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.

Chemotherapy of second stage human African trypanosomiasis: comparison between the parenteral diamidine DB829 and its oral prodrug DB868 in vervet monkeys.

Human African trypanosomiasis (HAT, sleeping sickness) ranks among the most neglected tropical diseases based on limited availability of drugs that are safe and efficacious, particularly against the second stage (central nervous system [CNS]) of infection. In response to this largely unmet need for new treatments, the Consortium for Parasitic Drug Development developed novel parenteral diamidines and corresponding oral prodrugs that have shown cure of a murine model of second stage HAT. As a rationale for selection of one of these compounds for further development, the pharmacokinetics and efficacy of intramuscular (IM) active diamidine 2,5-bis(5-amidino-2-pyridyl)furan (DB829; CPD-0802) and oral prodrug2,5-bis[5-(N-methoxyamidino)-2-pyridyl]furan (DB868) were compared in the vervet monkey model of second stage HAT. Treatment was initiated 28 days post-infection of monkeys with T. b. rhodesiense KETRI 2537. Results showed that IM DB829 at 5 mg/kg/day for 5 consecutive days, 5 mg/kg/day every other day for 5 doses, or 2.5 mg/kg/day for 5 consecutive days cured all monkeys (5/5). Oral DB868 was less successful, with no cures (0/2) at 3 mg/kg/day for 10 days and cure rates of 1/4 at 10 mg/kg/day for 10 days and 20 mg/kg/day for 10 days; in total, only 2/10 monkeys were cured with DB868 dose regimens. The geometric mean plasma Cmax of IM DB829 at 5 mg/kg following the last of 5 doses was 25-fold greater than that after 10 daily oral doses of DB868 at 20 mg/kg. These data suggest that the active diamidine DB829, administered IM, should be considered for further development as a potential new treatment for second stage HAT.

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.

CYP1A1 and CYP1B1-mediated biotransformation of the antitrypanosomal methamidoxime prodrug DB844 forms novel metabolites through intramolecular rearrangement.

DB844 (CPD-594-12), N-methoxy-6-{5-[4-(N-methoxyamidino)phenyl]-furan-2-yl}-nicotinamidine, is an oral prodrug that has shown promising efficacy in both mouse and monkey models of second stage human African trypanosomiasis. However, gastrointestinal (GI) toxicity was observed with high doses in a vervet monkey safety study. In the current study, we compared the metabolism of DB844 by hepatic and extrahepatic cytochrome P450s to determine whether differences in metabolite formation underlie the observed GI toxicity. DB844 undergoes sequential O-demethylation and N-dehydroxylation in the liver to form the active compound DB820 (CPD-593-12). However, extrahepatic CYP1A1 and CYP1B1 produced two new metabolites, MX and MY. Accurate mass and collision-induced dissociation mass spectrometry analyses of the metabolites supported proposed structures of MX and MY. In addition, MY was confirmed with a synthetic standard and detection of nitric oxide (NO) release when DB844 was incubated with CYP1A1. Taken altogether, we propose that MX is formed by insertion of oxygen into the amidine CN to form an oxaziridine, which is followed by intramolecular rearrangement of the adjacent O-methyl group and subsequent release of NO. The resulting imine ester, MX, is further hydrolyzed to form MY. These findings may contribute to furthering the understanding of toxicities associated with benzamidoxime- and benzmethamidoxime-containing molecules.

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.

Safety, pharmacokinetic, and efficacy studies of oral DB868 in a first stage vervet monkey model of human African trypanosomiasis.

There are no oral drugs for human African trypanosomiasis (HAT, sleeping sickness). A successful oral drug would have the potential to reduce or eliminate the need for patient hospitalization, thus reducing healthcare costs of HAT. The development of oral medications is a key objective of the Consortium for Parasitic Drug Development (CPDD). In this study, we investigated the safety, pharmacokinetics, and efficacy of a new orally administered CPDD diamidine prodrug, 2,5-bis[5-(N-methoxyamidino)-2-pyridyl]furan (DB868; CPD-007-10), in the vervet monkey model of first stage HAT. DB868 was well tolerated at a dose up to 30 mg/kg/day for 10 days, a cumulative dose of 300 mg/kg. Mean plasma levels of biomarkers indicative of liver injury (alanine aminotransferase, aspartate aminotransferase) were not significantly altered by drug administration. In addition, no kidney-mediated alterations in creatinine and urea concentrations were detected. Pharmacokinetic analysis of plasma confirmed that DB868 was orally available and was converted to the active compound DB829 in both uninfected and infected monkeys. Treatment of infected monkeys with DB868 began 7 days post-infection. In the infected monkeys, DB829 attained a median C(max) (dosing regimen) that was 12-fold (3 mg/kg/day for 7 days), 15-fold (10 mg/kg/day for 7 days), and 31-fold (20 mg/kg/day for 5 days) greater than the IC50 (14 nmol/L) against T. b. rhodesiense STIB900. DB868 cured all infected monkeys, even at the lowest dose tested. In conclusion, oral DB868 cured monkeys with first stage HAT at a cumulative dose 14-fold lower than the maximum tolerated dose and should be considered a lead preclinical candidate in efforts to develop a safe, short course (5-7 days), oral regimen for first stage HAT.

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.

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.

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.

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.

Prostatic acid phosphatase is required for the antinociceptive effects of thiamine and benfotiamine.

Thiamine (Vitamin B1) is an essential vitamin that must be obtained from the diet for proper neurological function. At higher doses, thiamine and benfotiamine (S-benzoylthiamine O-monophosphate, BT)-a phosphorylated derivative of thiamine-have antinociceptive effects in animals and humans, although how these compounds inhibit pain is unknown. Here, we found that Prostatic acid phosphatase (PAP, ACPP) can dephosphorylate BT in vitro, in dorsal root ganglia (DRG) neurons and in primary-afferent axon terminals in the dorsal spinal cord. The dephosphorylated product S-benzoylthiamine (S-BT) then decomposes to O-benzoylthiamine (O-BT) and to thiamine in a pH-dependent manner, independent of additional enzymes. This unique reaction mechanism reveals that BT only requires a phosphatase for conversion to thiamine. However, we found that the antinociceptive effects of BT, thiamine monophosphate (TMP) and thiamine-a compound that is not phosphorylated-were entirely dependent on PAP at the spinal level. Moreover, pharmacokinetic studies with wild-type and Pap(-/-) mice revealed that PAP is not required for the conversion of BT to thiamine in vivo. Taken together, our study highlights an obligatory role for PAP in the antinociceptive effects of thiamine and phosphorylated thiamine analogs, and suggests a novel phosphatase-independent function for PAP.

A mouse diversity panel approach reveals the potential for clinical kidney injury due to DB289 not predicted by classical rodent models.

DB289 is the first oral drug shown in clinical trials to have efficacy in treating African trypanosomiasis (African sleeping sickness). Mild liver toxicity was noted but was not treatment limiting. However, development of DB289 was terminated when several treated subjects developed severe kidney injury, a liability not predicted from preclinical testing. We tested the hypothesis that the kidney safety liability of DB289 would be detected in a mouse diversity panel (MDP) comprised of 34 genetically diverse inbred mouse strains. MDP mice received 10 days of oral treatment with DB289 or vehicle and classical renal biomarkers blood urea nitrogen (BUN) and serum creatinine (sCr), as well as urine biomarkers of kidney injury were measured. While BUN and sCr remained within reference ranges, marked elevations were observed for kidney injury molecule-1 (KIM-1) in the urine of sensitive mouse strains. KIM-1 elevations were not always coincident with elevations in alanine aminotransferase (ALT), suggesting that renal injury was not linked to hepatic injury. Genome-wide association analyses of KIM-1 elevations indicated that genes participating in cholesterol and lipid biosynthesis and transport, oxidative stress, and cytokine release may play a role in DB289 renal injury. Taken together, the data resulting from this study highlight the utility of using an MDP to predict clinically relevant toxicities, to identify relevant toxicity biomarkers that may translate into the clinic, and to identify potential mechanisms underlying toxicities. In addition, the sensitive mouse strains identified in this study may be useful in screening next-in-class compounds for renal injury.

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.

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.

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.

Pharmacologic and phenotypic study of docetaxel in patients with ovarian or primary peritoneal cancer.

PURPOSE: The objectives of this study were to determine whether the midazolam clearance predicted docetaxel pharmacokinetics, CA-125 change, and response and to assess the impact of cytochrome P450 (CYP) 3A5 and ATP-binding cassette, subfamily B, member 1 (ABCB1) genotypes on docetaxel pharmacokinetics and pharmacodynamics in ovarian or primary peritoneal cancer patients. METHODS: Thirty-four patients with advanced ovarian and primary peritoneal cancer were administered docetaxel at 75 mg/m(2) as a 1-h infusion in combination with carboplatin IV over 30 min at a target AUC of 5 mg/ml min. Cycles were repeated every 21 days for 6 cycles. Midazolam was administered at 2 mg as a 30-min IV infusion the day prior to cycle one of docetaxel administration. Pharmacokinetic studies of docetaxel and CYP3A5 and ABCB1 genotype studies were performed. RESULTS: There was an inverse relationship between midazolam clearance (CL) and CA-125 level after cycle 6 where a higher midazolam CL was associated with a CA-125 <10 U/ml (P = 0.007) and CA-125 <15 U/ml (P = 0.048). The CA-125 categories were associated with response achieved (complete response/partial response) (CR/PR), stable disease (SD), and progressive disease (PD) at the end of therapy (P = 0.0173). Docetaxel CL was not related to midazolam CL or genotype. Docetaxel exposure and genotypes were not related to toxicity or response (P > 0.05). CONCLUSIONS: The midazolam CL predicted CA-125 levels and response that was independent of other factors including docetaxel pharmacokinetics. Future studies need to evaluate the mechanism for the relationship between midazolam CL and response in patients with ovarian cancer.

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.