The Effect of Dolutegravir on the Pharmacokinetics of Metformin in Healthy Subjects.

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.

Drug interaction profile of the HIV integrase inhibitor cabotegravir: assessment from in vitro studies and a clinical investigation with midazolam.

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.

In vitro investigations into the roles of drug transporters and metabolizing enzymes in the disposition and drug interactions of dolutegravir, a HIV integrase inhibitor.

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.

Assessment of the drug interaction risk for remogliflozin etabonate, a sodium-dependent glucose cotransporter-2 inhibitor: evidence from in vitro, human mass balance, and ketoconazole interaction studies.

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-ß-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-ß-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.

Oral sulfasalazine as a clinical BCRP probe substrate: pharmacokinetic effects of genetic variation (C421A) and pantoprazole coadministration.

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.

Biopharmaceutics classification system: validation and learnings of an in vitro permeability assay.

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.

The role of efflux and uptake transporters in [N-{3-chloro-4-[(3-fluorobenzyl)oxy]phenyl}-6-[5-({[2-(methylsulfonyl)ethyl]amino}methyl)-2-furyl]-4-quinazolinamine (GW572016, lapatinib) disposition and drug interactions.

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.

Differential involvement of Mrp2 (Abcc2) and Bcrp (Abcg2) in biliary excretion of 4-methylumbelliferyl glucuronide and sulfate in the rat.

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.

In vitro p-glycoprotein inhibition assays for assessment of clinical drug interaction potential of new drug candidates: a recommendation for probe substrates.

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.

Multiple mechanisms are involved in the biliary excretion of acetaminophen sulfate in the rat: role of Mrp2 and Bcrp1.

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.

In vitro absorption and secretory quotients: practical criteria derived from a study of 331 compounds to assess for the impact of P-glycoprotein-mediated efflux on drug candidates.

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.

The systemic exposure of an N-methyl-D-aspartate receptor antagonist is limited in mice by the P-glycoprotein and breast cancer resistance protein efflux transporters.

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.

Predicting P-glycoprotein substrates by a quantitative structure-activity relationship model.

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.

P-glycoprotein influences the brain concentrations of cetirizine (Zyrtec), a second-generation non-sedating antihistamine.

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.

Passive permeability and P-glycoprotein-mediated efflux differentiate central nervous system (CNS) and non-CNS marketed drugs.

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.