Search

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.

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.

WHAT IS ALREADY KNOWN ABOUT THIS SUBJECT • The pharmacokinetics of cyclophosphamide and 4-hydroxycyclophosphamide in cancer patients have been well described. The current study was conducted to document the pharmacokinetics of cyclophosphamide and 4-hydroxycyclophosphamide in patients with glomerulonephritis. The study was necessary due to the numerous clinical abnormalities that are present in glomerulonephritis (altered glomerular filtration rate, proteinuria, hypoalbuminaemia) which can alter the disposition of drugs. WHAT THIS STUDY ADDS • This study documents the differences in pharmacokinetics of cyclophosphamide and 4-hydroxycyclophosphamide in glomerulonephritis vs. the cancer population. The current results demonstrate that clinical and pharmacogenetic covariates can both alter the disposition of cyclophosphamide and 4-hydroxycyclophosphamide in glomerulonephritis. 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 Cmax (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 Vd (P = 0.0271, 95% CI −57.5, −4.2) and decreased Cmax (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.

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.

Cancer drugs that can potentially treat Angelman syndrome are identified. Genomic imprinting disorders arise owing to a loss of function of non-imprinted alleles expressed from one parent. In Angelman syndrome, a neurodevelopmental disorder caused by dysfunction of the maternal allele of the Ube3a gene, the paternal allele remains intact but is epigenetically silenced. Benjamin Philpot and colleagues perform an unbiased drug screen on mouse cortical neurons expressing fluorescent Ube3a and identify topoisomerase inhibitors that are capable of activating paternal Ube3a, including topotecan, a cancer therapeutic approved by the US Food and Drug Administration. When the drug is delivered in vivo, paternal Ube3a is activated in multiple regions of the brain, and effects persist for several weeks after drug cessation. This demonstrates a potential method for reactivating dormant alleles of imprinted genes, which may be a therapeutic strategy in disorders such as Angelman syndrome. Angelman syndrome is a severe neurodevelopmental disorder caused by deletion or mutation of the maternal allele of the ubiquitin protein ligase E3A (UBE3A)1,2,3. In neurons, the paternal allele of UBE3A is intact but epigenetically silenced4,5,6, raising the possibility that Angelman syndrome could be treated by activating this silenced allele to restore functional UBE3A protein7,8. 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 Ube3a9,10,11. 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.

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.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.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).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.

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.

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.

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.

Diamidine 1 (pentamidine) and 65 analogues (2-66) have been tested for in vitro antiprotozoal activities against Trypanosoma brucei rhodesiense, Plasmodium falciparum, and Leishmania donovani, and for cytotoxicity against mammalian cells. Dications 32, 64, and 66 exhibited antitrypanosomal potencies equal or greater than melarsoprol (IC(50) = 4 nM). Nine congeners (2-4, 12, 27, 30, and 64-66) were more active against P. falciparum than artemisinin (IC(50) = 6 nM). Eight compounds (12, 32, 33, 44, 59, 62, 64, and 66) exhibited equal or better antileishmanial activities than 1 (IC(50) = 1.8 microM). Several congeners were more active than 1 in vivo, curing at least 2/4 infected animals in the acute mouse model of trypanosomiasis. The diimidazoline 66 was the most promising compound in the series, showing excellent in vitro activities and high selectivities against T. b. rhodesiense, P. falciparum, and L. donovani combined with high antitrypanosomal efficacy in vivo.

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.

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 (CVor=27%). The MS-based method strongly correlated with the immunoquantification method (r(2)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.

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.

Unprotected sexual intercourse is the predominant risk factor for acquiring HIV, with most transmission occur- ring from infected men to male and female partners.1 The efficiency of HIV transmission depends on a variety of factors, including the type of sex act, the viral burden of the infected partner, and the susceptibility of the uninfected partner to HIV infection.2–7 Although behavioral interventions, such as condom use, have been successful in reducing transmission,8–10 approximately 4 million adults and children are infected globally on a yearly basis.1 New and novel prevention methods are urgently needed to slow the spread of the epidemic. One potential mechanism to slow HIV transmission is the use of antiretroviral drugs (ARVs) to reduce HIV RNA concentrations in infectious secretions. ARV therapy has been shown reliably to decrease HIV RNA concentrations in the genital tract (GT),11–14 and lower semen HIV RNA concentrations are expected to decrease the efficiency of sexual transmission. Chakraborty and colleagues4 predict that a semen HIV RNA concentration of 100,000 copies/mL would result in transmission in 1 per 100 episodes of heterosexual intercourse, whereas a seminal viral load of 1000 copies/mL would decrease the probability of transmission to 3 per 10,000 acts. The relation between HIV RNA concentrations in blood plasma (BP) and semen is imperfect,15–18 and careful examination of viral sequences demonstrates that BP and GT can be viewed as separate viral compartments.19–21 Persistent GT HIV RNA shedding in subjects receiving ARVs has been reported,22 and long-lived resistant variants in the GT represent a particular problem for rebound viremia23 and transmitted resistance.19 It seems likely that poor penetration or altered metabolism of ARVs in the GT contribute to this problem.24 Nucleoside reverse transcriptase inhibitors (NRTIs) such as lamivudine (3TC) and zidovudine (ZDV) form the backbone of a typical ARV regimen, and this combination is currently recommended as an alternative option for initial treatment of HIV infection by the US Department of Health and Human Services.25 Random BP and seminal plasma (SP) concentrations of these drugs have been measured previously,14,26,27 and investigators have noted ZDV and 3TC concentrations to be 2 to 9 times higher in SP than in BP. As with all NRTIs, however, the active moiety is the triphosphate (TP) metabolite formed by intracellular enzymatic processes.28 Higher intracellular 3TC-TP and ZDV-TP concentrations in peripheral blood mononuclear cells (PBMCs) have been correlated with a faster decline in HIV RNA concentrations and an increase in CD4 T-cell counts.29 TP concentrations have not been previously characterized in GT mononuclear cells but are likely critical for NRTI efficacy locally. Here, we report on a comprehensive evaluation of extracellular and intracellular ZDV and 3TC concentrations in the male GT over a 12-hour dosing interval under steady-state conditions.

Heparan sulfate (HS) is a highly sulfated polysaccharide and is present in large quantities on the cell surface and in the extracellular matrix. Herpes simplex virus type 1 (HSV-1) utilizes a specialized cell surface HS, known as 3-O-sulfated HS, as an entry receptor to establish infection. Here, we exploit an approach to inhibiting HSV-1 infection by using a 3-O-sulfated octasaccharide, mimicking the active domain of the entry receptor. The 3-O-sulfated octasaccharide was synthesized by incubating a heparin octasaccharide (3-OH octasaccharide) with HS 3-O-sulfotransferase isoform 3. The resultant 3-O-sulfated octasaccharide has a structure of Delta UA2S-GlcNS6S-IdoUA2S-GlcNS6S-IdoUA2S-GlcNS3S6S-IdoUA2S-GlcNS6S (where Delta UA is 4-deoxy-alpha-L-threo-hex-4-enopyranosyluronic acid, GlcN is D-glucosamine, and IdoUA is L-iduronic acid). Results from cell-based assays revealed that the 3-O-sulfated octasaccharide has stronger activity in blocking HSV-1 infection than that of the 3-OH octasaccharide, suggesting that the inhibition of HSV-1 infection requires a unique sulfation moiety. Our results suggest the feasibility of inhibiting HSV-1 infection by blocking viral entry with a specific oligosaccharide.

Previous studies implicated P-glycoprotein (P-gp) as the major transport protein responsible for the biliary excretion of fexofenadine (FEX). However, FEX biliary excretion was not impaired in P-gp- or breast cancer resistance protein (Bcrp)-knockout mice or multidrug resistance-associated protein 2 (Mrp2)-deficient rats. The present study tested the hypothesis that species differences exist in the transport protein primarily responsible for FEX biliary excretion between mice and rats. Livers from Mrp2-knockout (Mrp2KO) mice and Mrp2-deficient (TR(-)) rats were perfused in a single-pass manner with 0.5 muM FEX. N-(4-[2-(1,2,3,4-Tetrahydro-6,7-dimethoxy-2-isoquinolinyl)ethyl]-phenyl)-9,10-dihydro-5-methoxy-9-oxo-4-acridine carboxamide (GF120918) (10 muM) was employed to inhibit P-gp and Bcrp. The biliary excretion rate of FEX was decreased 85% in Mrp2KO relative to wild-type mice (18.4 +/- 2.2 versus 122 +/- 34 pmol/min/g liver). In mice, more than 50% of FEX unbound intrinsic biliary clearance (CL(bile, int)(') = 3.0 ml/h/g liver) could be attributed to Mrp2 (Mrp2-dependent CL(bile, int)(') approximately 1.7 ml/h/g liver), with P-gp and Bcrp playing a minor role (P-gp- and Bcrp-dependent CL(bile, int)(') approximately 0.3 ml/h/g liver). Approximately one third of FEX CL(bile, int)(') was attributed to unidentified mechanisms in mice. In contrast to mice, FEX biliary excretion rate (245 +/- 38 and 250 +/- 25 pmol/min/g liver) and CL(bile, int)(') (9.72 +/- 2.47 and 6.49 +/- 0.68 ml/h/g liver) were comparable between TR(-) and control Wistar rats, respectively, suggesting that unidentified transport mechanism(s) can completely compensate for the loss of Mrp2 function in rats. Mrp2 clearly plays a major role in FEX biliary excretion in mice. In conclusion, remarkable species differences exist in FEX hepatobiliary transport mechanisms.

3,5-bis(4-amidinophenyl)isoxazole (3)-an analogue of 2,5-bis(4-amidinophenyl)furan (furamidine) in which the central furan ring is replaced by isoxazole-and 42 novel analogues were prepared by two general synthetic pathways. The 43 isoxazole derivatives were assayed against Trypanosoma brucei rhodesiense (T. brucei rhodesiense) STIB900, Plasmodium falciparum (P. falciparum) K1, and rat myoblast L6 cells (for cytotoxicity) in vitro. Eleven compounds (3, 13, 16-18, 22, 26, 29, 31, 37, and 41) exhibited antitrypanosomal IC50 values less than 10 nM, five of which displayed cytotoxic indices (ratios of cytotoxic IC50 to antiprotozoal IC50 values) at least 10 times higher than that of furamidine. Eighteen compounds (4-8, 12, 14, 18-22, 25, 26, 28, 29, 32, and 43) were more active against P. falciparum than furamidine, with IC50 values less than 15 nM. Fourteen of these compounds had cytotoxic indices ranging between 10 and 120 times higher than that of furamidine, and five analogues exhibited high selectivity for P. falciparum over T. brucei rhodesiense.

DB289 [2,5-bis(4-amidinophenyl)furan-bis-O-methylamidoxime] is biotransformed to the potent antiparasitic diamidine DB75 [2,5-bis(4-amidinophenyl) furan] by sequential oxidative O-demethylation and reductive N-dehydroxylation reactions. Previous work demonstrated that the N-dehydroxylation reactions are catalyzed by cytochrome b5/NADH-cytochrome b5 reductase. Enzymes responsible for catalyzing the DB289 O-demethylation pathway have not been identified. We report an in vitro metabolism study to characterize enzymes in human liver microsomes (HLMs) that catalyze the initial O-demethylation of DB289 (M1 formation). Potent inhibition by 1-aminobenzotriazole confirmed that M1 formation is catalyzed by P450 enzymes. M1 formation by HLMs was NADPH-dependent, with a Km and Vmax of 0.5 microM and 3.8 nmol/min/mg protein, respectively. Initial screening showed that recombinant CYP1A1, CYP1A2, and CYP1B1 were efficient catalysts of M1 formation. However, none of these three enzymes was responsible for M1 formation by HLMs. Further screening showed that recombinant CYP2J2, CYP4F2, and CYP4F3B could also catalyze M1 formation. An antibody against CYP4F2, which inhibited both CYP4F2 and CYP4F3B, inhibited 91% of M1 formation by HLMs. Two inhibitors of P450-mediated arachidonic acid metabolism, HET0016 (N-hydroxy-N'-(4-n-butyl-2-methylphenyl)formamidine) and 17-octadecynoic acid, effectively inhibited M1 formation by HLMs. Inhibition studies with ebastine and antibodies against CYP2J2 suggested that CYP2J2 was not involved in M1 formation by HLMs. Additionally, ketoconazole preferentially inhibited CYP4F2, but not CYP4F3B, and partially inhibited M1 formation by HLMs. We conclude that CYP4F enzymes (e.g., CYP4F2, CYP4F3B) are the major enzymes responsible for M1 formation by HLMs. These findings indicate that, in human liver, members of the CYP4F subfamily biotransform not only endogenous compounds but also xenobiotics.

Although glucuronide and sulfate conjugates of many drugs and endogenous compounds undergo appreciable hepatic basolateral excretion into sinusoidal blood, the mechanisms that govern basolateral translocation of these hydrophilic metabolites have not been completely elucidated. In the present study, the involvement in this process of Mrp3 and Mrp4, two basolateral efflux transporters, was evaluated by analyzing the hepatic basolateral excretion of the glucuronide and sulfate metabolites of acetaminophen, 4-methylumbelliferone, and harmol in Abcc3(-/-) and Abcc4(-/-) mice using a cassette dosing approach. In the livers of Abcc3(-/-) and Abcc4(-/-) mice, the basolateral excretory clearance of acetaminophen sulfate was reduced approximately 20 and approximately 20%, 4-methylumbelliferyl sulfate was reduced approximately 50 and approximately 65%, and harmol sulfate was decreased approximately 30 and approximately 45%, respectively. The basolateral excretory clearance of acetaminophen glucuronide, 4-methylumbelliferyl glucuronide, and harmol glucuronide was reduced by approximately 96, approximately 85, and approximately 40%, respectively, in the livers of Abcc3(-/-) mice. In contrast, basolateral excretory clearance of these glucuronide conjugates was unaffected by the absence of Mrp4. These results provide the first direct evidence that Mrp3 and Mrp4 participate in the hepatic basolateral excretion of sulfate conjugates, although additional mechanism(s) are likely involved. In addition, they reveal that Mrp3 mediates the hepatic basolateral excretion of diverse glucuronide conjugates.

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 (IC 50 ) (as established for PBMCs, assuming SMC IC 50 = PBMC IC 50 ) 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.

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., Author Summary Treatment of human African trypanosomiasis (HAT, sleeping sickness) suffers from a shortage of medicines that are both effective, especially against the second (late) stage of the disease, and safe for patients. The development of new HAT medicines also has been significantly influenced by the perceived need for easily administered oral medicines to reduce the need for hospitalization of patients in resource-poor settings where HAT typically occurs. However, the clinical status of second stage patients is likely to dictate the need for their hospitalization, thus both oral and parenterally administered medicines would be utilised effectively. Therefore, in an effort to develop new medicines that meet efficacy and safety requirements, we evaluated a novel injectable diamidine 2,5-bis(5-amidino-2-pyridyl)furan (DB829; CPD-0802) and its oral prodrug formulation 2,5-bis[5-(N-methoxyamidino)-2-pyridyl]furan (DB868) in the vervet monkey model of second stage HAT. Treatment with either compound was initiated 28 days post-infection of monkeys with T. b. rhodesiense KETRI 2537. DB829 was dosed 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 intramuscularly (IM) while DB868 was administered at 20, 10 or 3 mg/kg/day for 10 consecutive days orally. Clinical and parasitological monitoring was carried out for at least 300 days before the monkeys were declared cured. All IM DB829 and oral DB868 dose regimens were well tolerated. In addition, all monkeys (5/5) treated with IM DB829 were confirmed cured. In contrast, oral DB868 cured only 1/4 monkeys at either 10 or 20 mg/kg and did not cure any monkey when dosed at 3 mg/kg. These results indicate that IM DB829 is a suitable compound for further development as treatment for second stage HAT.

The functional impact of altered drug transport protein expression on the systemic pharmacokinetics of morphine, hepatically derived morphine glucuronide (morphine-3- and morphine-6-glucuronide), and fasting bile acids was evaluated in patients with biopsy-confirmed nonalcoholic steatohepatitis (NASH) compared to healthy subjects. The maximum concentration (Cmax ) and area under the concentration-time curve (AUC0-last ) of morphine glucuronide in serum were increased in NASH patients (343 vs. 225 nM and 58.8 vs. 37.2 µM*min, respectively; P ≤ 0.005); morphine pharmacokinetics did not differ between groups. Linear regression analyses detected an association of NASH severity with increased morphine glucuronide Cmax and AUC0-last (P < 0.001). Fasting serum glycocholate, taurocholate, and total bile acid concentrations were associated with NASH severity (P < 0.006). Increased hepatic basolateral efflux of morphine glucuronide and bile acids is consistent with altered hepatic transport protein expression in patients with NASH and may partially explain differences in efficacy and/or toxicity of some highly transported anionic drugs/metabolites in this patient population.

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.

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.

Purpose: To use genetically engineered mouse models (GEMM) and orthotopic syngeneic murine transplants (OST) to develop gene expression-based predictors of response to anticancer drugs in human tumors. These mouse models offer advantages including precise genetics and an intact microenvironment/immune system. Experimental Design: We examined the efficacy of 4 chemotherapeutic or targeted anticancer drugs, alone and in combination, using mouse models representing 3 distinct breast cancer subtypes: Basal-like (C3(1)-T-antigen GEMM), Luminal B (MMTV-Neu GEMM), and Claudin-low (T11/TP53−/− OST). We expression-profiled tumors to develop signatures that corresponded to treatment and response, and then tested their predictive potential using human patient data. Results: Although a single agent exhibited exceptional efficacy (i.e., lapatinib in the Neu-driven model), generally single-agent activity was modest, whereas some combination therapies were more active and life prolonging. Through analysis of RNA expression in this large set of chemotherapy-treated murine tumors, we identified a pair of gene expression signatures that predicted pathologic complete response to neoadjuvant anthracycline/taxane therapy in human patients with breast cancer. Conclusions: These results show that murine-derived gene signatures can predict response even after accounting for common clinical variables and other predictive genomic signatures, suggesting that mice can be used to identify new biomarkers for human patients with cancer. Clin Cancer Res; 19(17); 4889–99. ©2013 AACR.

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 trypanosomiasisHowever, gastrointestinal (GI) toxicity was observed with high doses in a vervet monkey safety studyIn 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 toxicityDB844 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 MYAccurate mass and collision-induced dissociation mass spectrometry analyses of the metabolites supported proposed structures of MX and MYIn addition, MY was confirmed with a synthetic standard and detection of nitric oxide (NO) release when DB844 was incubated with CYP1A1Taken 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 NOThe resulting imine ester, MX, is further hydrolyzed to form MYThese findings may contribute to furthering the understanding of toxicities associated with benzamidoxime- and benzmethamidoxime-containing molecules© 2013 Wiley Periodicals, Incand the American Pharmacists Association J Pharm Sci 103:337-349, 2014

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.

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.

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 Cmax (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., Author Summary Development of orally administered medicines for human African trypanosomiasis (HAT) would potentially reduce the need for patient hospitalization, thus lowering healthcare costs. In this study, we investigated the potential of a novel diamidine prodrug, DB868 (CPD-007-10), as an oral treatment for first stage HAT. When administered to uninfected monkeys by oral gavage, DB868 was well tolerated up to a maximum dose of 30 mg/kg/day for 10 days (cumulative dose [CD] = 300 mg/kg). DB868 was absorbed into the systemic circulation and was converted to the active compound DB829 in concentrations that were potentially therapeutic for blood trypanosomes. Subsequently, DB868 was evaluated for efficacy in the first stage vervet monkey model of HAT in which treatment was initiated at 7 days post-infection with T. b. rhodesiense KETRI 2537. All infected monkeys were cured, even at the lowest of the three dose regimens tested: 3 mg/kg/day for 7 days (CD = 21 mg/kg), 10 mg/kg/day for 7 days (CD = 70 mg/kg) and 20 mg/kg/day for 5 days (CD = 100 mg/kg). DB868 conversion to DB829 was comparable between uninfected and infected monkeys. In view of its favourable safety and oral efficacy profile, we conclude that DB868 is a suitable candidate for development as a new treatment for first stage HAT.

(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.

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.

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.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.

• 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.• 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.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.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.Rosiglitazone concentration was significantly correlated with paclitaxel exposure (P= 0.018) while ERMBT had no predictive value (P= 0.47).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, 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.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.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).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.

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.

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.

Three diamidines (DB 75, DB 867 and DB 1192) were selected and their ability to cure T. evansi experimentally infected goats was investigated. A toxicity assessment and pharmacokinetic analysis of these compounds were additionally carried out. Goats demonstrated no signs of acute toxicity, when treated with four doses of 1 mg/kg/day (total dose 4 mg/kg). Complete curative efficacy of experimentally infected goats was seen in the positive control group treated with diminazene at 5 mg/kg and in the DB 75 and DB 867 groups treated at 2.5 mg/kg. Drug treatment was administered once every second day for a total of seven days. Complete cure was also seen in the group of goats treated with DB 75 at 1.25 mg/kg. DB 1192 was incapable of curing goats at either four-times 2.5 mg/kg or 1.25 mg/kg. Pharmacokinetic analysis clearly demonstrated that the treatment failures of DB 1192 were due to sub-therapeutic compound levels in goat plasma, whilst compound levels for DB 75 and DB 867 remained well within the therapeutic window. In conclusion, two diamidine compounds (DB 75 and DB 867) presented comparable efficacy at lower doses than the standard drug diminazene and could be considered as potential clinical candidates against T. evansi infection.

This method provides a simple extraction procedure, as well as a validated, sensitive, and specific liquid chromatography-tandem mass spectrometry assay for the simultaneous quantification of ampicillin, piperacillin, tazobactam, meropenem, acyclovir, and metronidazole in human plasma. The method was validated over concentration ranges specific for each compound, with a lower limit of quantification of 50-300 ng/mL and a sample volume of 50 μL. The method is accurate and precise, with within- and between-day accuracy ranging from 85 to 110% and 92 to 110%, respectively, and within- and between-day precision of 89-111% and 91-109%, respectively. Simplicity, low plasma volume, and high throughput make this method suitable for clinical pharmacokinetic studies in premature infants.

To evaluate whether patients with human immunodeficiency virus (HIV) infection who were receiving protease inhibitor therapy had altered bile acid concentrations compared with noninfected control subjects, and whether bile acid concentrations could predict the onset of hepatotoxicity caused by protease inhibitors.Retrospective sample analysis from a prospectively conducted clinical trial.Academic research center.Eleven adults with advanced HIV disease who were taking protease inhibitor-based antiretroviral therapy, one of whom had developed protease inhibitor-induced hepatotoxicity.Plasma concentrations of cholic acid (CA), chenodeoxycholic acid (CDCA), deoxycholic acid (DCA), lithocholic acid (LCA), ursodeoxycholic acid (UDCA), and taurocholic acid (TC) were analyzed by using a novel high-performance liquid chromatography with tandem mass spectrometry detection method. Comparisons of the relative contribution of each bile acid to the total bile acid pool were made with previously published values and with bile acid concentrations contained in two pooled plasma samples from healthy, non-HIV-infected volunteers analyzed in our laboratory. Each pooled plasma sample used for this analysis contained contributions from three non-HIV-infected volunteers. The LCA and TC concentrations in HIV-infected patients were 3-4-fold higher than those previously reported for non-HIV-infected subjects; concentrations of other bile acids were similar to those of previous reports. The relative contribution of CDCA to the total bile acid pool was 9% in HIV-infected patients compared with 30-50% in noninfected subjects. Total and individual bile acid concentrations in the HIV-infected patient who developed hepatotoxicity were similar to the bile acid concentrations from the other study patients who did not develop hepatotoxicity.These data suggest that bile acid concentrations may be altered by HIV infection and/or protease inhibitor therapy. However, further investigations should be performed to assess whether antiretroviral-associated hepatotoxicity can be predicted by alterations in individual bile acid concentrations.

Forty-eight cationically substituted pentamidine congeners possessing benzofuran rings were synthesized by a copper mediated heteroannulation of substituted o-iodophenols with phenyl acetylenes. Activities of compounds 1-48 against Trypanosoma brucei rhodesiense, Plasmodium falciparum, and Leishmania donovani and cytotoxicities for mammalian cells were influenced by the nature of cationic substituents, placement of the benzofuran fragment, and the length of the carbon linker between aromatic moieties. Several dications exhibited superior antiplasmodial and antileishmanial potencies compared to pentamidine.

Paracellular transport through the tight junction shows selectivity for both ionic charge and solute size. It is known that charged residues on the extracellular loops of claudins control charge selectivity. It is also known that inducible expression of claudin-2, but not claudin-4, will selectively increase the permeability for polyethylene glycol (PEG) molecules which are

Previous reports have indicated that in vitro biliary clearance (Clbiliary) determined in sandwich-cultured hepatocytes correlates well with in vivo Clbiliary for limited sets of compounds. The purpose of this study was 1) to determine the in vitro Clbiliary 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 Clbiliary values with estimated in vivo hepatic clearance data in humans. The average biliary excretion index and in vitro intrinsic Clbiliary 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. Clbiliary 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 Clbiliary determined in sandwich-cultured human hepatocytes can be used to predict in vivo Clbiliary of compounds in humans.

Voriconazole is a potent second-generation triazole antifungal agent with broad-spectrum activity against clinically important fungi. It is cleared predominantly via metabolism in all species tested including humans. N-Oxidation of the fluoropyrimidine ring, its hydroxylation, and hydroxylation of the adjacent methyl group are the known pathways of voriconazole oxidative metabolism, with the N-oxide being the major circulating metabolite in human. In vitro studies have shown that CYP2C19, CYP3A4, and to a lesser extent CYP2C9 contribute to the oxidative metabolism of voriconazole. When cytochrome P450 (P450)-specific inhibitors and antibodies were used to evaluate the oxidative metabolism of voriconazole by human liver microsomes, the results suggested that P450-mediated metabolism accounted for approximately 75% of the total oxidative metabolism. The studies presented here provide evidence that the remaining approximately 25% of the metabolic transformations are catalyzed by flavin-containing monooxygenase (FMO). This conclusion was based on the evidence that the NADPH-dependent metabolism of voriconazole was sensitive to heat (45 degrees C for 5 min), a condition known to selectively inactivate FMO without affecting P450 activity. The role of FMO in the metabolic formation of voriconazole N-oxide was confirmed by the use of recombinant FMO enzymes. Kinetic analysis of voriconazole metabolism by FMO1 and FMO3 yielded K(m) values of 3.0 and 3.4 mM and V(max) values of 0.025 and 0.044 pmol/min/pmol, respectively. FMO5 did not metabolize voriconazole effectively. This is the first report of the role of FMO in the oxidative metabolism of voriconazole.

Whole cigarette smoke (WCS) is composed of approximately 5% particulates and 95% vapors by weight and is difficult to reproduce quantitatively in the laboratory, where typically, routine in vitro application of smoke normally only utilizes the particulate phase. In this study, we used a system for exposing epithelial cells cultured at an air-liquid interface to WCS. We hypothesized that the use of WSC in vitro was more relevant to what is seen in vivo than methods of cigarette smoke application that only use a small fraction of WCS [i.e., aqueous extract or cigarette smoke condensate (CSC)]. To test this hypothesis, we compared nicotine and cotinine concentrations (measured by mass spectrometry) in the airway surface liquid (ASL) of human primary bronchial epithelial cultures (HBECs) exposed to serial dilutions of WCS to the concentrations found in induced sputum of human subjects who had recently smoked a cigarette; this was also compared to the concentrations found after an exposure to a concentration of CSC commonly used in vitro. When measured by mass spectrometry, nicotine levels were not significantly different in induced sputum versus the ASL of HBECs exposed in vitro to a 1:30 exposure of WCS. However, HBECs that had been exposed to CSC returned significantly lower concentrations of ASL nicotine. These results suggest that nicotine is a good dosimetry marker of WCS exposure and provides direct evidence that the use of WCS is more relevant than the use of CSC for in vitro systems.

Studying the pharmacokinetics of antiretroviral drugs in breast milk has important implications for the health of both the mother and the infant, particularly in resource-poor countries. Breast milk is a highly complex biological matrix, yet it is necessary to develop and validate methods in this matrix, which simultaneously measure multiple analytes, as women may be taking any number of drug combinations to combat human immunodeficiency virus infection. Here, we report a novel extraction method coupled to high-performance liquid chromatography and tandem mass spectrometry for the accurate, precise, and specific measurement of 7 antiretroviral drugs currently prescribed to infected mothers. Using 200 microL of human breast milk, simultaneous quantification of lamivudine (3TC), stavudine (d4T), zidovudine (ZDV), nevirapine (NVP), nelfinavir (NFV), ritonavir, and lopinavir was validated over the range of 10-10,000 ng/mL. Intraday accuracy and precision for all analytes were 99.3% and 5.0 %, respectively. Interday accuracy and precision were 99.4 % and 7.8%, respectively. Cross-assay validation with UV detection was performed using clinical breast milk samples, and the results of the 2 assays were in good agreement (P = 0.0001, r = 0.97). Breast milk to plasma concentration ratios for the different antiretroviral drugs were determined as follows: 3TC = 2.96, d4T = 1.73, ZDV = 1.17, NVP = 0.82, and NFV = 0.21.

Objectives: To describe first dose and steady state antiretroviral drug exposure in the female genital tract. Design: Non-blinded, single center, open-label pharmacokinetic study in HIV-infected women. Method: Twenty-seven women initiating combination antiretroviral therapy underwent comprehensive blood plasma and cervicovaginal fluid sampling for drug concentrations during the first dose of antiretroviral therapy and at steady-state. Drug concentrations were measured by validated HPLC/UV or HPLC-MS/MS methods. Pharmacokinetic parameters were estimated for 11 drugs by non-compartmental analysis. Descriptive statistics and 95% confidence intervals were generated using Intercooled STATA Release 8.0 (Stata Corporation, College Station, Texas, USA). Results: For all antiretroviral drugs, genital tract concentrations were detected rapidly after the first dose. Drugs were stratified according to the genital tract concentrations achieved relative to blood plasma. Median rank order of highest to lowest genital tract concentrations relative to blood plasma at steady state were: lamivudine (concentrations achieved were 411% greater than blood plasma), emtricitabine (395%), zidovudine (235%) tenofovir (75%), ritonavir (26%), didanosine (21%), atazanavir (18%), lopinavir (8%), abacavir (8%), stavudine (5%), and efavirenz (0.4%). Conclusions: This is the first study to comprehensively evaluate antiretroviral drug exposure in the female genital tract. These findings support the use of lamivudine, zidovudine, tenofovir and emtricitabine as excellent pre-exposure/post-exposure prophylaxis (PrEP/PEP) candidates. Atazanavir and lopinavir might be useful agents for these applications due to favorable therapeutic indices, despite lower genital tract concentrations. Agents such as stavudine, abacavir, and efavirenz that achieve genital tract exposures less than 10% of blood plasma are less attractive PrEP/PEP candidates.

CYP4F enzymes, including CYP4F2 and CYP4F3B, were recently shown to be the major enzymes catalyzing the initial oxidative O-demethylation of the antiparasitic prodrug pafuramidine (DB289) by human liver microsomes. As suggested by a low oral bioavailability, DB289 could undergo first-pass biotransformation in the intestine, as well as in the liver. Using human intestinal microsomes (HIM), we characterized the enteric enzymes that catalyze the initial O-demethylation of DB289 to the intermediate metabolite, M1. M1 formation in HIM was catalyzed by cytochrome P450 (P450) enzymes, as evidenced by potent inhibition by 1-aminobenzotriazole and the requirement for NADPH. Apparent K(m) and V(max) values ranged from 0.6 to 2.4 microM and from 0.02 to 0.89 nmol/min/mg protein, respectively (n = 9). Of the P450 chemical inhibitors evaluated, ketoconazole was the most potent, inhibiting M1 formation by 66%. Two inhibitors of P450-mediated arachidonic acid metabolism, HET0016 (N-hydroxy-N'-(4-n-butyl-2-methylphenyl)formamidine) and 17-octadecynoic acid, inhibited M1 formation in a concentration-dependent manner (up to 95%). Immunoinhibition with an antibody raised against CYP4F2 showed concentration-dependent inhibition of M1 formation (up to 92%), whereas antibodies against CYP3A4/5 and CYP2J2 had negligible to modest effects. M1 formation rates correlated strongly with arachidonic acid omega-hydroxylation rates (r(2) = 0.94, P < 0.0001, n = 12) in a panel of HIM that lacked detectable CYP4A11 protein expression. Quantitative Western blot analysis revealed appreciable CYP4F expression in these HIM, with a mean (range) of 7 (3-18) pmol/mg protein. We conclude that enteric CYP4F enzymes could play a role in the first-pass biotransformation of DB289 and other xenobiotics.