The present IPL data confirmed the significant role of CLBL in RSV hepatic elimination, and demonstrated that both CLBL and CLBile influence RSV hepatic and systemic exposure.

Intestinal first-pass metabolism by cytochrome p450 and not p-glycoprotein is the major barrier to amprenavir absorption.

Recent studies showed that P-glycoprotein (P-gp) increases the portal bioavailability (FG) of loperamide by sparing its intestinal first-pass metabolism. Loperamide is a drug whose oral absorption is strongly attenuated by intestinal P-gp-mediated efflux and first-pass metabolism by cytochrome P450 3A (CYP3A). Here the effect of the interplay of P-gp and Cyp3a in modulating intestinal first-pass metabolism and absorption was investigated for another Cyp3a/P-gp dual substrate amprenavir, which is less efficiently effluxed by P-gp than loperamide. After oral administration of amprenavir, the portal concentrations and FG of amprenavir were approximately equal in P-gp competent and P-gp deficient mice. Mechanistic studies on the effect of P-gp on Cyp3a-mediated metabolism of amprenavir using intestinal tissue from P-gp competent and P-gp deficient mice (Ussing-type diffusion chamber) revealed that P-gp-mediated efflux caused only a slight reduction of oxidative metabolism of amprenavir. Studies in which portal concentrations and FG were measured in P-gp competent and P-gp deficient mice whose cytochrome P450 (P450) enzymes were either intact or inactivated showed that intestinal first-pass metabolism attenuates the oral absorption of amprenavir by approximately 10-fold, whereas P-gp efflux has a relatively small effect (approximately 2-fold) in attenuating the intestinal absorption. Cumulatively, these studies demonstrate that P-gp has little influence on the intestinal first-pass metabolism and FG of amprenavir and that intestinal P450-mediated metabolism plays the dominant role in attenuating the oral absorption of this drug.

P-glycoprotein increases portal bioavailability of loperamide in mouse by reducing first-pass intestinal metabolism.

P-glycoprotein (P-gp) and CYP3A (cytochrome P450 3A, generally; Cyp3a, rodent enzyme) in the intestine can attenuate absorption of orally administered drugs. While some suggest that P-gp enhances intestinal metabolism by CYP3A/Cyp3a during absorption of a dual substrate, others suggest that P-gp reduces the metabolism in the intestine when substrates are at subsaturating concentrations. Hence, to elucidate the cellular mechanisms that can address these divergent reports, we studied intestinal absorption of the dual substrate loperamide in portal vein-cannulated P-gp-competent and P-gp-deficient mice. These studies showed that at low doses of loperamide, which produced intestinal concentrations near the apparent K(m) for oxidative metabolism, the bioavailability across the intestine (F(G)) was 6-fold greater in the P-gp-competent mice than in P-gp-deficient mice. The higher F(G) of loperamide in the presence of P-gp was attributed to lower loperamide intestinal metabolism. However, at high doses of loperamide, the sparing of first-pass metabolism by P-gp was balanced against the attenuation of absorption by apical efflux, resulting in no net effect on F(G). In vitro studies with intestinal tissue from P-gp-competent and -deficient mice confirmed that P-gp reduced the metabolic rate of loperamide during absorptive flux at concentrations near K(m) but had little effect on metabolism at higher (saturating) concentrations. Further, studies in which Cyp3a was chemically inactivated by aminobenzotriazole in P-gp-competent and -deficient mice, showed that P-gp and Cyp3a individually attenuated F(G) by 8-fold and 70-fold, respectively. These results confirmed that P-gp effectively protects loperamide at low doses from intestinal first-pass metabolism during intestinal absorption.

Compartmental and enzyme kinetic modeling to elucidate the biotransformation pathway of a centrally acting antitrypanosomal prodrug.

DB868 [2,5-bis [5-(N-methoxyamidino)-2-pyridyl] furan], a prodrug of the diamidine DB829 [2,5-bis(5-amidino-2-pyridyl) furan], has demonstrated efficacy in murine models of human African trypanosomiasis. A cross-species evaluation of prodrug bioconversion to the active drug is required to predict the disposition of prodrug, metabolites, and active drug in humans. The phase I biotransformation of DB868 was elucidated using liver microsomes and sandwich-cultured hepatocytes from humans and rats. All systems produced four NADPH-dependent metabolites via O-demethylation (M1, M2) and N-dehydroxylation (M3, M4). Compartmental kinetic modeling of the DB868 metabolic pathway suggested an unusual N-demethoxylation reaction that was supported experimentally. A unienzyme Michaelis-Menten model described the kinetics of M1 formation by human liver microsomes (HLMs) (K(m), 11 µM; V(max), 340 pmol/min/mg), whereas a two-enzyme model described the kinetics of M1 formation by rat liver microsomes (RLMs) (K(m1), 0.5 µM; V(max1), 12 pmol/min/mg; K(m2), 27 µM; V(max2), 70 pmol/min/mg). Human recombinant CYP1A2, CYP3A4, and CYP4F2, rat recombinant Cyp1a2 and Cyp2d2, and rat purified Cyp4f1 catalyzed M1 formation. M2 formation by HLMs exhibited allosteric kinetics (S(50), 18 µM; V(max), 180 pmol/mg), whereas M2 formation by RLMs was negligible. Recombinant CYP1A2/Cyp1a2 catalyzed M2 formation. DB829 was detected in trace amounts in HLMs at the end of the 180-min incubation and was detected readily in sandwich-cultured hepatocytes from both species throughout the 24-h incubation. These studies demonstrated that DB868 biotransformation to DB829 is conserved between humans and rats. An improved understanding of species differences in the kinetics of DB829 formation would facilitate preclinical development of a promising antitrypanosomal prodrug.

Valproic acid reduces the tolerability of temsirolimus in children and adolescents with solid tumors.

A pediatric study has established a maximum tolerated dose (MTD) for temsirolimus (Tem) of more than 150 mg/m intravenously/week. A phase I trial was conducted to establish the MTD for Tem in combination with valproic acid (VPA) in children and adolescents with refractory solid tumors. The secondary aims included expression of mammalian target of rapamycin (mTOR) markers on archival tumor tissue; Tem pharmacokinetics; assessment of histone acetylation (HA); and tumor response. Patients were treated with VPA (5 mg/kg orally three times daily) with a target serum level of 75-100 mcg/ml. Tem was started at an initial dose of 60 mg/m/week. Pharmacokinetics and HA measurements were performed during weeks 1 and 5. Two of the first three patients experienced dose-limiting toxicity (grade 3 mucositis). Tem at 35 mg/m/week was found to be tolerable. Peak Tem concentrations were higher in all patients compared with those in previously published reports of single agent Tem. Increases in HA are correlated with VPA levels. All tumor samples expressed mTORC1 and mTORC2. An objective response was observed in one patient (melanoma), whereas transient stable disease was observed in four other patients (spinal cord ependymoma, alveolar soft part sarcoma, medullary thyroid carcinoma, and hepatocellular carcinoma). The MTD of Tem when administered with VPA is considerably lower than when used as a single agent, with mucositis the major dose-limiting toxicity. The combination merits further study and may have activity in melanoma. Attention to drug-drug interactions will be important in future multiagent trials including Tem.

Plasma, tumor and tissue pharmacokinetics of Docetaxel delivered via nanoparticles of different sizes and shapes in mice bearing SKOV-3 human ovarian carcinoma xenograft.

The particle fabrication technique PRINT® was used to fabricate monodisperse size and shape specific poly(lactide-co-glycolide) particles loaded with the chemotherapeutic Docetaxel. The pharmacokinetics of two cylindrical shaped particles with diameter=80nm; height=320nm (PRINT-Doc-80×320) and d=200nm; h=200nm (PRINT-Doc-200×200) were compared to Docetaxel in mice bearing human ovarian carcinoma SKOV-3 flank xenografts. The Docetaxel plasma exposure was ~20-fold higher for both particles compared to docetaxel. Additionally, the volume of distribution (Vd) of Docetaxel in PRINT formulations was ~18-fold (PRINT-Doc-80×320) and ~33-fold (PRINT-Doc-200×200) lower than Docetaxel. The prolonged duration of Docetaxel in plasma when dosed with PRINT formulations subsequently led to increased tumor exposure of Docetaxel from 0 to 168h (~53% higher for PRINT-Doc-80×320 and ~76% higher for PRINT-Doc-200×200 particles). PRINT-Doc-80×320 had lower exposures in the liver, spleen and lung compared with PRINT-Doc-200×200. Thus, the use of particles with smaller feature size may be preferred to decrease clearance by organs of the mononuclear phagocyte system. FROM THE CLINICAL EDITOR: In this study, the plasma, tumor, and tissue pharmacokinetics of different Docetaxel nanoparticles of precise shape and size were characterized in mice with human ovarian carcinoma xenograft. It is concluded that the use of particles with smaller feature size may be preferred to decrease clearance by organs of the mononuclear phagocyte system.

Directing the biological activities of heparan sulfate oligosaccharides using a chemoenzymatic approach.

Heparan sulfate (HS) and heparin are highly sulfated polysaccharides exhibiting essential physiological functions. The sulfation patterns determine the functional selectivity for HS and heparin. Chemical synthesis of HS, especially those larger than a hexasaccharide, remains challenging. Enzymatic synthesis of HS has recently gained momentum. Here we describe the divergent assembly of HS heptasaccharides and nonasaccharides from a common hexasaccharide precursor. The hexasaccharide precursor was synthesized via a chemical method. The subsequent elongation, sulfation and epimerization were completed by glycosyltransferases, HS sulfotransferases and epimerase. Using the synthesized heptasaccharides, we discovered that the iduronic acid is critical for binding to fibroblast growth factor-2. We also designed a synthetic path to prepare a nonasaccharide with an antithrombin-binding affinity of 3 nM. Our method demonstrated the feasibility of combining chemical and enzymatic synthesis to prepare structurally defined HS oligosaccharides with desired biological activities.

Impact of organic solvents on cytochrome P450 probe reactions: filling the gap with (S)-Warfarin and midazolam hydroxylation.

(S)-Warfarin 7-hydroxylation and midazolam 1'-hydroxylation are among the preferred probe substrate reactions for CYP2C9 and CYP3A4/5, respectively. The impact of solvents on enzyme activity, kinetic parameters, and predicted in vivo hepatic clearance (Cl(H)) associated with each reaction has not been evaluated. The effects of increasing concentrations [0.1-2% (v/v)] of six organic solvents (acetonitrile, methanol, ethanol, dimethyl sulfoxide, acetone, isopropanol) were first tested on each reaction using human liver microsomes (HLMs), human intestinal microsomes (midazolam 1'-hydroxylation only), and recombinant enzymes. Across enzyme sources, relative to water, acetonitrile and methanol had the least inhibitory effect on (S)-warfarin 7-hydroxylation (0-58 and 9-96%, respectively); acetonitrile, methanol, and ethanol had the least inhibitory effect on midazolam 1'-hydroxylation (0-29, 0-22, and 0-20%, respectively). Using HLMs, both acetonitrile and methanol (0.1-2%) decreased the V(max) (32-60 and 24-65%, respectively) whereas methanol (2%) increased the K(m) (100%) of (S)-warfarin-hydroxylation. (S)-Warfarin Cl(H) was underpredicted by 21-65% (acetonitrile) and 13-84% (methanol). Acetonitrile, methanol, and ethanol had minimal to modest impact on both the kinetics of midazolam 1'-hydroxylation (10-24%) and predicted midazolam Cl(H) (2-20%). In conclusion, either acetonitrile or methanol at ≤0.1% is recommended as the primary organic solvent for the (S)-warfarin 7-hydroxylation reaction; acetonitrile is preferred if higher solvent concentrations are required. Acetonitrile, methanol, and ethanol at ≤2% are recommended as primary organic solvents for the midazolam 1'-hydroxylation reaction. This information should facilitate optimization of experimental conditions and improve the interpretation and accuracy of in vitro-in vivo predictions involving these two preferred cytochrome P450 probe substrate reactions.

Pilot study of rosiglitazone as an in vivo probe of paclitaxel exposure.

WHAT IS ALREADY KNOWN ABOUT THIS SUBJECT: • Paclitaxel and rosiglitazone are primarily metabolized by CYP2C8 and their in vitro metabolism by human liver microsomes is correlated. Probe assays that quantify the in vivo activity of CYP enzymes which are important in drug metabolism have been developed for use in clinical pharmacology research. A probe of CYP2C8 that is easy to administer and interpret may be valuable for individualized dosing of paclitaxel. WHAT THIS STUDY ADDS: • This pilot study demonstrates for the first time that there is an in vivo correlation between paclitaxel and rosiglitazone exposure. The finding, that a single rosiglitazone plasma concentration after oral dosing may explain significant variance in paclitaxel exposure, suggests that rosiglitazone may satisfy the requirements of a clinically useful in vivo probe. However, it is acknowledged that there is a need for further studies evaluating the use of rosiglitazone as a CYP2C8 probe and quantifying the relationship, in order to guide dosing of narrow therapeutic index drugs metabolized primarily by CYP2C8, such as paclitaxel. AIMS: To evaluate the use of rosiglitazone and the erythromycin breath test (ERMBT), as probes of CYP2C8 and CYP3A4, respectively, to explain inter-individual variability in paclitaxel exposure. METHODS: The concentration of rosiglitazone at 3 h and ERMBT results were included in a regression model to explain the variability in paclitaxel exposure in 14 subjects. RESULTS: Rosiglitazone concentration was significantly correlated with paclitaxel exposure (P= 0.018) while ERMBT had no predictive value (P= 0.47). CONCLUSIONS: The correlation between the exposure of rosiglitazone and paclitaxel likely reflects mutual dependence on the activity of CYP2C8. Rosiglitazone or similar agents may have value as in vivo probes of CYP2C8 activity.

Cyclophosphamide and 4-hydroxycyclophosphamide pharmacokinetics in patients with glomerulonephritis secondary to lupus and small vessel vasculitis.

AIMS: Cyclophosphamide, the precursor to the active 4-hydroxycyclophosphamide, is used in active glomerulonephritis despite limited pharmacokinetics data. The pharmacokinetics of cyclophosphamide and 4-hydroxycyclophosphamide were evaluated. The influence of laboratory and pharmacogenomic covariates on pharmacokinetics was evaluated as a secondary aim. METHODS: Glomerulonephritis patients (n = 23) participated in a pharmacokinetic evaluation. Blood was serially collected and assayed for cyclophosphamide and 4-hydroxycyclophosphamide by LC/MS methods. Kidney function, serum albumin and polymorphisms in drug metabolism or transport genes were evaluated. Analyses included non-compartmental pharmacokinetics and parametric and non-parametric statistics. RESULTS: The mean area under the plasma concentration-time curve (AUC(0,∞)) data were 110,100 ± 42,900 ng ml(-1) h and 5388 ± 2841 ng ml(-1) h for cyclophosphamide and 4-hydroxycyclophosphamide, respectively. The mean metabolic ratio was 0.06 ± 0.04. A statistically significant relationship was found between increased serum albumin and increased half-life (0.584, P = 0.007, 95% CI 0.176, 0.820) and a borderline relationship with AUC(0,∞) (0.402, P = 0.079, 95% CI -0.064, 0.724) for 4-hydroxycyclophosphamide. Covariate relationships that trended toward significance for cyclophosphamide included decreased serum albumin and increased elimination rate constant (-0.427, P = 0.061, 95% CI 0.738, 0.034), increased urinary protein excretion and increased AUC(0,∞) (-0.392, P = 0.064, 95% CI -0.699 to 0.037), decreased C(max) (0.367, P = 0.085, 95% CI -0.067, 0.684) and decreased plasma clearance (-0.392, P = 0.064, 95% CI -0.699, 0.037). CYP2B6*9 variants vs. wildtype were found to have decreased elimination rate constant (P = 0.0005, 95% CI 0.033, 0.103), increased V(d) (P = 0.0271, 95% CI -57.5, -4.2) and decreased C(max) (P = 0.0176, 95% CI 0.696, 6179) for cyclophosphamide. ABCB1 C3435T variants had a borderline decrease in cyclophosphamide elimination rate constant (P = 0.0858; 95% CI -0.005, 0.102). CONCLUSIONS: Pharmacokinetics of cyclophosphamide and 4-hydroxycyclophosphamide in patients with lupus nephritis and small vessel vasculitis are similar. Clinical and pharmacogenetic covariates alter disposition of cyclophosphamide and 4-hydroxycyclophosphamide. Clinical findings of worsened glomerulonephritis lead to increased exposure to cyclophosphamide vs. the active 4-hydroxycyclophosphamide, which could have relevance in terms of clinical efficacy. The CYP2B6*9 and ABCB1 C3435T polymorphisms alter the pharmacokinetics of cyclophosphamide and 4-hydroxycyclophosphamide in glomerulonephritis.

Chemoenzymatic design of heparan sulfate oligosaccharides.

Heparan sulfate is a sulfated glycan that exhibits essential physiological functions. Interrogation of the specificity of heparan sulfate-mediated activities demands a library of structurally defined oligosaccharides. Chemical synthesis of large heparan sulfate oligosaccharides remains challenging. We report the synthesis of oligosaccharides with different sulfation patterns and sizes from a disaccharide building block using glycosyltransferases, heparan sulfate C(5)-epimerase, and sulfotransferases. This method offers a generic approach to prepare heparan sulfate oligosaccharides possessing predictable structures.

Use of sandwich-cultured human hepatocytes to predict biliary clearance of angiotensin II receptor blockers and HMG-CoA reductase inhibitors.

Previous reports have indicated that in vitro biliary clearance (Cl(biliary)) determined in sandwich-cultured hepatocytes correlates well with in vivo Cl(biliary) for limited sets of compounds. The purpose of this study was 1) to determine the in vitro Cl(biliary) in sandwich-cultured human hepatocytes of angiotensin II receptor blockers and HMG-CoA reductase inhibitors that undergo limited metabolism and 2) to compare the predicted Cl(biliary) values with estimated in vivo hepatic clearance data in humans. The average biliary excretion index and in vitro intrinsic Cl(biliary) values of olmesartan, valsartan, pravastatin, rosuvastatin, and pitavastatin in sandwich-cultured human hepatocytes were 35, 23, 31, 25, and 16%, respectively, and 0.943, 1.20, 0.484, 3.39, and 5.48 ml/min/kg, respectively. Cl(biliary) values predicted from sandwich-cultured human hepatocytes correlated with estimated in vivo hepatic clearance values based on published data (no in vivo data in humans was available for pitavastatin), and the rank order was also consistent. In conclusion, in vitro Cl(biliary) determined in sandwich-cultured human hepatocytes can be used to predict in vivo Cl(biliary) of compounds in humans.

Fexofenadine brain exposure and the influence of blood-brain barrier P-glycoprotein after fexofenadine and terfenadine administration.

P-glycoprotein (P-gp) plays an important role in determining net brain uptake of fexofenadine. Initial in vivo experiments with 24-h subcutaneous osmotic minipump administration demonstrated that fexofenadine brain penetration was 48-fold higher in mdr1a(-/-) mice than in mdr1a(+/+) mice. In contrast, the P-gp efflux ratio at the blood-brain barrier (BBB) for fexofenadine was only approximately 4 using an in situ brain perfusion technique. Pharmacokinetic modeling based on the experimental results indicated that the apparent fexofenadine P-gp efflux ratio is time-dependent due to low passive permeability at the BBB. Fexofenadine brain penetration after terfenadine administration was approximately 25- to 27-fold higher than after fexofenadine administration in both mdr1a(+/+) and mdr1a(-/-) mice, consistent with terfenadine metabolism to fexofenadine in murine brain tissue. The fexofenadine formation rate after terfenadine in situ brain perfusion was comparable with that in a 2-h brain tissue homogenate in vitro incubation. The fexofenadine formation rate increased approximately 5-fold during a 2-h brain tissue homogenate incubation with hydroxyl-terfenadine, suggesting that the hydroxylation of terfenadine is the rate-limiting step in fexofenadine formation. Moreover, regional brain metabolism seems to be an important factor in terfenadine brain disposition and, consequently, fexofenadine brain exposure. Taken together, these results indicate that the fexofenadine BBB P-gp efflux ratio has been underestimated previously due to the lack of complete equilibration of fexofenadine across the blood-brain interface under typical experimental paradigms.

Breast cancer resistance protein interacts with various compounds in vitro, but plays a minor role in substrate efflux at the blood-brain barrier.

Expression of breast cancer resistance protein (Bcrp) at the blood-brain barrier (BBB) has been revealed recently. To investigate comprehensively the potential role of Bcrp at the murine BBB, a chemically diverse set of model compounds (cimetidine, alfuzosin, dipyridamole, and LY2228820) was evaluated using a multiexperimental design. Bcrp1 stably transfected MDCKII cell monolayer transport studies demonstrated that each compound had affinity for Bcrp and that polarized transport by Bcrp was abolished completely by the Bcrp inhibitor chrysin. However, none of the compounds differed in brain uptake between Bcrp wild-type and knockout mice under either an in situ brain perfusion or a 24-h subcutaneous osmotic minipump continuous infusion experimental paradigm. In addition, alfuzosin and dipyridamole were shown to undergo transport by P-glycoprotein (P-gp) in an MDCKII-MDR1 cell monolayer model. Alfuzosin brain uptake was 4-fold higher in mdr1a(-/-) mice than in mdr1a(+/+) mice in in situ and in vivo studies, demonstrating for the first time that it undergoes P-gp-mediated efflux at the BBB. In contrast, P-gp had no effect on dipyridamole brain penetration in situ or in vivo. In fact, in situ BBB permeability of these solutes appeared to be primarily dependent on their lipophilicity in the absence of efflux transport, and in situ brain uptake clearance correlated with the intrinsic transcellular passive permeability from in vitro transport and cellular accumulation studies. In summary, Bcrp mediates in vitro transport of various compounds, but seems to play a minimal role at the BBB in vivo.

Sex-dependent disposition of acetaminophen sulfate and glucuronide in the in situ perfused mouse liver.

Breast cancer resistance protein (BCRP, ABCG2) is expressed in the hepatic canalicular membrane and mediates biliary excretion of xenobiotics including sulfate and glucuronide metabolites of some compounds. Hepatic Bcrp expression is sex-dependent, with higher expression in male mice. The hypothesis that sex-dependent Bcrp expression influences the hepatobiliary disposition of phase II metabolites was tested in the present study using acetaminophen (APAP) and the generated APAP glucuronide (AG) and sulfate (AS) metabolites in single-pass in situ perfused livers from male and female wild-type and Abcg(-/-) (Bcrp-deficient) mice. Pharmacokinetic modeling was used to estimate parameters governing the hepatobiliary disposition of APAP, AG, and AS. In wild-type mice, the biliary excretion rate constant was 2.5- and 7-fold higher in males than in females for AS and AG, respectively, reflecting male-predominant Bcrp expression. Sex-dependent differences in AG biliary excretion were not observed in Bcrp-deficient mice, and AS biliary excretion was negligible. Interestingly, sex-dependent basolateral excretion of AG (higher in males) and AS (higher in females) was noted in wild-type mice with a similar trend in Bcrp-deficient mouse livers, reflecting an increased rate constant for AG formation in male and AS formation in female mouse livers. In addition, the rate constant for AS basolateral excretion was increased significantly in female mouse livers compared with that in male mouse livers. It is interesting to note that multidrug resistance-associated protein 4 was higher in female than in male mouse livers. In conclusion, sex-dependent differences in conjugation and transporter expression result in profound differences in the hepatobiliary disposition of AG and AS in male and female mouse livers.

A gel-free MS-based quantitative proteomic approach accurately measures cytochrome P450 protein concentrations in human liver microsomes.

The human cytochrome P450 (P450) superfamily consists of membrane-bound proteins that metabolize a myriad of xenobiotics and endogenous compounds. Quantification of P450 expression in various tissues under normal and induced conditions has an important role in drug safety and efficacy. Conventional immunoquantification methods have poor dynamic range, low throughput, and a limited number of specific antibodies. Recent advances in MS-based quantitative proteomics enable absolute protein quantification in a complex biological mixture. We have developed a gel-free MS-based protein quantification strategy to quantify CYP3A enzymes in human liver microsomes (HLM). Recombinant protein-derived proteotypic peptides and synthetic stable isotope-labeled proteotypic peptides were used as calibration standards and internal standards, respectively. The lower limit of quantification was approximately 20 fmol P450. In two separate panels of HLM examined (n = 11 and n = 22), CYP3A, CYP3A4 and CYP3A5 concentrations were determined reproducibly (CV or=0.87) and marker activities (r(2)>or=0.88), including testosterone 6beta-hydroxylation (CYP3A), midazolam 1'-hydroxylation (CYP3A), itraconazole 6-hydroxylation (CYP3A4) and CYP3A5-mediated vincristine M1 formation (CYP3A5). Taken together, our MS-based method provides a specific, sensitive and reliable means of P450 protein quantification and should facilitate P450 characterization during drug development, especially when specific substrates and/or antibodies are unavailable.

In vitro biliary clearance of angiotensin II receptor blockers and 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors in sandwich-cultured rat hepatocytes: comparison with in vivo biliary clearance.

Previous reports have indicated that in vitro biliary clearance (Cl(biliary)) determined in sandwich-cultured hepatocytes correlates well with in vivo Cl(biliary) for limited sets of compounds. This study was designed to estimate the in vitro Cl(biliary) in sandwich-cultured rat hepatocytes (SCRHs) of angiotensin II receptor blockers and HMG-CoA reductase inhibitors that undergo limited metabolism, to compare the estimated Cl(biliary) values with published in vivo Cl(biliary) data in rats, and to characterize the mechanism(s) of basolateral uptake and canalicular excretion of these drugs in rats. The average biliary excretion index (BEI) and in vitro Cl(biliary) values of olmesartan, valsartan, pravastatin, rosuvastatin, and pitavastatin were 15, 19, 43, 45, and 20%, respectively, and 1.7, 3.2, 4.4, 46.1, and 34.6 ml/min/kg, respectively. Cl(biliary) predicted from SCRHs, accounting for plasma unbound fraction, correlated with reported in vivo Cl(biliary) for these drugs. The rank order of Cl(biliary) values predicted from SCRHs was consistent with in vivo Cl(biliary) values. Bromosulfophthalein inhibited the uptake of all drugs. BEI and Cl(biliary) values of olmesartan, valsartan, pravastatin, and rosuvastatin, known multidrug resistance-associated protein (Mrp) 2 substrates, were reduced in SCRHs from Mrp2-deficient (TR(-)) compared with wild-type (WT) rats. Although Mrp2 plays a minor role in pitavastatin biliary excretion, pitavastatin BEI and Cl(biliary) were reduced in TR(-) compared with WT SCRHs; Bcrp expression in SCRHs from TR(-) rats was decreased. In conclusion, in vitro Cl(biliary) determined in SCRHs can be used to estimate and compare in vivo Cl(biliary) of compounds in rats and to characterize transport proteins responsible for their hepatic uptake and excretion.