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Background: Dolutegravir is an integrase strand transfer inhibitor (INSTI) licensed for use in HIV-1 infection and is an inhibitor of organic cation transporter 2 (OCT2). This study assessed the effect of dolutegravir on the pharmacokinetics of metformin, an OCT2 substrate., Design: This was an open-label, parallel-group, 3-period crossover study in healthy adult subjects. Subjects were enrolled into 1 of 2 treatment cohorts (15 subjects/cohort) receiving metformin 500 mg q12h for 5 days in period 1; metformin 500 mg q12h plus dolutegravir 50 mg q24h (cohort 1) or 50 mg q12h (cohort 2) for 7 days in period 2; and metformin 500 mg q12h for 10 days in period 3. There were no washout periods between treatments. Effects of dolutegravir on metformin transport and paracellular permeability were evaluated in vitro., Results: Co-administration of dolutegravir 50 mg q24h increased metformin area under the curve(0-τ) by 79% and Cmax by 66%, whereas dolutegravir 50 mg q12h increased metformin area under the curve(0-τ) and Cmax by 145% and 111%, respectively. Metformin t(1/2) remained unchanged. Increased metformin exposure during dolutegravir co-administration returned to period 1 levels after dolutegravir discontinuation in period 3. Co-administration of dolutegravir and metformin was well tolerated. In vitro, dolutegravir was not a clinically relevant inhibitor of OCT1, OCT3, multidrug and toxin extrusion protein 1, multidrug and toxin extrusion protein 2-K, or plasma membrane monoamine transporter, and it did not affect metformin paracellular permeability or uptake into an intestinal cell line., Conclusions: Dolutegravir significantly increased metformin plasma exposure, which can be partially explained by OCT2 inhibition. It is recommended that dose adjustments of metformin be considered to maintain optimal glycemic control when patients are starting/stopping dolutegravir while taking metformin.

1. Cabotegravir (CAB; GSK1265744) is a potent HIV integrase inhibitor in clinical development as an oral lead-in tablet and long-acting injectable for the treatment and prevention of HIV infection. 2. This work investigated if CAB was a substrate for efflux transporters, the potential for CAB to interact with drug-metabolizing enzymes and transporters to cause clinical drug interactions, and the effect of CAB on the pharmacokinetics of midazolam, a CYP3A4 probe substrate, in humans. 3. CAB is a substrate for Pgp and BCRP; however, its high intrinsic membrane permeability limits the impact of these transporters on its intestinal absorption. 4. At clinically relevant concentrations, CAB did not inhibit or induce any of the CYP or UGT enzymes evaluated in vitro and had no effect on the clinical pharmacokinetics of midazolam. 5. CAB is an inhibitor of OAT1 (IC50 0.81 µM) and OAT3 (IC50 0.41 µM) but did not or only weakly inhibited Pgp, BCRP, MRP2, MRP4, MATE1, MATE2-K, OATP1B1, OATP1B3, OCT1, OCT2 or BSEP. 6. Based on regulatory guidelines and quantitative extrapolations, CAB has a low propensity to cause clinically significant drug interactions, except for coadministration with OAT1 or OAT3 substrates.

Dolutegravir (DTG; S/GSK1349572) is a potent HIV-1 integrase inhibitor with a distinct resistance profile and a once-daily dose regimen that does not require pharmacokinetic boosting. This work investigated the in vitro drug transport and metabolism of DTG and assessed the potential for clinical drug-drug interactions. DTG is a substrate for the efflux transporters P-glycoprotein (Pgp) and human breast cancer resistance protein (BCRP). Its high intrinsic membrane permeability limits the impact these transporters have on DTG's intestinal absorption. UDP-glucuronosyltransferase (UGT) 1A1 is the main enzyme responsible for the metabolism of DTG in vivo, with cytochrome P450 (P450) 3A4 being a notable pathway and UGT1A3 and UGT1A9 being only minor pathways. DTG demonstrated little or no inhibition (IC(50) values > 30 μM) in vitro of the transporters Pgp, BCRP, multidrug resistance protein 2, organic anion transporting polypeptide 1B1/3, organic cation transporter (OCT) 1, or the drug metabolizing enzymes CYP1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 3A4, UGT1A1, or 2B7. Further, DTG did not induce CYP1A2, 2B6, or 3A4 mRNA in vitro using human hepatocytes. DTG does inhibit the renal OCT2 (IC(50) = 1.9 μM) transporter, which provides a mechanistic basis for the mild increases in serum creatinine observed in clinical studies. These in vitro studies demonstrate a low propensity for DTG to be a perpetrator of clinical drug interactions and provide a basis for predicting when other drugs could result in a drug interaction with DTG.

Remogliflozin etabonate is the ester prodrug of remogliflozin, a selective sodium-dependent glucose cotransporter-2 inhibitor. This work investigated the absorption, metabolism, and excretion of [(14)C]remogliflozin etabonate in humans, as well as the influence of P-glycoprotein (Pgp) and cytochrome P450 (P450) enzymes on the disposition of remogliflozin etabonate and its metabolites to understand the risks for drug interactions. After a single oral 402 ± 1.0 mg (106 ± 0.3 μCi) dose, [(14)C]remogliflozin etabonate is rapidly absorbed and extensively metabolized. The area under the concentration-time curve from 0 to infinity [AUC((0-∞))] of plasma radioactivity was approximately 14-fold higher than the sum of the AUC((0-∞)) of remogliflozin etabonate, remogliflozin, and 5-methyl-4-({4-[(1-methylethyl)oxy]phenyl}methyl)-1H-pyrazol-3-yl-β-d-glucopyranoside (GSK279782), a pharmacologically active N-dealkylated metabolite. Elimination half-lives of total radioactivity, remogliflozin etabonate, and remogliflozin were 6.57, 0.39, and 1.57 h, respectively. Products of remogliflozin etabonate metabolism are eliminated primarily via renal excretion, with 92.8% of the dose recovered in the urine. Three glucuronide metabolites made up the majority of the radioactivity in plasma and represent 67.1% of the dose in urine, with 5-methyl-1-(1-methylethyl)-4-({4-[(1-methylethyl)oxy]phenyl}methyl)-1H-pyrazol-3-yl-β-d-glucopyranosiduronic acid (GSK1997711) representing 47.8% of the dose. In vitro studies demonstrated that remogliflozin etabonate and remogliflozin are Pgp substrates, and that CYP3A4 can form GSK279782 directly from remogliflozin. A ketoconazole clinical drug interaction study, along with the human mass balance findings, confirmed that CYP3A4 contributes less than 50% to remogliflozin metabolism, demonstrating that other enzyme pathways (e.g., P450s, UDP-glucuronosyltransferases, and glucosidases) make significant contributions to the drug's clearance. Overall, these studies support a low clinical drug interaction risk for remogliflozin etabonate due to the availability of multiple biotransformation pathways.

This study evaluated the utility of oral sulfasalazine as a probe substrate for Breast Cancer Resistance Protein (BCRP; ABCG2) activity by assessing the impact of genetic variation or coadministration of an inhibitor (pantoprazole) on plasma and urine pharmacokinetics of sulfasalazine and metabolites. Thirty-six healthy male subjects prescreened for ABCG2 421CC (reference activity), CA, and AA (lower activity) genotypes (N = 12 each) received a single 500 mg oral dose of enteric coated sulfasalazine alone, with 40 mg pantoprazole, or with 40 mg famotidine (gastrointestinal pH control) in a 3-period, single fixed sequence, crossover design. No significant difference in sulfasalazine or metabolite pharmacokinetics in 421AA or CA compared to 421CC subjects was found; however, high inter-subject variability was observed. Geometric mean (95% CI) sulfasalazine plasma AUC((0-infinity)) values were 32.1 (13.2, 78.1), 16.8 (7.15, 39.6) and 62.7 (33.4, 118) microg h/mL, and C(max) were 4.01 (1.62, 9.92), 1.70 (0.66, 4.40), and 6.86 (3.61, 13.0) microg/mL for CC, CA, and AA subjects, respectively. Pantoprazole and famotidine did not affect sulfasalazine pharmacokinetics in any genotypic cohort. These results suggest that the pharmacokinetics of oral, enteric-coated 500 mg sulfasalazine are not sufficiently sensitive to ABCG2 genetic variation or inhibitors to be useful as a clinical probe substrate of BCRP activity., ((c) 2009 Wiley-Liss, Inc. and the American Pharmacists Association.)

The Biopharmaceutics Classification System (BCS) is the scientific basis for classifying drugs based on their aqueous solubility and intestinal permeability that supports in vivo bioavailability and bioequivalence waivers for immediate-release solid dosage form drugs. One requirement of the BCS is that the permeability method must be validated. In order to accommodate the variety of in vitro/in situ permeability models, the BCS Guidance gives a general framework for the validation requirements, necessitating implemented experimental details to be selected by the applicant laboratory. The objective of this work was to define the parameters for a cell based in vitro permeability method (e.g., cell type, pH, transport direction, time, and concentration) and validate the method to support formal BCS classification of drugs. Twenty reference drugs were selected and permeability values determined using the Madin-Darby canine kidney type II cell line heterologously expressing the human P-glycoprotein transporter (MDCKII-MDR1). A rank order relationship was established between the in vitro permeability value and human intestinal absorption values. This relationship was as predicted and validates the MDCKII-MDR1 permeability method as defined by the BCS Guidance. The final validated in vitro permeability method employs the MDCKII-MDR1 cell line incubated with the Pgp inhibitor GF120918. It is a unidirectional apical-to-basolateral transport assay performed at apical pH values of 5.5 and 7.4 and a basolateral pH of 7.4. Four reference standards (metoprolol, pindolol, labetalol and ranitidine) dosed and analyzed as a single cassette are included in each experiment. A strategy on selection of drug concentrations and on how to deal with problematic compounds (i.e., those suffering from poor mass balance) is discussed.

Lapatinib [N-{3-chloro-4-[(3-fluorobenzyl)oxy]phenyl}-6-[5-({[2-(methylsulfonyl)ethyl]amino}methyl)-2-furyl]-4-quinazolinamine, GW572016, Tykerb] is a tyrosine kinase inhibitor approved for use in combination with capecitabine to treat advanced or metastatic breast cancers overexpressing HER2 (ErbB2). In this work we investigated the role of efflux and uptake transporters in lapatinib disposition and drug interactions. In vitro studies evaluated whether lapatinib is a substrate for efflux transporters or an inhibitor of efflux/uptake transporters. In vivo studies included whole-body autoradiography and an evaluation of the role of efflux transporters on the intestinal absorption and brain penetration of lapatinib using chemical or genetic knockout animals. Lapatinib is a substrate for the efflux transporters P-glycoprotein (Pgp) and breast cancer resistance protein (BCRP). Furthermore, lapatinib is an inhibitor (IC(50) values 0.025-5 muM) of Pgp, BCRP, and organic anion transporting polypeptide 1B1 (a hepatic uptake transporter). In contrast, lapatinib yielded little inhibition on renal transporters (organic anion transporters, organic cation transporters, and uric acid transporter). In vivo studies demonstrated that brain concentrations of lapatinib were low and influenced by efflux transporters at the blood-brain barrier. In contrast, systemic exposure of lapatinib after oral dosing was unchanged when efflux by Pgp and BCRP was absent from the gastrointestinal tract. These in vitro and in vivo preclinical investigations provide a mechanistic basis for elucidating clinical drug interactions.

The hepatic excretion of hydrophilic conjugates, end products of phase II metabolism, is not completely understood. In the present studies, transport mechanism(s) responsible for the biliary excretion of 4-methylumbelliferyl glucuronide (4MUG) and 4-methylumbelliferyl sulfate (4MUS) were studied. Isolated perfused livers (IPLs) from Mrp2-deficient (TR(-)) Wistar rats were used to examine the role of Mrp2 in the biliary excretion of 4MUG and 4MUS. After a 30-micromol dose of 4-methylumbelliferone, cumulative biliary excretion of 4MUG was extensive in wild-type rat IPLs (25 +/- 3 micromol) but was negligible in TR(-) livers (0.4 +/- 0.1 micromol); coadministration of the Bcrp and P-glycoprotein inhibitor GF120918 [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] had no effect on 4MUG biliary excretion in wild-type rat IPLs. In contrast, biliary excretion of 4MUS was partially maintained in Mrp2-deficient rat IPLs. Recovery of 4MUS in bile was approximately 50 to 60% lower in both control TR(-) (149 +/- 8 nmol) and wild-type IPLs with GF120918 coadministration (176 +/- 30 nmol) relative to wild-type control livers (378 +/- 37 nmol) and was nearly abolished in TR(-) IPLs in the presence of GF120918 (13 +/- 8 nmol). These changes were the result of decreased rate constants governing 4MUG and 4MUS biliary excretion. In vitro assays and perfused livers from Bcrp and P-glycoprotein gene-knockout mice indicated that 4MUS did not interact with P-glycoprotein but was transported by Bcrp in a GF120918-sensitive manner. In the rat liver, Mrp2 mediates the biliary excretion of 4MUG, whereas both Mrp2 and Bcrp contribute almost equally to the transport of 4MUS into bile.

Because modulation of P-glycoprotein (Pgp) through inhibition or induction can lead to drug-drug interactions by altering intestinal, central nervous system, renal, or biliary efflux, it is anticipated that information regarding the potential interaction of drug candidates with Pgp will be a future regulatory expectation. Therefore, to be able to utilize in vitro Pgp inhibition findings to guide clinical drug interaction studies, the utility of five probe substrates (calcein-AM, colchicine, digoxin, prazosin, and vinblastine) was evaluated by inhibiting their Pgp-mediated transport across multidrug resistance-1-transfected Madin-Darby canine kidney cell type II monolayers with 20 diverse drugs having various degrees of Pgp interaction (e.g., efflux ratio, ATPase, and calcein-AM inhibition). Overall, the rank order of inhibition was generally similar with IC(50) values typically within 3- to 5-fold of each other. However, several notable differences in the IC(50) values were observed. Digoxin and prazosin were the most sensitive probes (e.g., lowest IC(50) values), followed by colchicine, vinblastine, and calcein-AM. Inclusion of other considerations such as a large dynamic range, commercially available radiolabel, and a clinically meaningful probe makes digoxin an attractive probe substrate. Therefore, it is recommended that digoxin be considered as the standard in vitro probe to investigate the inhibition profiles of new drug candidates. Furthermore, this study shows that it may not be necessary to generate IC(50) values with multiple probe substrates for Pgp as is currently done for cytochrome P450 3A4. Finally, a strategy integrating results from in vitro assays (efflux, inhibition, and ATPase) is provided to further guide clinical interaction studies.

Previous reports have demonstrated that sulfate metabolites may be excreted into bile by the multidrug resistance-associated protein 2 (Mrp2, Abcc2). Although recombinant human breast cancer resistance protein (BCRP, ABCG2) has affinity for sulfated xenobiotics and endobiotics, its relative importance in biliary excretion of sulfate metabolites in the intact liver is unknown. In the present studies, the potential contribution of Bcrp1 to the biliary excretion of acetaminophen sulfate (AS) was examined following acetaminophen administration (66 micromol, bolus) to isolated perfused livers (IPLs) from wild-type Wistar and Mrp2-deficient (TR(-)) Wistar rats in the presence or absence of the Bcrp1 and P-glycoprotein inhibitor, GF120918 [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]. Recovery of AS in bile of TR(-) rat livers was approximately 5-fold lower relative to wild-type controls (0.3 +/- 0.1 versus 1.5 +/- 0.3 micromol). In the presence of GF120918, biliary excretion of AS was decreased approximately 2-fold in both TR(-) (0.16 +/- 0.09 micromol) and wild-type (0.8 +/- 0.3 micromol) rat IPLs. These changes were primarily due to alterations in the rate constant governing biliary excretion of AS, which was decreased approximately 90% in TR(-) relative to wild-type rat IPLs (0.02 +/- 0.01 versus 0.2 +/- 0.1 h(-1)) and was further decreased in the presence of GF120918 (0.010 +/- 0.003 and 0.12 +/- 0.05 h(-1); TR(-) and wild-type, respectively). In vitro assays indicated that impaired AS biliary excretion in the presence of GF120918 was due to inhibition of Bcrp1, and not P-glycoprotein. In conclusion, Mrp2 and, to a lesser extent, Bcrp1 mediate biliary excretion of AS in the intact liver.

The absorptive (AQ) and secretory (SQ) quotients have been proposed as a novel experimental approach to quantify the modulation of intestinal absorption and secretion by P-glycoprotein (Pgp). Because these unidirectional assays inherently assess for the impact of Pgp, conclusions as to whether a compound is a Pgp substrate will be made from the data. Therefore, the objective of this study was to establish the relationship between AQ/SQ and the bidirectional efflux assay and to derive criteria to classify a compound as a Pgp substrate. AQ and SQ parameters were calculated for 331 compounds that had previously been evaluated in the bidirectional assay and the concordance of Pgp substrate classification between these methods assessed by establishing AQ/SQ criteria of increasing magnitude. The AQ and SQ values correctly identified 80 and 85% of the compounds as Pgp substrates/nonsubstrates relative to the bidirectional efflux assay. This study demonstrates that the optimal AQ and SQ value to classify compounds as Pgp substrates was 0.3 and provides a basis to deploy unidirectional efflux assays in the early stages of drug discovery, which would benefit from the twofold increase in throughput over current bidirectional transport assays.

GV196771 [E-4,6-dichloro-3-(2-oxo-1-phenyl-pyrrolidin-3-glydenemethyl)-1H-indole-2 carboxylic acid] is a potent antagonist of the modulatory glycine site of the N-methyl-d-aspartate receptor. GV196771 has low oral bioavailability (<10%) and plasma clearance ( approximately 2 ml/min/kg) in rats. P-Glycoprotein (Pgp) and breast cancer resistance protein (Bcrp) are ATP-binding cassette (ABC) transporters that limit the oral absorption of drugs and dietary constituents. The objective of this work was to assess the involvement of Pgp and/or Bcrp on the systemic exposure of GV196771 in mice. In vitro, GV196771 was a Bcrp substrate [basolateral-to-apical/apical-to-basolateral (B-->A/A-->B) ratio = 5.1] with high passive membrane permeability (P(app) = 64-170 nm/s); however, GV196771 was not an in vitro Mdr1a substrate (B-->A/A-->B ratio = 1.9; no effect of GF120918 on efflux ratio). The role of Pgp and Bcrp on the systemic exposure of GV196771 was assessed by pretreatment of wild-type and Pgp-deficient mdr1a/1b(-/-) mice with a single oral dose of GF120918 (50 mg/kg; a dual Pgp and Bcrp inhibitor) or vehicle (0.5% hydroxypropylmethylcellulose and 1% Tween 80) 2 h before administration of a single oral dose of GV196771 (2 mg/kg). Compared with wild-type animals, the GV196771 area under the plasma concentration-time curve [AUC((0-->6 h))] increased 6.2-fold in Pgp-deficient mice, 10.3-fold in GF120918-pretreated wild-type mice, and 16.4-fold in GF120918-pretreated Pgp-deficient mice. C(max) values changed in parallel with the AUC((0-->6 h)) values; however, t(max) remained relatively unchanged. This study supports a role for Pgp and Bcrp in attenuating the systemic exposure of GV196771 in mice and demonstrates that two ABC efflux transporters can have nonredundant roles in attenuating the disposition of a compound.

Purpose: G17DT is a gastrin immunogen, raising antibodies that blockade gastrin-stimulated growth. The aim of the study was to characterise antibody response and assess safety and tolerability of G17DT given to patients with gastric cancer., Experimental Design: G17DT was administered to 52 patients with gastric adenocarcinoma at weeks 0, 2 and 6 by intramuscular injection at doses of 10, 100 and 250 microg. Antibody levels were measured by an ELISA assay. A radioligand displacement assay determined the ability of G17DT-immunised patients' sera to inhibit binding of 125IG17 to cholecystokinin (CCK)-2 receptors., Results: By week 12 of the study, 6/12 evaluable stage I-III patients achieved an antibody response in the 10 microg group, 7/11 in the 100 microg group, and 11/12 in the 250 microg group. Stage IV patients dosed at 250 microg achieved a similar response rate to stage I-III patients dosed at 10 or 100 microg. G17DT was well tolerated in 47/52 patients. Two patients suffered significant adverse reactions including injection site pain and abscess. G17DT antibodies displaced iodinated gastrin from CCK-2 receptors, with the level of displacement correlating with antibody titre., Conclusions: G17DT immunisation is a well-tolerated method of raising functional antibodies to 17 amino acid gastrin forms in patients with gastric carcinomas.

A quantitative structure-activity relationship (QSAR) model has been developed to predict whether a given compound is a P-glycoprotein (Pgp) substrate or not. The training set consisted of 95 compounds classified as substrates or non-substrates based on the results from in vitro monolayer efflux assays. The two-group linear discriminant model uses 27 statistically significant, information-rich structure quantifiers to compute the probability of a given structure to be a Pgp substrate. Analysis of the descriptors revealed that the ability to partition into membranes, molecular bulk, and the counts and electrotopological values of certain isolated and bonded hydrides are important structural attributes of substrates. The model fits the data with sensitivity of 100% and specificity of 90.6% in the jackknifed cross-validation test. A prediction accuracy of 86.2% was obtained on a test set of 58 compounds. Examination of the eight "mispredicted" compounds revealed two distinct categories. Five mispredictions were explained by experimental limitations of the efflux assay; these compounds had high permeability and/or were inhibitors of calcein-AM transport. Three mispredictions were due to limitations of the chemical space covered by the current model. The Pgp QSAR model provides an in silico screen to aid in compound selection and in vitro efflux assay prioritization., (Copyright 2004 Wiley-Liss, Inc. and the American Pharmacists Association.)

Objectives: We have previously reported the ability of D17DT (formerly GnRH-DT) vaccination to produce castrate levels of androgens in men with advanced prostate cancer. This study examines the efficacy and tolerability of 3 and 15 micrograms of D17DT in 12 patients with advanced prostate cancer to establish a dose-response relationship., Methods: 12 patients received either 3 or 15 micrograms of D17DT as 3 deep intramuscular injections over 6 weeks. Outcome was assessed in terms of physical and biochemical evaluations of clinical progression and antibody titres., Results: Significant titres of anti-GnRH antibodies were detected in 2 out of 6 subjects who received 15 micrograms of D17DT; suppression of testosterone to castrate levels accompanied by a significant and prolonged reduction in PSA was also demonstrated. No responses were seen following treatment with 3 micrograms of D17DT., Conclusion: The induction of anti-GnRH antibodies through vaccination with 15 micrograms D17DT can produce and sustain castrate levels of testosterone in men with advanced prostate cancer.

Recent in vitro studies have suggested that P-glycoprotein (Pgp) and passive membrane permeability may influence the brain concentrations of non-sedating (second-generation) antihistamines. The purpose of this study was to determine the importance of Pgp-mediated efflux on the in vivo brain distribution of the non-sedating antihistamine cetirizine (Zyrtec), and the structurally related sedating (first-generation) antihistamine hydroxyzine (Atarax). In vitro MDR1-MDCKII monolayer efflux assays demonstrated that cetirizine was a Pgp substrate (B-->A/A-->B + GF120918 ratio = 5.47) with low/moderate passive permeability (PappB-->A = 56.5 nm/s). In vivo, the cetirizine brain-to-free plasma concentration ratios (0.367 to 4.30) were 2.3- to 8.7-fold higher in Pgp-deficient mice compared with wild-type mice. In contrast, hydroxyzine was not a Pgp substrate in vitro (B-->A/A-->B ratio = 0.86), had high passive permeability (PappB-->A + GF120918 = 296 nm/s), and had brain-to-free plasma concentration ratios >73 in both Pgp-deficient and wild-type mice. These studies demonstrate that Pgp-mediated efflux and passive permeability contribute to the low cetirizine brain concentrations in mice and that these properties account for the differences in the sedation side-effect profiles of cetirizine and hydroxyzine., (Copyright 2003 Wiley-Liss, Inc. and the American Pharmacists Association)

Membrane permeability and P-glycoprotein (Pgp) can be limiting factors for blood-brain barrier penetration. The objectives of this study were to determine whether there are differences in the in vitro permeability, Pgp substrate profiles, and physicochemical properties of drugs for central nervous system (CNS) and non-CNS indications, and whether these differences are useful criteria in selecting compounds for drug development. Apparent permeability (P(app)) and Pgp substrate profiles for 93 CNS (n = 48) and non-CNS (n = 45) drugs were determined by monolayer efflux. Calcein-AM inhibition assays were used to supplement the efflux results. The CNS set (2 of 48, 4.2%) had a 7-fold lower incidence of passive permeability values <150 nm/s compared with the non-CNS set (13 of 45, 28.9%). The majority of drugs (72.0%, 67 of 93) were not Pgp substrates; however, 49.5% (46 of 93) were positive in the calcein-AM assay when tested at 100 microM. The CNS drug set (n = 7 of 48, 14.6%) had a 3-fold lower incidence of Pgp-mediated efflux than the non-CNS drug set (n = 19 of 45, 42.2%). Analysis of 18 physicochemical properties revealed that the CNS drug set had fewer hydrogen bond donors, fewer positive charges, greater lipophilicity, lower polar surface area, and reduced flexibility compared with the non-CNS group (p < 0.05), properties that enhance membrane permeability. This study on a large, diverse set of marketed compounds clearly demonstrates that permeability, Pgp-mediated efflux, and certain physicochemical properties are factors that differentiate CNS and non-CNS drugs. For CNS delivery, a drug should ideally have an in vitro passive permeability >150 nm/s and not be a good (B --> A/A --> B ratio <2.5) Pgp substrate.

P-glycoprotein (Pgp) affects the absorption, distribution, and clearance of a variety of compounds. Thus, identification of compounds that are Pgp substrates can aid drug candidate selection and optimization. Our goal was to evaluate three assays used to determine whether compounds are Pgp substrates. Sixty-six compounds were tested in monolayer efflux, ATPase, and calcein-AM assays. Assay results yielded two categories of compounds. Category I (n = 35) exhibited concordance across the assays. Category II (n = 31) revealed differences among the assays that related to the apparent permeability (P(app)) of the compounds. Within category II, two groups were discerned based on the absence (group IIA, n = 10, nontransported substrates) or presence (group IIB, n = 21, transported substrates) of monolayer efflux. Detection of efflux (group IIB) was associated with compounds having low/moderate P(app) values (mean = 16.6 nm/s), whereas inability to detect efflux (group IIA) was associated with compounds having high P(app) values (mean = 535 nm/s). The calcein-AM and ATPase assays revealed Pgp interactions for highly permeable group IIA compounds but were less responsive than monolayer efflux for low/moderate P(app) compounds of group IIB. All assays detected substrates across a broad range of P(app), but the efflux assay was more prone to fail at high P(app), whereas the calcein-AM and ATPase assays were more prone to fail at low P(app). When P(app) is low, efflux is a greater factor in the disposition of Pgp substrates. The efflux assay is more reliable at low/moderate P(app) and is the method of choice for evaluating drug candidates despite low throughput and reliance on liquid chromatography with tandem mass spectrometry.

Purpose: The objectives of this work were to evaluate the importance of moderate passive permeability on apparent P-glycoprotein (P-gp) kinetics, and demonstrate that inspection of basolateral to apical and apical to basolateral (BL-AP/AP-BL) permeability ratios may result in a compound being overlooked as a P-gp substrate and inhibitor of another drug's transport via P-gp inhibition., Methods: The permeability ratios of nicardipine, vinblastine, cimetidine, and ranitidine were determined across Caco-2 monolayers that express P-gp, in the presence and absence of the specific P-gp inhibitor, GF120918. In addition, the permeability ratio of vinblastine was studied after pretreatment of Caco-2 monolayers with nicardipine, ranitidine, or cimetidine. Similar studies were repeated with hMDRI-MDCK monolayers., Results: The permeability ratios for cimetidine and vinblastine were >2. The permeability ratios for nicardipine and ranitidine were close to unity, and were not affected by the addition of GF120918. Based solely on ratios, only compounds with moderate transcellular permeability (vinblastine and cimetidine) would be identified as P-gp substrates. Although the permeability ratios appeared to be unity for nicardipine and ranitidine, both compounds affected the permeability of vinblastine, and were identified as substrates and inhibitors of P-gp. Studies performed in hMDR1-MDCK cells confirmed these experimental results. Data were explained in the context of a kinetic model, where passive permeability and P-gp efflux contribute to overall drug transport., Conclusions: Moderate passive permeability was necessary for P-gp to reduce the AP-BL drug permeability. Inspection of the permeability ratio after directional transport studies did not effectively identify P-gp substrates that affected the P-gp kinetics of vinblastine. Because of the role of passive permeability, drug interaction studies with known P-gp substrates, rather than directional permeability studies, are needed to elucidate a more complete understanding of P-gp kinetics.

D17DT consists of the GnRH decapeptide linked to diphtheria toxoid. The aim of this pilot study was to assess the tolerance of D17DT and the production of anti-GnRH antibodies from two doses, 30 and 100 microg, in patients with locally advanced prostate cancer. Twelve patients with histologically proven prostate cancer in whom hormonal therapy was indicated were recruited. Patients received either 30 or 100 microg given intramuscularly on three separate occasions over six weeks. Patients were followed up and blood was taken for estimation of serum testosterone, PSA and anti-GnRH antibody titre. Overall the drug was well tolerated. In 5 patients a significant reduction in serum testosterone and PSA was seen. Castrate levels of testosterone were achieved in 4 and maintained for up to 9 months. Patients with the highest antibody titre had the best response in terms of testosterone suppression. This study shows that it is possible to immunize a patient with prostate cancer against GnRH to induce castrate levels of testosterone. This state appears to be reversible. This novel form of immunotherapy may have advantages over conventional forms of hormonal therapy and further studies are warranted in order to try and increase the proportion of responders.

Cell accumulation, transendothelial permeability, and efflux studies were conducted in bovine brain capillary endothelial cells (BBCECs) to assess the role of P-glycoprotein (P-gp) in the blood-brain barrier (BBB) transport of morphine in the presence and absence of P-gp inhibitors. Cellular accumulation of morphine and rhodamine 123 was enhanced by the addition of the P-gp inhibitors N-{4-[2-(1,2,3,4-tetrahydro-6,7dimethoxy-2-isoquinolinyl)-ethyl]-phenyl}-9,10-dihydro-5-methoxy-9- carboxamide (GF120918), verapamil, and cyclosporin A. Positive (rhodamine 123) and negative (sucrose and propranolol) controls for P-gp transport also were assessed. Morphine glucuronidation was not detected, and no alterations in the accumulation of propranolol or sucrose were observed. Transendothelial permeability studies of morphine and rhodamine 123 demonstrated vectorial transport. The basolateral to apical (B:A) fluxes of morphine (50 microM) and rhodamine (1 microM) were approximately 50 and 100% higher than the fluxes from the apical to the basolateral direction (A:B), respectively. Decreasing the extracellular concentration of morphine to 0.1 microM resulted in a 120% difference between the B:A and A:B permeabilities. The addition of GF120918 abolished any significant directionality in transport rates across the endothelial cells. Efflux studies showed that the loss of morphine from BBCECs was temperature- and energy-dependent and was reduced in the presence of P-gp inhibitors. These observations indicate that morphine is transported by P-gp out of the brain capillary endothelium and that the BBB permeability of morphine may be altered in the presence of P-gp inhibitors.

Purpose: To determine the role of P-glycoprotein (Pgp) on the CNS penetration of the HIV protease inhibitor (PI) amprenavir (141W94) and to test the hypothesis that co-administration of a second HIV PI (ritonavir) could enhance amprenavir's brain penetration in vivo., Methods: Pgp-mediated efflux was investigated in vitro with Caco-2 cells and in vivo by whole-body autoradiography (WBA). "Genetic" mdr1a/1b double knockout mice, "chemical" Pgp knockout mice generated by administration of the Pgp inhibitor GF120918, and mice pretreated with ritonavir were used in WBA studies to investigate the effects of Pgp modulation on the CNS penetration of amprenavir., Results: Amprenavir, indinavir, ritonavir, and saquinavir had 2- to 23-fold higher transport rates from the basolateral to apical direction than from the apical to basolateral direction across Caco-2 monolayers. Incubation with GF120918 negated this difference, suggesting that the efflux was Pgp-mediated. WBA studies demonstrated a 13- and 27-fold increase in the brain and a 3.3-fold increase in the CSF concentrations of amprenavir in mice pretreated with GF120918 and in mdr1a/1b double knockout mice. In contrast, pretreatment with ritonavir did not alter the CNS exposure of amprenavir., Conclusions: These results provide evidence that amprenavir and other HIV PIs are Pgp substrates and that co-administration of a specific Pgp inhibitor will enhance amprenavir's CNS penetration in vivo. These results will have an important therapeutic impact in the treatment of AIDS dementia.

Terazosin is a selective alpha 1-adrenoceptor antagonist; its actions on the serum lipoprotein profile were compared with those of the cardioselective beta-adrenoceptor antagonist atenolol in 40 patients with mild to moderate hypertension. Atenolol and terazosin were titrated over six weeks until blood pressure control (diastolic blood pressure less than 90 mmHg or greater than 10 mmHg fall in blood pressure) or a maximum dose of atenolol 100 mg or terazosin 10 mg had been achieved. Patients not controlled were then prescribed additional diuretic therapy (cyclopenthiazide 0.5 mg and potassium 1200 mg). At each visit blood pressure and adverse events were recorded; plasma lipids were measured at baseline, six and 12 weeks. During titration there was a linear decrease in systolic (-29 mmHg on atenolol and -24 mmHg on terazosin) and diastolic blood pressure (-17 and -12 mmHg) in both groups without subsequent change over the next six weeks; atenolol reduced heart rate (-11 bpm) without change on terazosin. During the initial six weeks the total cholesterol fell in both groups; however, there were significant between-treatment differences in triglyceride responses with a fall on terazosin and a rise on atenolol. Comparing atenolol and terazosin over the total 12 week study (irrespective of thiazide treatment) increased triglyceride levels and reduced cholesterol ratios and HDL were demonstrated on atenolol, contrasting with reduced triglyceride levels and elevated HDL and cholesterol ratios of terazosin.(ABSTRACT TRUNCATED AT 250 WORDS)

The relative cost and cost-effectiveness of different methods of screening diabetic patients for sight-threatening retinopathy are assessed. The resource costs per screening visit, both to the health service and to patients, of ophthalmoscopic examination by primary screeners including general practitioners, hospital physicians, and ophthalmic opticians are estimated together with those of a similar screening test by ophthalmological clinical assistants. The total resource cost per screen of screening using non-mydriatic photography is also estimated. Using estimates of sensitivity, specificity, and prevalence generated in the screening of 3318 diabetic patients in three UK centres, the relative cost-effectiveness of screening methods is estimated in terms of their cost per true positive case detected. On the assumption that a patient makes a special trip for eye screening, the cost per true positive case detected for primary screeners ranges from 633 pounds for a GP-screened group in one centre to 1079 pounds for another GP-screened group in a second centre; the cost per true positive case detected of photography ranges from 497 pounds for a camera that is taken to general practices in one centre to 1546 pounds for a hospital-based camera. Relative cost-effectiveness changes if, in some contexts, the screening can take place without requiring an additional patient visit, and is strongly related to the relative sensitivity of the screening methods and to the prior probability (prevalence or incidence) of retinopathy in the diabetic population.

The results of the screening of 3318 diabetic patients for sight-threatening diabetic retinopathy in three UK centres are reported. The aims of the study were to determine the extent of diabetic retinopathy in the screened population and to assess the relative effectiveness of different screening methods in appropriately referring cases from a diabetic population, in a context very close to a routine clinical service. Patients were assessed by ophthalmoscopic examination by an ophthalmological clinical assistant. The clinical assistants' referral grades formed the reference standard against which to assess the effectiveness of other screening methods including ophthalmoscopy by primary screeners who were general practitioners (GPs), ophthalmic opticians and hospital physicians, and the assessment by consultant ophthalmologists of non-mydriatic Polaroid fundus photography. The performance of primary screeners based on ophthalmoscopy ranged from a sensitivity of 0.41, with a specificity of 0.89, for one of the GP groups, to a sensitivity of 0.67, with a specificity of 0.96, for the hospital physician group. The performance of the non-mydriatic camera ranged from a sensitivity of 0.35, with a specificity of 0.95, to a sensitivity of 0.67, with a specificity of 0.98.

Differences in the sensitivity of human and Fischer 344 rat tissues to cadmium sulphate (CdSO(4)) toxicity were investigated in an in vitro model using human and rat nasal turbinate epithelial (NTE) cells. Both rat and human NTE cells were obtained from fresh, normal tissue. Methods were developed for isolating and culturing NTE cells from rat and human tissue using identical procedures, and for measuring the cellular nucleotides by high-performance liquid chromatography. Changes in adenylate energy charge and nucleotide levels were used as toxicity endpoints. Cellular Cd levels were measured by graphite-furnace atomic absorption spectrometry and expressed per unit DNA. Cd uptake was significantly greater in human NTE cells than in rat cells, particularly at the highest exposure concentration (4.8 mm-CdSO(4)). The effects of CdSO(4) on the adenylate energy charge of human and rat NTE cells were similar except at high exposure concentrations and after long exposure times; after a 2-4-hr exposure to 4.8 mm CdSO(4) the adenylate energy charge of human cells was significantly less than that of the rat cells.

Platelet damage during cardiopulmonary bypass (CPB), although proportional to the duration of bypass, may result in significant dysfunction after the initial contact with an extracorporeal circuit, the so-called 'first pass' phenomenon. The platelet sparing effect of prostacyclin (PGI2) infusion was studied in a double-blind randomized trial on male patients undergoing coronary artery bypass grafts to assess the effect of the 'first pass' through the CPB circuit. Prostacyclin infusion was begun before the onset of CPB or during CPB in two groups which were compared to a placebo control group. A standardized anaesthetic, surgical and perfusion technique were used. Preoperatively and during surgery at pre-set intervals, whole blood platelet aggregation was studied using ADP and collagen agonists. Platelet numbers and function measured by ADP aggregation were conserved in the two PGI2 groups. There was no significant difference between the treated groups. We conclude, therefore, that the initial contact of platelets with the CPB circuit, in the absence of PGI2 did not irreversibly affect platelet function. In addition, the hypotensive action of PGI2 was easier to control once on bypass. It may therefore be preferable to delay PGI2 infusion until CPB has been established.

Alpha-1 blockers have certain disadvantages over conventional and antihypertensive therapies in their haemodynamic profile and metabolic effects. This paper reviews the development of alpha-blockade, the therapeutic efficacy of prazosin, the prototype alpha-1 blocker, and the rationale for the once-daily antihypertensive compounds, terazosin and doxazosin. These drugs offer a useful alternative to first- or second-line therapy in suitable hypertensive patients, particularly with their potentially beneficial effects on serum lipids.

Flusoxolol (Ro 31-1411) is the pharmacologically active optical isomer of Ro 31-1118, a potent cardioselective beta-adrenoceptor antagonist with partial agonist activity. It was given to 6 healthy volunteers in a single dose, 40 mg, and then in multiple doses, 40 mg daily for 8 days. Plasma concentration data were best described by a linear two-compartment pharmacokinetic model with first order absorption, and the results confirmed linear kinetics. Pharmacokinetic data for flusoxolol were comparable to those for the racemate Ro 31-1118.

The myofibrillar regulatory proteins, troponin-I and tropomyosin were isolated from human and bovine atria and ventricles and studied in the adult and during foetal development. A number of analytical electrophoretic procedures were employed to detect possible atrial, ventricular or foetal specific forms of these proteins. Biological activity of the regulatory protein complex during development was monitored by measuring the calcium sensitivity of the myofibrillar Mg2+ activated ATPase. No evidence was obtained for unique or foetal specific forms of either troponin I or the alpha and beta subunits of tropomyosin. Although the atria and ventricles possess markedly different contractile properties, no difference was observed between the two chambers at any developmental stage in the relative amounts of alpha and beta tropomyosin present. However, the relative amount of beta tropomyosin increased by 50% in both atria and ventricles during the transition from foetus to adult. A strong inverse correlation existed (r = -0.92) between beta tropomyosin content and heart rate in different species and at different developmental stages in both cardiac chambers. The relative invariance of tropomyosin and troponin I forms in the myocardium was reflected in the high and constant level of calcium sensitivity of myofibrillar Mg2+ ATPase retained in the atria and ventricles throughout development. The implications of these results in relation to control of cardiac contraction are discussed.