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Vincristine is a cytotoxic chemotherapeutic agent used as first-line therapy for pediatric acute lymphocytic leukemia. It is cleared by hepatic oxidative metabolism by CYP3A4 and CYP3A5 and via hepatic (biliary) efflux mediated by P-glycoprotein (P-gp) transporter. Bottom-up physiologically based pharmacokinetic (PBPK) models were developed to predict vincristine disposition in pediatric and adult populations. The models incorporated physicochemical properties, metabolism by CYP3A4/5, efflux by P-gp, and intracellular binding to β-tubulin. The adult and pediatric PBPK models predicted pharmacokinetics (PK) within twofold of the observed PK parameters (area under the curve, terminal half-life, volume of distribution, and clearance). Simulating a higher hypothetical (4.9-fold) pediatric expression of β-tubulin relative to adult improved predictions of vincristine PKs. To our knowledge, this is the first time that intracellular binding has been incorporated into a pediatric PBPK model. Utilizing this PBPK modeling approach, safe and effective doses of vincristine could be predicted., (© 2019 The Authors. CPT: Pharmacometrics & Systems Pharmacology published by Wiley Periodicals, Inc. on behalf of the American Society for Clinical Pharmacology and Therapeutics.)

Background and Purpose: It has been extensively reported that the leading anti-diabetic drug, metformin, exerts significant anticancer effects. This hydrophilic, cationic drug requires cation transporters for cellular entry where it activates its intracellular target, the AMPK signalling pathway. However, clinical results on metformin therapy (used at antidiabetic doses) for breast cancer are ambiguous. It is likely that the antidiabetic dose is inadequate in patients that have breast tumours with low cation transporter expression, resulting in non-responsiveness to the drug. We postulate that cation transporter expression and metformin dose are key determinants in its antitumour efficacy in breast cancer., Experimental Approach: Antitumour efficacy of metformin was compared between low cation transporter-expressing MCF-7 breast tumours and MCF-7 tumours overexpressing organic cation transporter 3 (OCT3-MCF7). A dose-response relationship of metformin in combination with standard-of-care paclitaxel (for oestrogen receptor-positive MCF-7 breast tumours) or carboplatin (for triple-negative MDA-MB-468 breast tumours) was investigated in xenograft mice., Key Results: Metformin had greater efficacy against tumours with higher cation transporter expression, as observed in OCT3-MCF7 versus MCF-7 tumours and MDA-MB-468 versus MCF-7 tumours. In MCF-7 tumours, a threefold higher metformin dose was required to achieve intratumoural exposure that was comparable to exposure in MDA-MB-468 tumours and enhance antitumour efficacy of standard-of-care in MCF-7 tumours versus MDA-MB-468 tumours. Antitumour efficacy correlated with intratumoural AMPK activation and metformin concentration., Conclusions and Implications: An efficacious metformin dose for breast cancer varies among tumour subtypes based on cation transporter expression, which provides a useful guide for dose selection., (© 2019 The British Pharmacological Society.)

BackgroundAge-dependent differences in pharmacokinetics exist for metabolically cleared medications. Differential contributions in the cytochrome P450 3A (CYP3A), CYP2C, and flavin-containing monooxygenases (FMOs) families have an important role in the metabolic clearance of a large number of drugs administered to children.MethodsUnlike previous semiquantitative characterization of age-dependent changes in the expression of genes and proteins (western blot analysis), this study quantifies both gene and absolute protein expression in the same fetal, pediatric, and adult hepatic tissue. Expression was then correlated with the corresponding functional activities in the same samples.ResultsCYP3A and FMO families showed a distinct switch from fetal (CYP3A7 and FMO1) to adult isoforms (CYP3A4 and FMO3) at birth, whereas CYP2C9 showed a linear maturation from birth into adulthood. In contrast, analysis of CYP2C19 revealed higher expression and catalytic efficiency in pediatric samples compared with that in fetal and adult samples. Further, CYP3A and FMO enzymes exhibited an unexpectedly higher functional activity in fetal samples not entirely explained by protein expression.ConclusionThese surprising findings suggest that CYP and FMO enzymes may encounter development-related differences in their microenvironments that can influence the enzyme activity in addition to protein expression levels.

Background and Purpose: The potential for therapeutic antibody treatment of neurological diseases is limited by poor penetration across the blood-brain barrier. I.c.v. delivery is a promising route to the brain; however, it is unclear how efficiently antibodies delivered i.c.v. penetrate the cerebrospinal spinal fluid (CSF)-brain barrier and distribute throughout the brain parenchyma., Experimental Approach: We evaluated the pharmacokinetics and pharmacodynamics of an inhibitory monoclonal antibody against β-secretase 1 (anti-BACE1) following continuous infusion into the left lateral ventricle of healthy adult cynomolgus monkeys., Key Results: Animals infused with anti-BACE1 i.c.v. showed a robust and sustained reduction (~70%) of CSF amyloid-β (Aβ) peptides. Antibody distribution was near uniform across the brain parenchyma, ranging from 20 to 40 nM, resulting in a ~50% reduction of Aβ in the cortical parenchyma. In contrast, animals administered anti-BACE1 i.v. showed no significant change in CSF or cortical Aβ levels and had a low (~0.6 nM) antibody concentration in the brain., Conclusion and Implications: I.c.v. administration of anti-BACE1 resulted in enhanced BACE1 target engagement and inhibition, with a corresponding dramatic reduction in CNS Aβ concentrations, due to enhanced brain exposure to antibody., (© 2017 The British Pharmacological Society.)

Flavin-containing monooxygenases (FMOs) have a significant role in the metabolism of small molecule pharmaceuticals. Among the five human FMOs, FMO1, FMO3, and FMO5 are the most relevant to hepatic drug metabolism. Although age-dependent hepatic protein expression, based on immunoquantification, has been reported previously for FMO1 and FMO3, there is very little information on hepatic FMO5 protein expression. To overcome the limitations of immunoquantification, an ultra-performance liquid chromatography (UPLC)-multiple reaction monitoring (MRM)-based targeted quantitative proteomic method was developed and optimized for the quantification of FMO1, FMO3, and FMO5 in human liver microsomes (HLM). A post-in silico product ion screening process was incorporated to verify LC-MRM detection of potential signature peptides before their synthesis. The developed method was validated by correlating marker substrate activity and protein expression in a panel of adult individual donor HLM (age 39-67 years). The mean (range) protein expression of FMO3 and FMO5 was 46 (26-65) pmol/mg HLM protein and 27 (11.5-49) pmol/mg HLM protein, respectively. To demonstrate quantification of FMO1, a panel of fetal individual donor HLM (gestational age 14-20 weeks) was analyzed. The mean (range) FMO1 protein expression was 7.0 (4.9-9.7) pmol/mg HLM protein. Furthermore, the ontogenetic protein expression of FMO5 was evaluated in fetal, pediatric, and adult HLM. The quantification of FMO proteins also was compared using two different calibration standards, recombinant proteins versus synthetic signature peptides, to assess the ratio between holoprotein versus total protein. In conclusion, a UPLC-MRM-based targeted quantitative proteomic method has been developed for the quantification of FMO enzymes in HLM., (Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.)

The antidiabetic drug metformin exerts antineoplastic effects against breast cancer and other cancers. One mechanism by which metformin is believed to exert its anticancer effect involves activation of its intracellular target, adenosine monophosphate-activated protein kinase (AMPK), which is also implicated in the antidiabetic effect of metformin. It is proposed that in cancer cells, AMPK activation leads to inhibition of the mammalian target of rapamycin (mTOR) and the downstream pS6K that regulates cell proliferation. Due to its hydrophilic and cationic nature, metformin requires cation-selective transporters to enter cells and activate AMPK. This study demonstrates that expression levels of cation-selective transporters correlate with the antiproliferative and antitumor efficacy of metformin in breast cancer. Metformin uptake and antiproliferative activity were compared between a cation-selective transporter-deficient human breast cancer cell line, BT-20, and a BT-20 cell line that was engineered to overexpress organic cation transporter 3 (OCT3), a representative of cation-selective transporters and a predominant transporter in human breast tumors. Metformin uptake was minimal in BT-20 cells, but increased by >13-fold in OCT3-BT20 cells, and its antiproliferative potency was >4-fold in OCT3-BT20 versus BT-20 cells. This increase in antiproliferative activity was associated with greater AMPK phosphorylation and decreased pS6K phosphorylation in OCT3-BT20 cells. In vitro data were corroborated by in vivo observations of significantly greater antitumor efficacy of metformin in xenograft mice bearing OCT3-overexpressing tumors versus low transporter-expressing wildtype tumors. Collectively, these findings establish a clear relationship between cation-selective transporter expression, the AMPK-mTOR-pS6K signaling cascade, and the antiproliferative activity of metformin in breast cancer., (© 2015 UICC.)

Transport proteins in intestinal epithelial cells facilitate absorption of nutrients/compounds that are organic anions, cations, and zwitterions. For two decades, we have studied intestinal absorption and transport of hydrophilic ionic compounds, with specific focus on transport properties of organic cations and their interactions with intestinal transporters and tight junction proteins. Our data reveal how complex interactions between a compound and transporters in intestinal apical/basolateral (BL) membranes and tight junction proteins define oral absorption, and that the BL membrane lacks an efflux transporter that can transport positively charged compounds. Based on our investigations of transport mechanisms of zwitterionic, anionic, and cationic compounds, we postulate that physicochemical properties of these ionic species, in relation to the intestinal micro pH environment, have exerted evolutionary pressure for development of transporters that can handle apical uptake/efflux of all 3 ionic species and BL efflux of anions and zwitterions, but such evolutionary pressure is lacking for development of a BL efflux transporter for cationic compounds. This review provides an overview of intestinal uptake/efflux transporters and describes our studies on intestinal transport of cationic, anionic, and zwitterionic drugs that led to hypothesize that there are no cation-selective BL efflux transporters in the intestine., (Copyright © 2016 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.)

Multiple small-molecule inhibitors of the β-secretase enzyme (BACE1) are under preclinical or clinical investigation for Alzheimer's disease (AD). Prior work has illustrated robust lowering of central amyloid β (Aβ) after acute administration of BACE1 inhibitors. However, very few studies have assessed the overall impact of chronically administered BACE1 inhibitors on brain amyloid burden, neuropathology, and behavioral function in aged preclinical models. We investigated the effects of a potent nonbrain-penetrant BACE1 inhibitor, delivered directly to the brain using intracerebroventricular infusion in an aged transgenic mouse model. Intracerebroventricular infusion of the BACE1 inhibitor (0.3-23.5 μg/d) for 8 weeks, initiated in 17-month-old Tg2576 mice, produced dose-dependent increases in brain inhibitor concentrations (0.2-13 μm). BACE1 inhibition significantly reversed the behavioral deficit in contextual fear conditioning, and reduced brain Aβ levels, plaque burden, and associated pathology (e.g., dystrophic neurites), with maximal effects attained with ∼1 μg/d dose. Strikingly, the BACE1 inhibitor also reversed amyloid pathology below baseline levels (amyloid burden at the start of treatment), without adversely affecting cerebral amyloid angiopathy, microhemorrhages, myelination, or neuromuscular function. Inhibitor-mediated decline in brain amyloid pathology was associated with an increase in microglial ramification. This is the first demonstration of chronically administered BACE1 inhibitor to activate microglia, reverse brain amyloid pathology, and elicit functional improvement in an aged transgenic mouse model. Thus, engagement of novel glial-mediated clearance mechanisms may drive disease-modifying therapeutic benefit with BACE1 inhibition in AD., (Copyright © 2015 the authors 0270-6474/15/356931-06$15.00/0.)

Metformin is the frontline therapy for type II diabetes mellitus. The oral bioavailability of metformin is unexpectedly high, between 40 and 60%, given its hydrophilicity and positive charge at all physiologic pH values. Previous studies in Caco-2 cell monolayers, a cellular model of the human intestinal epithelium, showed that during absorptive transport metformin is taken up into the cells via transporters in the apical (AP) membrane; however, predominant transport to the basolateral (BL) side occurs via the paracellular route because intracellular metformin cannot egress across the BL membrane. Furthermore, these studies have suggested that the AP transporters can contribute to intestinal accumulation and absorption of metformin. Transporter-specific inhibitors as well as a novel approach involving a cocktail of transporter inhibitors with overlapping selectivity were used to identify the AP transporters that mediate metformin uptake in Caco-2 cell monolayers; furthermore, the relative contributions of these transporters in metformin AP uptake were also determined. The organic cation transporter 1, plasma membrane monoamine transporter (PMAT), serotonin reuptake transporter, and choline high-affinity transporter contributed to approximately 25%, 20%, 20%, and 15%, respectively, of the AP uptake of metformin. PMAT-knockdown Caco-2 cells were constructed to confirm the contribution of PMAT in metformin AP uptake because a PMAT-selective inhibitor is not available. The identification of four intestinal transporters that contribute to AP uptake and potentially intestinal absorption of metformin is a significant novel finding that can influence our understanding of metformin pharmacology and intestinal drug-drug interactions involving this highly prescribed drug., (Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics.)

Background and Objectives: The effect of ontogeny in drug-metabolizing enzymes on pediatric pharmacokinetics is poorly predicted. Voriconazole, a potent antifungal, is cleared predominantly via oxidative metabolism and exhibits vastly different pharmacokinetics between adults and children. A physiologically based pharmacokinetic (PBPK) model was developed integrating hepatic in vitro metabolism data with physiologic parameters to predict pharmacokinetic parameters of voriconazole in adult and pediatric populations., Methods: Adult and pediatric PBPK models integrated voriconazole physicochemical properties with hepatic in vitro data into the models. Simulated populations contained 100 patients (10 trials with 10 patients each). Trial design and dosing was based on published clinical trials. Simulations yielded pharmacokinetic parameters that were compared against published values and visual predictive checks were employed to validate models., Results: All adult models and the pediatric intravenous model predicted pharmacokinetic parameters that corresponded with observed values within a 20% prediction error, whereas the pediatric oral model predicted an oral bioavailability twofold higher than observed ranges. After incorporating intestinal first-pass metabolism into the model, the prediction of oral bioavailability improved substantially, suggesting that voriconazole is subject to intestinal first-pass metabolism in children, but not in adults., Conclusions: The PBPK approach used in this study suggests a mechanistic reason for differences in bioavailability between adults and children. If verified, this would be the first example of differential first-pass metabolism in children and adults.

Objective: The objective of this commentary is to explore the potential role of the pharmacist in the field of global health by understanding the definition of global health and how it can be applied to the profession of pharmacy., Summary: While the role of the pharmacist in public health has been defined, the literature is limited with respect to the role of the pharmacist in global health. It is suggested that the "global" in global health should refer to the scope of a problem and not necessarily the geographic location. Through this lens, pharmacists have the opportunity to play an important role in both public health and global health. In particular, pharmacists can look at the varied global health careers established in medicine and use this as a framework to understand the potential role of the pharmacist within global health practice and program delivery, research, and policy., Conclusions: To further define the role of the pharmacist in global health, pharmacists may have to change their perception of what services they can provide and where these services can be applied.

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.

Our knowledge regarding the molecular pathophysiology underlying anxiety disorders remains incomplete. Increasing evidence points to a role of glutamate in anxiety. The group III metabotropic glutamate receptors (mGlu4, mGlu6, mGlu7 and mGlu8 receptors) remain the least investigated glutamate receptor subtypes partially due to a delay in the development of specific pharmacological tools. Early work using knockout animals and pharmacological tools aimed at investigating the role of mGlu7 receptor in the pathophysiology of anxiety disorders has yielded exciting yet not always consistent results. To further investigate the role this receptor plays in anxiety-like behaviour, we knocked down mGlu7 receptor mRNA levels in the adult mouse brain using siRNA delivered via an osmotic minipump. This reduced anxiety-like behaviour in the light-dark box coupled with an attenuation of stress-induced hyperthermia (SIH) and a reduction of the acoustic startle response (ASRs) in the fear-potentiated startle paradigm (FPS). These effects on anxiety-like behaviour were independent of any impairment of locomotor activity and surprisingly, no behavioural changes were observed in the forced swim test (FST), which is in contrast to mGlu7 receptor knockout animals. Furthermore, the previously reported epilepsy-prone phenotype seen in mGlu7 receptor knockout animals was not observed following siRNA-induced knockdown of the receptor. These data suggest targeting mGlu7 receptors with selective antagonist drugs may be an effective and safe strategy for the treatment of anxiety disorders., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Organic cation transporters (OCTs) are members of the solute carrier 22 family of transporter proteins that are involved in absorption, distribution, and excretion of organic cations. OCT3 is localized in the apical (AP) membrane of enterocytes, but the literature is ambiguous about OCT1 (mOct1) localization, with some evidence suggesting a basolateral (BL) localization in human and mouse enterocytes. This is contrary to our preliminary findings showing AP localization of OCT1 in Caco-2 cell monolayers, an established model of human intestinal epithelium. Therefore, this study aims at determining the localization of OCT1 (mOct1) in Caco-2 cells, and human and mouse enterocytes. Functional studies using OCT1-specific substrate pentamidine showed transporter-mediated AP but not BL uptake in Caco-2 cells and human and mouse intestinal tissues. OCT1 inhibition decreased AP uptake of pentamidine by ∼50% in all three systems with no effect on BL uptake. A short hairpin RNA-mediated OCT1 knockdown in Caco-2 cells decreased AP uptake of pentamidine by ∼50% but did not alter BL uptake. Immunostaining and confocal microscopy in all three systems confirmed AP localization of OCT1 (mOct1). Our studies unequivocally show AP membrane localization of OCT1 (mOct1) in Caco-2 cells and human and mouse intestine. These results are highly significant as they will require reinterpretation of previous drug disposition and drug-drug interaction studies where conclusions were drawn assuming BL localization of OCT1 in enterocytes. Most importantly, these results will require revision of the regulatory guidance for industry in the United States and elsewhere because it has stated that OCT1 is basolaterally localized in enterocytes.

Sorafenib is an orally active tyrosine kinase inhibitor used in the treatment of renal and hepatocellular carcinoma. This study was designed to establish whether transport proteins are involved in the hepatic uptake of sorafenib and to determine the extent of biliary excretion of sorafenib and its metabolites in human hepatocytes. Initial uptake was assessed in freshly isolated, suspended human hepatocytes in the presence of inhibitors and modulators. [(14)C]Sorafenib (1 µM) uptake at 4°C was reduced by about 61-63% of the uptake at 37°C, suggesting a high degree of passive diffusion. Hepatocyte uptake of [(14)C]sorafenib was not Na(+) dependent or influenced by the organic anion transporter 2 inhibitor ketoprofen. However, initial [(14)C]sorafenib hepatocyte uptake was reduced by 46 and 30% compared with control values in the presence of the organic anion transporting polypeptide inhibitor rifamycin SV and the organic cation transporter (OCT) inhibitor decynium 22, respectively. [(14)C]Sorafenib (0.5-5 µM) uptake was significantly higher in hOCT1-transfected Chinese hamster ovary cells compared with mock cells, and inhibited by the general OCT inhibitor, 1-methyl-4-phenylpryidinium. OCT1-mediated uptake was saturable with a Michaelis-Menten constant of 3.80 ± 2.53 µM and a V(max) of 116 ± 42 pmol/mg/min. The biliary excretion index and in vitro biliary clearance of sorafenib (1 µM) in sandwich-cultured human hepatocytes were low (∼11% and 11 ml/min/kg, respectively). Results suggest that sorafenib uptake in human hepatocytes occurs via passive diffusion, by OCT1, and by organic anion transporting polypeptide(s). Sorafenib undergoes modest biliary excretion, predominantly as a glucuronide conjugate(s).

The nociceptin/orphanin FQ (N/OFQ) peptide (NOP) receptor is the fourth and most recently discovered member of the opioid receptor superfamily that also includes μ, δ, and κ opioid receptor subtypes (MOR, DOR, and KOR, respectively). The widespread anatomic distribution of the NOP receptor enables the modulation of several physiologic processes by its endogenous agonist, N/OFQ. Accordingly, the NOP receptor has gained a lot of attention as a potential target for the development of ligands with therapeutic use in several pathophysiological states. NOP receptor activation frequently results in effects opposing classic opioid receptor action; therefore, regulation of the NOP receptor and conditions affecting its modulatory tone are important to understand. Mounting evidence reveals a heterologous interaction of the NOP receptor with other G protein-coupled receptors, including MOR, DOR, and KOR, which may subsequently influence their function. Our focus in this review is to summarize and discuss the findings that delineate the cellular mechanisms of NOP receptor signaling and regulation and the regulation of other receptors by N/OFQ and the NOP receptor.

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.

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.

Huntington's disease is caused by expression of a mutant form of Huntingtin protein containing an expanded polyglutamine repeat. One possible treatment for Huntington's disease may be to reduce expression of mutant Huntingtin in the brain via RNA interference. Unless the therapeutic molecule is designed to be allele-specific, both wild-type and mutant protein will be suppressed by an RNA interference treatment. A key question is whether suppression of wild-type as well as mutant Huntingtin in targeted brain regions can be tolerated and result in a net benefit to patients with Huntington's disease. Whether Huntingtin performs essential functions in the adult brain is unclear. Here, we tested the hypothesis that the adult primate brain can tolerate moderately reduced levels of wild-type Huntingtin protein for an extended period of time. A serotype 2 adeno-associated viral vector encoding for a short hairpin RNA targeting rhesus huntingtin messenger RNA (active vector) was bilaterally injected into the striatum of four adult rhesus monkeys. Four additional animals received a comparable vector encoding a scrambled control short hairpin RNA (control vector). General health and motor behaviour were monitored for 6 months. Upon termination, brain tissues were sampled and assessed blindly for (i) huntingtin messenger RNA knockdown; (ii) Huntingtin protein expression; and (iii) neuropathological changes. Reduction in wild-type huntingtin messenger RNA levels averaging ∼30% was measured in the striatum of active vector recipients 6 months post-injection. A widespread reduction in Huntingtin protein levels was also observed by immunohistochemistry in these animals, with an average protein reduction of ∼45% relative to controls measured by western blot analysis in the putamen of active vector recipients. As with control vector recipients, no adverse effects were observed behaviourally, and no neurodegeneration was found on histological examination of active vector recipients. Our results suggest that long-term partial suppression of wild-type Huntingtin may be safe, and thus if a comparable level of suppression of mutant Huntingtin is beneficial, then partial suppression of both wild-type and mutant Huntingtin may result in a net benefit in patients with heterozygous Huntington's disease.

Fexofenadine is a nonsedative antihistamine that exhibits good oral bioavailability despite its zwitterionic chemical structure and efflux by P-gp. Evidence exists that multiple uptake and efflux transporters play a role in hepatic disposition of fexofenadine. However, the roles of specific transporters and their interrelationship in intestinal absorption of this drug are unclear. This study was designed to elucidate vectorial absorptive transport of fexofenadine across Caco-2 cells involving specific apical uptake and efflux transporters as well as basolateral efflux transporters. Studies with cellular models expressing single transporters showed that OATP2B1 expression stimulated uptake of fexofenadine at pH 6.0. Apical uptake of fexofenadine into Caco-2 cells was decreased by 45% by pretreatment with estrone 3-sulfate, an OATP inhibitor, at pH 6.0 but not at pH 7.4, indicating that OATP2B1 mediates apical uptake of fexofenadine into these cells. Examination of fexofenadine efflux from preloaded Caco-2 cells in the presence or absence of (i) the MRP inhibitor MK-571 and (ii) the P-gp inhibitor GW918 showed that apical efflux is predominantly mediated by P-gp, with a small contribution by MRP2, whereas basolateral efflux is predominantly mediated by MRP3. These results also showed that while OSTαβ is functionally active in the basolateral membrane of Caco-2 cells, it does not play a role in the export of fexofenadine. MK-571 decreased the absorptive transport of fexofenadine by 17%. However, the decrease in absorptive transport by MK-571 was 42% when P-gp was inhibited by GW918. The results provide a novel insight into a vectorial transport system mainly consisting of apical OATP2B1 and basolateral MRP3 that may play an important role in delivering hydrophilic anionic and zwitterionic drugs such as pravastatin and fexofenadine into systemic circulation upon oral administration.

Micafungin, a new echinocandin antifungal agent, has been used widely for the treatment of various fungal infections in human populations. Micafungin is predominantly cleared by biliary excretion and it binds extensively to plasma proteins. Micafungin body weight-adjusted clearance is higher in neonates than in adults, but the mechanisms underlying this difference are not understood. Previous work had revealed the roles of sinusoidal uptake (Na(+) -taurocholate co-transporting peptide, NTCP; organic anion transporting polypeptide, OATP) as well as canalicular efflux (bile salt export pump, BSEP; breast cancer resistance protein, BCRP) transporters in micafungin hepatobiliary elimination. In the present study, the relative protein expression of hepatic transporters was compared between liver homogenates from neonates and adults. Also, the extent of micafungin binding to serum from neonates and adults was measured in vitro. The results indicate that relative expression levels of NTCP, OATP1B1/3, BSEP, BCRP and MRP3 were similar in neonates and in adults. However, the micafungin fraction unbound (f(u) ) in neonatal serum was about 8-fold higher than in the adult serum (0.033±0.012 versus 0.004±0.001, respectively). While there was no evidence for different intrinsic hepatobiliary clearance of micafungin between neonates and adults, our data suggest that age-dependent serum protein binding of micafungin is responsible for its higher clearance in neonates compared with adults., (Copyright © 2011 John Wiley & Sons, Ltd.)

Phospholipase C (PLC) enzymes are an important family of regulatory proteins involved in numerous cellular functions, primarily through hydrolysis of the polar head group from inositol-containing membrane phospholipids. U73122 (1-(6-((17β-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione), one of only a few small molecules reported to inhibit the activity of these enzymes, has been broadly applied as a pharmacological tool to implicate PLCs in diverse experimental phenotypes. The purpose of this study was to develop a better understanding of molecular interactions between U73122 and PLCs. Hence, the effects of U73122 on human PLCβ3 (hPLCβ3) were evaluated in a cell-free micellar system. Surprisingly, U73122 increased the activity of hPLCβ3 in a concentration- and time-dependent manner; up to an 8-fold increase in enzyme activity was observed with an EC50=13.6±5 μm. Activation of hPLCβ3 by U73122 required covalent modification of cysteines as evidenced by the observation that enzyme activation was attenuated by thiol-containing nucleophiles, l-cysteine and glutathione. Mass spectrometric analysis confirmed covalent reaction with U73122 at eight cysteines, although maximum activation was achieved without complete alkylation; the modified residues were identified by LC/MS/MS peptide sequencing. Interestingly, U73122 (10 μm) also activated hPLCγ1 (>10-fold) and hPLCβ2 (∼2-fold); PLCδ1 was neither activated nor inhibited. Therefore, in contrast to its reported inhibitory potential, U73122 failed to inhibit several purified PLCs. Most of these PLCs were directly activated by U73122, and a simple mechanism for the activation is proposed. These results strongly suggest a need to re-evaluate the use of U73122 as a general inhibitor of PLC isozymes.

The purpose of the present study was to elucidate the transport mechanisms responsible for elimination of micafungin, a new semisynthetic echinocandin antifungal agent, which is predominantly cleared by biliary excretion in humans and rats. In vitro studies using sandwich-cultured rat and human hepatocytes were conducted. Micafungin uptake occurred primarily (∼75%) by transporter-mediated mechanisms in rat and human. Micafungin uptake into hepatocytes was inhibited by taurocholate (K(i) = 61 μM), Na(+) depletion (45-55% reduced), and 10 μM rifampin (20-25% reduced); these observations support the involvement of Na(+)-taurocholate-cotransporting polypeptide (NTCP/Ntcp) and, to a lesser extent, organic anion-transporting polypeptides in the hepatic uptake of micafungin. The in vitro biliary clearance of micafungin, as measured by the B-CLEAR technique, amounted to 14 and 19 μl/(min · mg protein) in human and rat, respectively. In vitro biliary excretion of micafungin was reduced by 80 and 75% in the presence of the bile salt export pump (BSEP) inhibitors taurocholate (100 μM) and nefazodone (25 μM), respectively. Biliary excretion of micafungin also was reduced in the presence of breast cancer resistance protein inhibitors [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 μM) and fumitremorgin C (10 μM)]. In vitro biliary excretion of micafungin was not significantly altered by coincubation with P-glycoprotein or multidrug resistance-associated protein 2 inhibitors. These results suggest that NTCP/Ntcp and BSEP/Bsep are primarily responsible for hepatobiliary disposition of micafungin in human and rat. Interference with hepatic bile acid disposition could be one mechanism underlying hepatotoxicity associated with micafungin in some patients.

Adefovir dipivoxil is a diester prodrug of the antiviral drug adefovir, with much greater oral bioavailability than adefovir. Evidence shows that the prodrug is metabolized to adefovir in the enterocytes during intestinal absorption. However, it is unknown how the highly charged and hydrophilic adefovir crosses the basolateral membrane in the intestine. This study determines the role of specific basolateral transporter(s) in the egress of adefovir across the basolateral membrane when formed from adefovir dipivoxil in Caco-2 cells, a model for intestinal epithelium. Multidrug resistance-associated protein 4 (MRP4) plays an important role in renal secretion of adefovir. Immunofluorescence images showed that MRP4 is localized in the basolateral membrane of Caco-2 cells. This localization was further confirmed by Western blotting of the apical and basolateral membrane fractions that were isolated by a novel method involving biotinylation of respective membrane proteins and affinity enrichment. MRP4-knockdown Caco-2 cells were produced by stable transfection with MRP4-specific siRNA expression plasmid. These cells showed reduced MRP4 protein expression and corresponding reduction in the basolateral egress of adefovir when adefovir dipivoxil was dosed on the apical side. A comparison of these data with the reduction in the basolateral egress of adefovir by the general MRP inhibitor indomethacin established that MRP4, among MRPs, plays a predominant role in the basolateral egress of adefovir in Caco-2 cells. The results highlight the importance of MRP4 in oral absorption of adefovir dipivoxil, and suggest that significant drug-drug interactions can occur if an MRP4 inhibitor is co-administered with adefovir dipivoxil.

Voriconazole is a broad spectrum antifungal agent for treating life-threatening fungal infections. Its clearance is approximately 3-fold higher in children compared with adults. Voriconazole is cleared predominantly via hepatic metabolism in adults, mainly by CYP3A4, CYP2C19, and flavin-containing monooxygenase 3 (FMO3). In vitro metabolism of voriconazole by liver microsomes prepared from pediatric and adult tissues (n = 6/group) mirrored the in vivo clearance differences in children versus adults, and it showed that the oxidative metabolism was significantly faster in children compared with adults as indicated by the in vitro half-life (T(1/2)) of 33.8 + or - 15.3 versus 72.6 + or - 23.7 min, respectively. The K(m) for voriconazole metabolism to N-oxide, the major metabolite formed in humans, by liver microsomes from children and adults was similar (11 + or - 5.2 versus 9.3 + or - 3.6 microM, respectively). In contrast, apparent V(max) was approximately 3-fold higher in children compared with adults (120.5 + or - 99.9 versus 40 + or - 13.9 pmol/min/mg). The calculated in vivo clearance from in vitro data was found to be approximately 80% of the observed plasma clearance values in both populations. Metabolism studies in which CYP3A4, CYP2C19, or FMO was selectively inhibited provided evidence that contribution of CYP2C19 and FMO toward voriconazole N-oxidation was much greater in children than in adults, whereas CYP3A4 played a larger role in adults. Although expression of CYP2C19 and FMO3 is not significantly different in children versus adults, these enzymes seem to contribute to higher metabolic clearance of voriconazole in children versus adults.

Although immunization against amyloid-beta (Abeta) holds promise as a disease-modifying therapy for Alzheimer disease (AD), it is associated with an undesirable accumulation of amyloid in the cerebrovasculature [i.e., cerebral amyloid angiopathy (CAA)] and a heightened risk of micro-hemorrhages. The central and peripheral mechanisms postulated to modulate amyloid with anti-Abeta immunotherapy remain largely elusive. Here, we compared the effects of prolonged intracerebroventricular (i.c.v.) versus systemic delivery of anti-Abeta antibodies on the behavioral and pathological changes in an aged Tg2576 mouse model of AD. Prolonged i.c.v. infusions of anti-Abeta antibodies dose-dependently reduced the parenchymal plaque burden, astrogliosis, and dystrophic neurites at doses 10- to 50-fold lower than used with systemic delivery of the same antibody. Both i.c.v. and systemic anti-Abeta antibodies reversed the behavioral impairment in contextual fear conditioning. More importantly, unlike systemically delivered anti-Abeta antibodies that aggravated vascular pathology, i.c.v.-infused antibodies globally reduced CAA and associated micro-hemorrhages. We present data suggesting that the divergent effects of i.c.v.-delivered anti-Abeta antibodies result from gradually engaging the local (i.e., central) mechanisms for amyloid clearance, distinct from the mechanisms engaged by high doses of anti-Abeta antibodies that circulate in the vasculature following systemic delivery. With robust efficacy in reversing AD-related pathology and an unexpected benefit in reducing CAA and associated micro-hemorrhages, i.c.v.-targeted passive immunotherapy offers a promising therapeutic approach for the long-term management of AD.

The antiparasitic activity of aromatic diamidine drugs, pentamidine and furamidine, depends on their entry into the pathogenic protozoa via membrane transporters. However, no such diamidine transporter has been identified in mammalian cells. The goal of this study is to investigate whether these dicationic drugs are substrates for human organic cation transporters (hOCTs, solute carrier family 22A1-3) and whether hOCTs play a role in their tissue distribution, elimination, and toxicity. Inhibitory and substrate activities of pentamidine and furamidine were studied in stably transfected Chinese hamster ovary (CHO) cells. The results of [(3)H]1-methyl-4-phenylpyridinium uptake study showed that pentamidine is a potent inhibitor for all three OCT isoforms (IC50 < 20 microM), whereas furamidine is a potent inhibitor for hOCT1 and hOCT3 (IC50 < 21 microM) but a less potent inhibitor for hOCT2 (IC50 = 189.2 microM). Both diamidines are good substrates for hOCT1 (Km = 36.4 and 6.1 microM, respectively), but neither is a substrate for hOCT2 or hOCT3. The cytotoxicity of pentamidine and furamidine was 4.4- and 9.3-fold greater, respectively, in CHO-hOCT1 cells compared with the mock cells. Ranitidine, an hOCT1 inhibitor, reversed this hOCT1-mediated potentiation of cytotoxicity. This is the first finding that dicationic drugs, such as pentamidine and furamidine, are substrates for hOCT1. In humans, aromatic diamidines are primarily eliminated in the bile but are distributed and cause toxicity in both liver and kidney. These transporters may play important roles in the disposition of aromatic diamidines in humans, as well as resultant drug-drug interactions and toxicity involving diamidine drugs.

Formation and extinction of aversive memories in the mammalian brain are insufficiently understood at the cellular and molecular levels. Using the novel metabotropic glutamate receptor 7 (mGluR7) agonist AMN082, we demonstrate that mGluR7 activation facilitates the extinction of aversive memories in two different amygdala-dependent tasks. Conversely, mGluR7 knockdown using short interfering RNA attenuated the extinction of learned aversion. mGluR7 activation also blocked the acquisition of Pavlovian fear learning and its electrophysiological correlate long-term potentiation in the amygdala. The finding that mGluR7 critically regulates extinction, in addition to acquisition of aversive memories, demonstrates that this receptor may be relevant for the manifestation and treatment of anxiety disorders.

The purpose of the study was to elucidate mechanisms of metformin absorptive transport to explain the dose-dependent absorption observed in humans. Apical (AP) and basolateral (BL) uptake and efflux as well as AP to BL (absorptive) transport across Caco-2 cell monolayers were evaluated over a range of concentrations. Transport was concentration-dependent and consisted of saturable and nonsaturable components (K(m) approximately 0.05 mM, J(max) approximately 1.0 pmol min(-1) cm(-2), and K(d, transport) approximately 10 nl min(-1) cm(-2)). AP uptake data also revealed the presence of saturable and nonsaturable components (K(m) approximately 0.9 mM, V(max) approximately 330 pmol min(-1) mg of protein(-1), and K(d, uptake) approximately 0.04 microl min(-1) mg of protein(-1)). BL efflux was rate-limiting to transcellular transport of metformin; AP efflux was 7-fold greater than BL efflux and was not inhibited by 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 (GW918), a P-glycoprotein inhibitor. AP efflux was trans-stimulated by metformin and prototypical substrates of organic cation transporters, suggesting that a cation-specific bidirectional transport mechanism mediated the AP efflux of metformin. BL efflux of intracellular metformin was much less efficient in comparison with the overall transport, with BL efflux clearance accounting for approximately 7 and approximately 13% of the overall transport clearance at 0.05 and 10 mM metformin concentrations, respectively. Kinetic modeling of cellular accumulation and transport processes supports the finding that transport occurs almost exclusively via the paracellular route (approximately 90%) and that the paracellular transport is saturable. This report provides strong evidence for a saturable mechanism in the paracellular space and provides insight into possible mechanisms for the dose dependence of metformin absorption in vivo.

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.

Epithelial cell monolayers are routinely used to evaluate efficacy of paracellular permeability enhancers (PPEs). The purpose of the present work was to investigate how biorelevant refinements to the Caco-2 cell model impact in vitro efficacy (decrease in transepithelial electrical resistance and increase in mannitol permeability) of PPEs. Standard transport buffer was replaced by fasted-state simulated intestinal fluid (FaSSIF) or serum; or stirring was performed to decrease the unstirred water layer thickness. Apical FaSSIF significantly reduced the efficacy of amphiphilic PPEs palmitoylcarnitine and hexadecylphosphocholine and reduced the amount of these PPEs associated with cells. In contrast, FaSSIF did not affect efficacy of nonamphiphilic PPEs, ethylenediaminetetraacetic acid or 3-nitrocoumarin. Basolateral serum increased the transepithelial flux of PPEs, but did not lessen their potency. Stirring increased the flux of all PPEs, and also enhanced the potency of the amphiphilic PPEs. These results show that inclusion of FaSSIF and agitation in the cellular models significantly alter the efficacy of amphiphilic PPEs but not of hydrophilic or lipophilic PPEs. Future studies should be directed at evaluating the ability to these refined in vitro systems to predict in vivo effects of PPEs.

Psychiatric and neurological disorders are among the most complex, poorly understood and debilitating diseases in medicine. Abrogating gene function using knockout animals is one of the primary means of examining the pathophysiological significance of a given gene product and has been used successfully in models of neuropsychiatric disorders. However, the developmental compensations that may potentially arise from such approaches are problematic and difficult to assess. The recent discovery of RNAi (RNA interference), as a highly efficient method for gene knockdown, has opened up the possibility for its application in examining the potential role of genes in adult brain function and/or disorders. Recent efforts have focused on applying RNAi-based knockdown to understand the genes implicated in neuropsychiatric disorders. We have developed a method of gene knockdown involving chronic infusion of siRNA (short interfering RNA) using osmotic minipumps. We have silenced a number of genes including those for the serotonin and dopamine transporter. Such tailoring of tools that deliver RNAi in the brain will significantly aid in our understanding of the complex pathophysiology of neuropsychiatric disorders where there is an immensely unmet medical need.

Metabolism by cytochrome P4503A (CYP3A) and P-glycoprotein (P-gp)-mediated efflux are two important biochemical barriers to drug absorption from the intestine. CYP3A, the most important family of drug-metabolizing enzymes, shares many substrates with the efflux transporter P-gp. Although the individual impact of these two systems on drug disposition is routinely assessed, the effect of both systems acting together during intestinal absorption is difficult to ascertain. Pharmacokinetic theory predicts that the effect of efflux on overall metabolism depends on substrate concentrations relative to the respective kinetic parameters of these processes (i.e. affinities for transport and metabolism, as well as the capacities of these processes). Researchers have published conflicting findings on how efflux affects metabolism. Furthermore, the in vitro parameters that have been used to explain or predict this interation are more relevant for describing overall changes in extraction efficiency of the system (intestinal epithelium), rather than deconvoluting the effect of P-gp on CYP3A-mediated metabolism. Developing a more refined way to understand this interplay and its potential relevance to drug absorption is an important goal, as a large proportion of marketed drugs and many modern drug discovery candidates are known to be affected by one or both of these proteins.

Purpose: The purpose of this work was to investigate the involvement of carrier-mediated apical (AP) uptake and efflux mechanisms in the absorptive intestinal transport of the hydrophilic cationic drug ranitidine in Caco-2 cells., Methods: Absorptive transport and AP uptake of ranitidine were determined in Caco-2 cells as a function of concentration. Permeability of ranitidine in the absorptive and secretory directions was assessed in the absence or presence of the P-glycoprotein (P-gp) inhibitor, GW918. Characterization of the uptake mechanism was performed with respect to inhibitor specificity, pH, energy, membrane potential, and Na+ dependence. Efflux from preloaded monolayers was evaluated over a range of concentrations and in the absence or presence of high extracellular ranitidine concentrations., Results: Saturable absorptive transport and AP uptake of ranitidine were observed with Km values of 0.27 and 0.45 mM, respectively. The ranitidine absorptive permeability increased and secretory permeability decreased upon inhibition of P-gp. AP ranitidine uptake was inhibited in a concentration-dependent fashion by a diverse set of organic cations including tetraethylammonium, 1-methyl-4-phenylpyridinium, famotidine, and quinidine. AP ranitidine uptake was pH and membrane potential dependent and reduced under conditions that deplete metabolic energy. Efflux of [3H]ranitidine across the basolateral membrane was neither saturable as a function of concentration nor trans stimulated by unlabeled ranitidine., Conclusions: Saturable absorptive transport of ranitidine in Caco-2 cells is partially mediated via a pH-dependent uptake transporter for organic cations and is subject to attenuation by P-gp. Inhibition and driving force studies suggest the uptake carrier exhibits similar properties to cloned human organic cation transporters. The results also imply ranitidine transport is not solely restricted to the paracellular space.

Purpose: The mechanism of intestinal drug transport for hydrophilic cations such as ranitidine is complex, and evidence suggests a role for carrier-mediated apical (AP) uptake and saturable paracellular mechanisms in their overall absorptive transport. The purpose of this study was to develop a model capable of describing the kinetics of cellular accumulation and transport of ranitidine in Caco-2 cells, and to assess the relative contribution of the transcellular and paracellular routes toward overall ranitidine transport., Methods: Cellular accumulation and absorptive transport of ranitidine were determined in the absence or presence of uptake and efflux inhibitors and as a function of concentration over 60 min in Caco-2 cells. A three-compartment model was developed, and parameter estimates were utilized to assess the expected relative contribution from transcellular and paracellular transport., Results: Under all conditions, ranitidine absorptive transport consisted of significant transcellular and paracellular components. Inhibition of P-glycoprotein decreased the AP efflux rate constant (k21) and increased the relative contribution of the transcellular transport pathway. In the presence of quinidine, both the AP uptake rate constant (k12) and k21 decreased, resulting in a predominantly paracellular contribution to ranitidine transport. Increasing the ranitidine donor concentration decreased k12 and the paracellular rate constant (k13). No significant changes were observed in the relative contribution of the paracellular and transcellular routes as a function of ranitidine concentration., Conclusions: These results suggest the importance of uptake and efflux transporters as determinants of the relative contribution of transcellular and paracellular transport for ranitidine, and provide evidence supporting a concentration-dependent paracellular transport mechanism. The modeling approach developed here may also be useful in estimating the relative contribution of paracellular and transcellular transport for a wide array of drugs expected to utilize both pathways.

Psychiatric and neurological disorders are among the most complex, poorly understood, and debilitating diseases in medicine. The burgeoning advances in functional genomic technologies have led to the identification of a vast number of novel genes that are potentially implicated in the pathophysiology of such disorders. However, many of these candidate genes have not yet been functionalized and require validation in vivo. Traditionally, abrogating gene function is one of the primary means of examining the physiological significance of a given gene product. Several methods have been developed for gene ablation or knockdown, however, with limited levels of success. The recent discovery of RNA interference (RNAi), as a highly efficient method for gene knockdown, has been one of the major breakthroughs in molecular medicine. In vivo application of RNAi is further demonstrating the promise of this technology. Recent efforts have focused on applying RNAi-based knockdown to understand the genes implicated in neuropsychiatric disorders. However, the greatest challenge with this approach is translating the success of RNAi from mammalian cell cultures to the brain in animal models of disease and, subsequently, in patients. In this review, we describe the various methods that are being developed to deliver RNAi into the brain for down-regulating gene expression and subsequent phenotyping of genes in vivo. We illustrate the utility of various approaches with a few successful examples and also discuss the potential benefits and pitfalls associated with the use of each delivery approach. Appropriate tailoring of tools that deliver RNAi in the brain may not only aid our understanding of the complex pathophysiology of neuropsychiatric disorders, but may also serve as a valuable therapy for disorders, where there is an immense unmet medical need.

RNA interference (RNAi) is a natural mechanism for regulating gene expression, which exists in plants, invertebrates, and mammals. We investigated whether non-viral infusion of short interfering RNA (siRNA) by the intracerebroventricular route would enable a sequence-specific gene knockdown in the mouse brain and whether the knockdown translates into disease-relevant behavioral changes. Initially, we targeted enhanced green fluorescent protein (EGFP) in mice overexpressing EGFP. A selective knockdown of both EGFP protein and mRNA was observed throughout the brain, with lesser down-regulation in regions distal to the infusion site. We then targeted endogenous genes, encoding the dopamine (DAT) and serotonin transporters (SERT). DAT-siRNA infusion in adult mice produced a significant down-regulation of DAT mRNA and protein and elicited hyperlocomotion similar, but delayed, to that produced on infusion of GBR-12909, a potent and selective DAT inhibitor. Similarly, SERT-siRNA infusion resulted in significant knockdown of SERT mRNA and protein and elicited reduced immobility in the forced swim test similar to that obtained on infusion of citalopram, a very selective and potent SSRI. Application of this non-viral RNAi approach may accelerate target validation for neuropsychiatric disorders that involve a complex interplay of gene(s) from various brain regions.

Human organic cation transporters (hOCTs) are expressed in organs of drug absorption and elimination and play an important role in the uptake and elimination of xenobiotics. The purpose of this study was to evaluate the substrate and inhibitory activity of the H2-receptor antagonists ranitidine and famotidine toward hOCTs and to determine the hOCT isoforms involved in the absorption and elimination of these compounds in humans. Inhibition and substrate specificity of hOCT1, hOCT2, and hOCT3 for ranitidine and famotidine were elucidated in cRNA-injected Xenopus laevis oocytes. Ranitidine and famotidine exhibited similarly potent inhibition of [3H]1-methyl-4-phenyl pyridinium uptake into hOCT1-expressing (IC50= 33 and 28 microM, respectively) and hOCT2-expressing oocytes (IC50= 76 and 114 microM, respectively). Famotidine exhibited potent inhibition of hOCT3; in contrast, ranitidine was a moderately weak inhibitor (IC50= 6.7 and 290 microM, respectively). [3H]Ranitidine uptake was stimulated by hOCT1 (Km= 70 +/- 9 microM) and to a much smaller extent by hOCT2. No stimulation of [3H]ranitidine uptake was observed in hOCT3-expressing oocytes. trans-Stimulation and electrophysiology studies suggested that famotidine also is an hOCT1 substrate and exhibits poor or no substrate activity toward hOCT2 and hOCT3. Thus, hOCT1, which is expressed in the intestine and liver, is likely to play a major role in the intestinal absorption and hepatic disposition of ranitidine and famotidine in humans, whereas hOCT2, the major isoform present in the kidney, may play only a minor role in their renal elimination. Famotidine seems to be one of the most potent inhibitors of hOCT3 yet identified.

In this study, we investigate the molecular mechanisms by which acute orphanin FQ/nociceptin (OFQ/N), acting through the nociceptin opioid peptide (NOP) receptor, desensitizes the mu-opioid receptor. We described previously the involvement of protein kinase C and G-protein-coupled receptor kinases (GRK) 2 and 3 in OFQ/N-induced mu receptor desensitization. Because phosphorylation of the mu receptor triggers the successive regulatory mechanisms responsible for desensitization, such as receptor uncoupling, internalization, and down-regulation, we investigated the ability of OFQ/N to modulate [d-Ala(2),N-Me-Phe(4),Gly(5)-ol]-enkephalin (DAMGO)-induced mu receptor phosphorylation in BE(2)-C human neuroblastoma cells transfected with epitope-tagged mu receptors. OFQ/N treatment (100 nM, 60 min) potentiated DAMGO-induced mu receptor phosphorylation; inhibition of GRK2 or protein kinase C concomitant with OFQ/N treatment blocked the OFQ/N-mediated increase in DAMGO-induced phosphorylation. Inclusion of the NOP antagonist peptide III-BTD during OFQ/N pretreatment blocked the potentiation of DAMGO-induced phosphorylation by OFQ/N, which is consistent with the potentiation being mediated via actions of the NOP receptor. In addition, in cells expressing mu receptors in which the GRK-mediated phosphorylation site Ser(375) was mutated to alanine, OFQ/N treatment failed to potentiate DAMGO-induced mu receptor phosphorylation and failed to desensitize the mu receptor. However, DAMGO-induced mu receptor phosphorylation and OFQ/N-induced mu receptor desensitization occurred in cells expressing mu receptors lacking non-GRK phosphorylation sites. These data suggest that OFQ/N binds to NOP receptors and activates protein kinase C, which then increases the ability of GRK2 to phosphorylate the agonist-occupied mu receptor, heterologously regulating homologous mu receptor desensitization.

Selective serotonin reuptake inhibitors (SSRIs) are widely used antidepressant drugs that increase the extracellular levels of serotonin by blocking the reuptake activity of the serotonin transporter (SERT). Although SSRIs elevate brain serotonergic neurotransmission acutely, their full therapeutic effects involve neurochemical adaptations that emerge following chronic drug administration. The adaptive downregulation of SERT has recently been implicated in the therapeutic response of SSRIs. Interestingly, studies using SERT-knockout mice reveal somewhat paradoxical depression-related effects, probably specific to the downregulation of SERT during early development. However, the behavioral significance of SSRI-mediated downregulation of SERT during adulthood is still unknown. We investigated whether somatic gene manipulation, triggered by infusing short interfering RNA (siRNA) into the ventricular system, would enable the downregulation of SERT in the adult mouse brain. Infusing the SERT-targeting siRNA, for 2 weeks, significantly reduced the mRNA levels of SERT in raphe nuclei. Further, a significant, specific and widespread downregulation of SERT-binding sites was achieved in the brain. In contrast, 2-week infusion of the SSRI, citalopram, produced a widespread downregulation of SERT-binding sites, independent of any alterations at the mRNA level. Irrespective of their mechanisms for downregulating SERT in the brain, infusions of SERT-siRNA or citalopram elicited a similar antidepressant-related behavioral response in the forced swim test. These results signify a role for the downregulation of SERT in mediating the antidepressant action of SSRIs in adults. Further, these data demonstrate that siRNA-induced widespread knockdown of gene expression serves as a powerful tool for assessing the function of endogenous genes in the adult brain.

Gene expression analysis implicates an increasing number of novel genes in the brain as potential targets for the treatment of neurological and psychiatric disorders. Frequently, these genes are ubiquitously expressed in the brain and, thus, may contribute to a pathophysiological state through actions in several brain nuclei. Current strategies employing genetically modified animals for in vivo validation of such targets are time-consuming and often limited by developmental adaptations. Somatic gene manipulation using viral-mediated RNA interference (RNAi) has emerged recently, although restricting the target validation to specific brain nuclei. We investigated whether nonviral infusion of short interfering RNA (siRNA) into the ventricular system would enable a sequence-specific gene knockdown. The temporality and extent of siRNA-induced down-regulation were analyzed by targeting a transgene, EGFP, in mice overexpressing EGFP. Extensive knockdown of EGFP was observed, especially in regions adjacent or dorsoventrally and mediolaterally distant to the infusion site (dorsal third ventricle), with lesser knockdown in more distal regions. We challenged our RNAi approach to generate a specific knockdown of an endogenous gene, encoding the dopamine transporter (DAT) in regions (ventral midbrain) far distal to the infusion site. DAT-siRNA infusion in adult mice produced a significant down-regulation of DAT mRNA and protein in the brain and also elicited a temporal hyperlocomotor response similar to that (but delayed) obtained upon infusion of GBR-12909, a pharmacologically selective DAT inhibitor. Application of this nonviral RNAi approach may accelerate target validation for neuropsychiatric disorders that involve a complex interplay of gene(s) from various brain regions.

The H(2) antagonists, ranitidine and famotidine, exhibit saturable absorptive transport across Caco-2 cell monolayers and human intestine via a yet unidentified mechanism. A photoreactive derivative of famotidine has been synthesized and evaluated as a photoaffinity probe for the putative transporter protein(s). The probe irreversibly inhibited ranitidine transport across Caco-2 cell monolayers and irreversibly increased the transepithelial electrical resistance (TEER) after UV activation. Photoaffinity labeling was protected by a molar excess of famotidine.

DB75 (2,5-bis(4-amidinophenyl)furan) is a promising antimicrobial agent against African trypanosomiasis and Pneumocystis carinii pneumonia. However, it suffers from poor oral activity in rodent models for both infections. In contrast, a novel prodrug of DB75, 2,5-bis(4-amidinophenyl)furan-bis-O-methylamidoxime (DB289), has excellent oral activity. DB289 is currently undergoing clinical investigation as a candidate drug to treat primary stage African trypanosomiasis and Pneumocystis carinii pneumonia. In this study, metabolites of DB289 formed after incubation with freshly isolated rat hepatocytes were characterized using liquid chromatography/ion trap mass spectrometry. Administration of DB289 and octadeuterated DB289 in a 1 : 1 mixture greatly facilitated metabolite identification by providing isotope patterns with twin ions separated by 8 m/z units in the ratio 1 : 1, in the extracted ion chromatograms of molecular ions and in the product ion mass spectra of metabolites. Ten metabolites were identified. Series of O-demethylations and N-dehydroxylations led to the metabolic activation of DB289 to DB75 with the production of four intermediate phase I metabolites. Phase II glucuronidation and sulfation led to the formation of four glucuronide and one sulfate metabolites., (Copyright 2004 John Wiley & Sons, Ltd.)