Utilization of in vitro Caco-2 permeability and liver microsomal half-life screens in discovering...

PHARMACOKINETICS, PHARMACODYNAMICS ANDDRUG METABOLISM

Utilization of In Vitro Caco-2 Permeability and LiverMicrosomal Half-Life Screens in Discovering BMS-488043,a Novel HIV-1 Attachment Inhibitor with ImprovedPharmacokinetic Properties

ZHENG YANG,1 LISA M. ZADJURA,1 ANTHONY M. MARINO,1 CELIA J. D’ARIENZO,2 JACEK MALINOWSKI,2

CHRISTOPH GESENBERG,3 PIN-FANG LIN,4 RICHARD J. COLONNO,4 TAO WANG,5 JOHN F. KADOW,5

NICHOLAS A. MEANWELL,5 STEVEN B. HANSEL1

1Department of Metabolism and Pharmacokinetics, Bristol-Myers Squibb Research and Development,Wallingford, Connecticut 06492

2Department of Bioanalytical Research, Bristol-Myers Squibb Research and Development, New Brunswick, NJ 08903

3Department of Discovery Pharmaceutics, Bristol-Myers Squibb Research and Development, Wallingford,Connecticut 06492

4Department of Virology, Bristol-Myers Squibb Research and Development, Wallingford, Connecticut 06492

5Department of Discovery Chemistry, Bristol-Myers Squibb Research and Development, Wallingford, Connecticut 06492

Received 17 June 2009; revised 16 August 2009; accepted 17 August 2009

Published online 24 September 2009 in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jps.21948

Steven B. Hamacokinetics, DCT 06340.

CorrespondenCo., Rt. 206 an

ABSTRACT: Optimizing pharmacokinetic properties to improve oral exposure is acommon theme in modern drug discovery. In the present work, in vitro Caco-2 perme-ability and microsomal half-life screens were utilized in an effort to guide the structure–activity relationship in order to improve the pharmacokinetic properties of novelHIV-1 attachment inhibitors. The relevance of the in vitro screens to in vivo pharma-cokinetic properties was first demonstrated with a number of program compounds at theearly stage of lead optimization. The Caco-2 permeability, tested at 200mM, wasquantitatively predictive of in vivo oral absorption, with complete absorption occurringat a Caco-2 permeability of 100 nm/s or higher. The liver microsomal half-life screen,conducted at 1mM substrate concentration, can readily differentiate low-, intermediate-,and high-clearance compounds in rats, with a nearly 1:1 correlation in 12 out of13 program compounds tested. Among the >100 compounds evaluated, BMS-488043emerged as a lead, exhibiting a Caco-2 permeability of 178 nm/s and a microsomal half-life predictive of a low clearance (4 mL/min/kg) in humans. These in vitro characteristicstranslated well to the in vivo setting. The oral bioavailability of BMS-488043 in rats,

nsel’s present address is Department of Phar-ynamics and Metabolism, Pfizer, Inc., Groton,

ce to: Zheng Yang (Bristol-Myers Squibbd Province Line Rd., Princeton, NJ 08543.

Telephone: 609-252-4595; Fax: 609-252-6802;E-mail: [email protected])

Journal of Pharmaceutical Sciences, Vol. 99, 2135–2152 (2010)

� 2009 Wiley-Liss, Inc. and the American Pharmacists Association

JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 4, APRIL 2010 2135

Figure 1. S378806.

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2136 YANG ET AL.

dogs, and monkeys was 90%, 57%, and 60%, respectively. The clearance was low in allthree species tested, with a terminal half-life ranging from 2.4 to 4.7 h. Furthermore, theoral exposure of BMS-488043 was significantly improved (6- to 12-fold in rats andmonkeys) compared to the prototype compound BMS-378806 that had a suboptimalCaco-2 permeability (51 nm/s) and microsomal half-life. More importantly, the improve-ments in preclinical pharmacokinetics translated well to humans, leading to a >15-foldincrease in the human oral exposure of BMS-488043 than BMS-378806 and enabling aclinical proof-of-concept for this novel class of anti-HIV agents. The current studiesdemonstrated the valuable role of in vitro ADME screens in improving oral pharma-cokinetics at the lead optimization stage. � 2009 Wiley-Liss, Inc. and the American Pharma-

cists Association J Pharm Sci 99:2135–2152, 2010

Keywords: preclinical pharmacokine
tics; HIV-1 attachment inhibitor; Caco-2permeability; liver microsomal half-life
INTRODUCTION

Human immunodeficiency virus (HIV) infectionremains a disease that affects millions of peopleworldwide, with around 30.8 million adults and2 million children estimated to be living with HIVat the end of 2007.1 While viral resistance,tolerance/toxicity, and patient compliance repre-sent major issues to the current antiretroviraltherapy,2 a number of new classes of anti-HIVagents are emerging.3,4 HIV-1 attachment inhi-bitors are one promising class of new agents thatblock an early step of the viral entry process.5,6

These attachment inhibitors bind directly to theviral gp120 envelope protein, thus interferingwith the interaction between gp120 and host CD4receptors found on T-cells.

Early pharmacokinetic work with a prototypeattachment inhibitor BMS-378806 (Fig. 1) hasshown that the compound had a modest Caco-2cell permeability (51 nm/s) and less than optimalhuman liver microsomal half-life that predicted

tructures of BMS-488043 and BMS-

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an intermediate clearance in humans.7 Thesein vitro characteristics were also reflected in vivoin the preclinical setting, where incomplete oralabsorption was noted in rats and monkeys with ashort half-life.7 Therefore, it was highly desirableto further improve upon these pharmacokineticcharacteristics so that an HIV-1 attachmentinhibitor could achieve adequate oral exposurein humans that would allow for testing a proof-of-concept in the clinic. Moreover, optimizing phar-macokinetic properties in drug discovery is ofparticular importance for identifying a novel anti-HIV agent, since animal models are not availablefor testing antiviral efficacy and sustaining highdrug levels in the circulation is critical tocircumventing HIV-1.

In the present work, we describe the utilizationof the in vitro Caco-2 permeability and livermicrosomal half-life assays as a first-tier screen inan effort to drive structure–activity relationships(SAR) for improving the oral pharmacokineticproperties of HIV-1 attachment inhibitors. Therelevance of the in vitro screens to in vivopharmacokinetic properties was demonstratedwith program compounds. The difficulty of balan-cing metabolic stability with Caco-2 permeabilityand/or antiviral potency was illustrated by over100 compounds evaluated in the two assays. Thelead molecule BMS-488043 (Fig. 1), identifiedthrough this screening process, was characterizedextensively for its metabolism and pharmacoki-netic properties in the preclinical setting. Sig-nificant improvements in the oral exposure ofBMS-488043 over the prototype compound BMS-377806 were demonstrated in animal species andtranslated well to humans. This work illustratesthe important role of in vitro ADME screens inimproving pharmacokinetic properties at the leadoptimization stage by conserving in vivo resources

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ROLE OF IN VITRO ADME SCREENS IN IMPROVING ORAL PHARMACOKINETICS 2137

and providing quantitative information to driveSAR.

MATERIALS AND METHODS

Chemicals and Reagents

HIV-1 attachment inhibitors, including BMS-488043 and BMS-378806, were synthesized atBristol-Myers Squibb Research and Development(Wallingford, CT). Furafylline, sulfaphenazole,quinidine, ketoconazole, b-nicotinamide adeninedinucleotide phosphase (b-NADPH), glucose-6-phosphate, and glucose-6-phosphate dehydrogen-ase were purchased from Sigma-Aldrich Co. (St.Louis, MO). 3-Cyano-7-ethoxycoumarin (CEC), 7-methoxy-4-trifluoromethylcoumarin (MFC), 3-[2-(N,N-diethyl-N-methylamino)ethyl]-7-methoxy-4-methylcoumarin (AMMC), benzoylresorufin(BzRes), and 7-benzyloxy-4-trifluoromethylcou-marin (BFC) were obtained from BD Biosciences(Woburn, MA). All other solvents and chemicalswere of HPLC or analytical grade.

In Vitro Screens

Caco-2 Cell Permeability

Caco-2 cells, obtained from the American TypeCulture Collection (Rockville, MD), were seededonto a collagen coated polycarbonate filter mem-brane at a density of 80,000 cells/cm2 and wereused between 14 and 21 days in culture. Theintegrity of Caco-2 cells was assessed based onthe trans-epithelial electrical resistance (TEER)value and the permeability of 14C-mannitolconducted in individual Caco-2 permeabilitystudies. The TEER value was measured usingMillicell-ERS (Millipore, Billerica, MA), and theconcentrations of mannitol in both the receiverand donor compartments were determined byscintillation counting (Packard Liquid Scintilla-tion Analyzer 2500 TR, PerkinElmer, Waltham,MA). In addition, prior to all the Caco-2 perme-ability studies, the cytotoxicity of HIV-1 attach-ment inhibitors was evaluated in Hela 67 cellsusing a 72-h XTT (2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide)assay. The viability of cells was determined bythe extent of the conversion of the tetrazoliumsalt to an orange-colored formazan product inthe presence of an electron coupling agentphenazine methosulfate at a wavelength of

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450 nm. The cytotoxic concentration CC50 thatcauses a 50% decrease in cell viability wasestimated.

The medium for permeability studies wasmodified Hank’s balanced salt solution containingeither 10 mM N-2-hydroxyethylpiperazine-N0-2-ethanesulfonic acid (HEPES) or 25 mM 2-(N-morpholino)ethanesulfonic acid (MES) dependingon the intended pH (i.e., pH 6.5 at the apical sideand pH 7.4 at the basolateral side). A compound ofinterest was added to the apical side of themonolayer at an initial concentration of 200mM(diluted from 20 mM dimethyl sulfoxide (DMSO)stock with a final DMSO concentration of 1%), andthen incubated at 378C for 4 h. After incubations,samples were collected from both the receiver anddonor compartments, and drug concentrationswere determined by an HPLC/UC methoddescribed in the Sample Analysis Section. Theapparent Caco-2 permeability coefficient (Pc) wascalculated using the equation Pc¼ (dA/dt)/(SC0),where dA/dt is the rate of permeation across themonolayer, S the surface area of the monolayer(0.33 cm2), and C0 the initial concentration in thedonor compartment.

Microsomal Stability Half-Life

The metabolism of HIV-1 attachment inhibitorswas evaluated in the presence of NADPH-fortifiedpooled human and rat liver microsomes purchasedfrom BD Biosciences and In Vitro Technologies(Baltimore, MD), respectively. The concentrationsof cytochrome P450 (CYP) enzymes in thepreparations used were 0.48–0.55 and 0.52–0.79 nmol/mg protein in humans and rats, respec-tively.

An incubation mixture of 3 mL was prepared ina 0.1 M potassium phosphate buffer (pH 7.4)containing 1mM test compound, 0.5mM CYPenzymes, 1.8mM glucose-6-phosphate, and 0.4units/mL glucose-6-phosphate dehydrogenase. The finalorganic content in the incubation mixture was0.1% that contained 98% acetonitrile and 2%DMSO. The reaction, after a 5-min preincubationat 378C, was initiated by adding b-NADPH(0.15 mM final concentration). Aliquots of0.25 mL were taken at 0, 2, 5, 10, 20, and30 min, and placed to 3 volumes of ice-coldacetonitrile (containing an internal standard—IS)to terminate the reaction. The samples werevortexed and centrifuged at 10,000g for 5 min tocollect supernatant for sample analysis by LC/MSas described in the Sample Analysis Section. The

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turnover rate constant (k) in liver microsomes wasdetermined by nonlinear regression of the per-centage of the compound remaining (y)–time (t)curve using the equation of y¼ y0exp(�kt), wherey0 is percentage of the compound remaining attime zero estimated from the equation. Themicrosomal stability half-life (T1/2) was calculatedusing the equation T1/2¼ 0.693/k.

Evaluation of Metabolism and PharmacokineticProperties of BMS-488043

Bi-Directional Caco-2 Cell Permeability

The same study protocol as the Caco-2 perme-ability screen was employed for the bi-directionalCaco-2 study with BMS-488043, except that thecompound was added to either the apical orbasolateral side of the monolayer at an initialconcentration of 100mM. To assess the role ofefflux transporters such as P-glycoprotein (P-gp),a known P-gp substrate/inhibitor verapamil(100mM) was added to both sides of the monolayerinitially.

Blood Stability and Blood-to-PlasmaConcentration Ratios

The blood stability of BMS-488043 was evaluatedin freshly collected and pooled blood from humans,rats, dogs, and monkeys, with K2EDTA as ananticoagulant. The compound was spiked in bloodat a concentration of 1mg/mL, and the mixturewas incubated at 378C for 2 h. Serial bloodsamples (0.5 mL each) were taken at 0, 15, 30,45, 60, 90, and 120 min during the incubation. Ahalf volume of the blood sample (0.25 mL) wasimmediately placed into a microtainer (BectonDickinson, Franklin Lakes, NJ) and centrifugedto obtain plasma; the other half was transferred tothe tube containing 0.125 mL water and vortexedfor hemolysis. The plasma and hemolyzed bloodsamples were analyzed by LC/MS as described inthe Sample Analysis Section.

Serum Protein Binding

The extent of serum protein binding of BMS-488043 was determined at 378C using an ultra-filtration method. The compound, at 1mg/mL, wasspiked in serum of humans, rats, dogs, andmonkeys in triplicate. After incubating themixture at 378C for 30 min, an aliquot of 0.5 mLwas transferred to the Amicon Centrifree micro-

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partition device containing a 30,000-Dalton cutoffYMT membrane (Millipore, Bedford, MA). Thedevice was centrifuged at 2,000g for 20 minto generate ultrafiltrate (Jouan MR 1822, FixedAngle Rotor 85 mm, Winchester, VA). Thesamples were analyzed by LC/MS as describedin the Sample Analysis Section. The unboundfraction in serum was determined from the ratioof ultrafiltrate to serum concentrations.

The nonspecific binding of BMS-488043 toultrafiltration devices was determined by addingthe compound to water at 0.5mg/mL. An aliquot of0.5 mL was transferred to an ultrafiltration deviceand centrifuged at 2,000g for 5 min. Nonspecificbinding was estimated by comparing the chroma-tographic peak height ratios of BMS-488043 inwater before and after ultrafiltration.

Intrinsic Clearance in Liver Microsomes

The intrinsic clearance of BMS-488043 wasevaluated in the presence of pooled liver micro-somes of rats, dogs, monkeys, and humans. Thehuman and rat liver microsomes were purchasedfrom BD Biosciences and In Vitro Technologies,respectively. The dog and monkey liver micro-somes were obtained from XenoTech LLC (KansasCity, KS). The concentrations of cytochrome P450(CYP) enzymes in the preparations used were0.48–0.55, 0.52–0.79, 0.89, and 1.4 nmol/mg pro-tein in humans, rats, dogs, and monkeys, respec-tively.

The same study protocol as the microsomal half-life screen was employed for determination of theintrinsic clearance of BMS-488043, except thatthe experiment was run in triplicate for eachspecies. The turnover rate constant k in livermicrosomes (k) was determined as describedabove, and the intrinsic clearance was calculatedas described in the Data Analysis Section.

Intrinsic Clearance in Human Hepatocytes

The metabolic stability of BMS-488043 wasevaluated in cryopreserved human hepatocytes(In Vitro Technologies) at a cellular concentrationof 0.33� 106 or 0.67� 106 cells/mL. Cells werethawed in a stepwise manner and washed in asuspension buffer (Krebs-Henseleit buffer, pH7.4). The cells were subjected to Percoll purifica-tion by centrifuging a mixture of cell suspension(12.5 mL) and Percoll solution (12.5 mL) at 50gfor 5 min at 48C. After a further wash in thesuspension buffer, the viability and cell density

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were measured by trypan-blue exclusion method.The compound (3mM) was incubated at 378C for1 h, and aliquots of samples (0.2 mL) was taken at0, 20, 40, and 60 min. The reaction was stoppedby adding an equal volume of acetonitrile. Thesamples were then stored at �208C and analyzedby LC/MS. The turnover rate constant k forthe metabolism of BMS-488043 in hepatocyteswas determined by nonlinear regression of thepercentage of the compound remaining (y)–time(t) curve using the equation of y¼ 100exp(�kt),and the intrinsic clearance in human hepatocyteswas calculated as described in the Data AnalysisSection. The positive controls included in thestudies were 7-ethoxycoumarin (100mM), 7-hydro-xycoumarin (100mM), and midazolam (10mM).

Inhibition of Human CYP Enzymes

The potential of BMS-488043 to inhibit majorhuman cytochrome P450 (CYP) enzymes wasevaluated using a recombinant CYP system(BD SupersomesTM Enzymes, BD Biosciences).The IC50 values of BMS-488043 for each CYPisoform was determined from the inhibition ofdeethylation of CEC (CYP1A2 and CYP2C19),dealkylation of MFC (CYP2C9), demethylation ofAMMC (CYP2D6), and dealkylation of BzRes(CYP3A4) and of BFC (CYP3A4) by fluorescencedetection in a 96-well format. The studies wereconducted in duplicate over a 45-min incubationperiod, with substrate concentrations equal to theapparent Km (BFC concentration below the Km)and eight inhibitor concentrations ranging from18 nM to 40mM. The IC50 values were estimatedusing XlfitTM curve-fitting software (ID BusinessSolutions Ltd., Guildford, UK).

Reaction Phenotyping in Human Liver Microsomes

The role of CYP enzymes in the metabolism ofBMS-488043 was studied in human liver micro-somes using isoform-selective chemical inhibitors.The inhibitors used were furafylline (20mM) forCYP1A2, sulfaphenazole (30mM) for CYP2C9,quinidine (1mM) for CYP2D6, and ketoconazole(3mM) for CYP3A4. The stock solutions ofinhibitors and BMS-488043 were prepared inmethanol or acetonitrile, with a final organicconcentration of 0.1% in the incubation mixture.The studies were conducted in triplicate for eachinhibitor along with the vehicle controls, usingthe incubation procedure described above.Furafylline was preincubated in the incubation

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mixture for 15 min in the presence of b-NADPH,with the reaction initiated by the addition of BMS-488043 (1mM final concentration). For the otherinhibitors, they were added along with BMS-488043 for a 5-min preincubation, with thereaction initiated by the addition of b-NADPH.Aliquots of samples were taken at 0 and60 min. The extent of inhibition was determinedby comparing the percentage of BMS-488043remaining at 60 min in the presence and absenceof inhibitors.

Pharmacokinetics in Rats

The animal testing methods used in the studiesdescribed below were approved by the Bristol-Myers Squibb Institutional Animal Care and UseCommittee and adhered to the ‘‘Principles ofLaboratory Animal Care’’ (NIH publication #85-23, revised in 1985). The animals were housed in a12-h light cycle, with free access to food and water.

Male Sprague–Dawley rats (300–350 g) withcatheters placed in the jugular vein and/or bileduct were purchased from Hilltop Lab Animals,Inc. (Scottdale, PA) and used in the pharmacoki-netic studies with BMS-488043. In the studiesdescribed below, the compound was dosed in apolyethylene glycol 400 (PEG400)/ethanol solu-tion (90/10, v/v) unless noted otherwise. Plasmasamples were harvested from blood (0.2–0.3 mL)collected from the jugular vein using K2EDTA asan anticoagulant, and stored at�208C until analysis.

In an intravenous (IV) study, a dose of 1 mg/kgwas administered to three rats as a bolus dose,and serial plasma samples were collected beforedosing and 0.033, 0.17, 0.25, 0.5, 0.75, 1, 2, 4, 6, 8,and 24 h after dosing. In an oral (PO) study, rats(n¼ 3) received a dose of 5 mg/kg. Serial plasmasamples were taken before dosing and 0.25, 0.5,0.75, 1, 2, 4, 6, 8, and 24 h after dosing. In aseparate study, a PO dose of 5 mg/kg was alsoadministered to rats as a crystalline suspension(0.75% Methocel1 A4M Premium, Dow ChemicalCo., Midland, MI). The average particle size insuspension was 6.9mm.

In a bile duct-cannulated (BDC) rat study, fourrats were placed in metabolic cages and received aPO dose of 5 mg/kg. An additional rat was givenwater as a control to provide drug-free urine andbile. Plasma samples were obtained as the POsampling scheme listed above. Urine sampleswere collected in the intervals of 0–2, 2–7, and 7–24 h, and bile samples were collected in theintervals of 0–2, 2–5, 5–7, and 7–24 h. Both urine


2140 YANG ET AL.

and bile samples were collected on ice and storedat �208C until analysis.

In a brain uptake study, 18 animals were givenan IV dose of 5 mg/kg and sacrificed at 0.033, 0.17,0.33, 0.67, 1, 2, 4, 6, and 8 h after dosing (n¼ 2animals per time point). At each time point, braintissues and plasma samples were collected. Brainsamples were blotted dry, weighed, and stored at�208C until analysis.

Pharmacokinetics in Dogs

Three male beagle dogs (BW 11.5� 1.0 kg, Mar-shall Farms USA, Inc., North Rose, NY) werefasted overnight and used in the oral bioavail-ability study with BMS-488043 in a crossoverstudy design. In the first phase, an IV dose of 1 mg/kg was given. After a 1-week washout, a PO doseof 5 mg/kg was administered. The dosing vehiclewas a PEG400/ethanol solution (90/10, v/v). For IVadministration, the dose was given via thecephalic vein as a 5-min infusion at a rate of0.1 mL/min/kg; samples were taken before dosingand 0.083, 0.17, 0.25, 0.5, 0.75, 1, 2, 4, 6, 8, and24 h after starting the infusion. For PO dosing,samples were taken prior to dosing and 0.17, 0.25,0.5, 0.75, 1, 2, 4, 6, 8, and 24 h postdosing. Plasmasamples were harvested from blood (0.5 mL)drawn from the femoral artery using K2EDTAas an anticoagulant and stored at �208C untilanalysis.

Pharmacokinetics in Monkeys

Three male cynomolgus monkeys (BW 9.1� 1.1 kg,Charles River Biomedical Research Foundation,Houston, TX) were fasted overnight and used inthe oral bioavailability studies with BMS-488043in a crossover study design. In the first phase, anIV dose of 1 mg/kg was given. After a 1-weekwashout, a PO dose of 5 mg/kg was administered.BMS-488043 was administered in a PEG400/ethanol solution (90/10, v/v). For IV dosing, thedose was given via the femoral vein as a 5-mininfusion at a rate of 0.1 mL/min/kg; the plasmasamples were taken before dosing and 0.083, 0.17,0.25, 0.5, 0.45, 1, 2, 4, 6, 8, and 24 h after startinginfusion. For PO dosing, the samples were takenprior to dosing and 0.25, 0.5, 0.75, 1, 2, 4, 6, 8, and24 h postdosing. Plasma samples were harvestedfrom blood (0.5 mL) drawn from the femoral arteryusing K2EDTA as an anticoagulant, and stored at�208C until analysis.


Sample Analysis

HPLC/UV Method for In Vitro Caco-2Permeability Screen

The HPLC system consisted of a Waters Alliance2690 separations module and a Waters 996photodiode array detector (Waters Co., Milford,MA). A C18 YMC ODS AQ reverse phase column(4.6� 50 mm2, 5mm particles, Waters Co.) wasused at a flow rate of 1.0 mL/min. The mobilephase, consisting of solvent A (water/acetonitrile/trifluoroacetic acid, 95:5:0.1% v/v) and solvent B(water/acetonitrile/trifluoroacetic acid, 20:80:0.1%v/v). The initial mobile phase composition was95%A/5%B. After sample injection, the mobilephase was changed to 60%A/40%B over 1 min andthen changed to 1%A/99%B for another min. Themobile phase was then held at that compositionfor an additional 2.5 min before reverting back toinitial conditions over 0.1 min. The total analysistime was �5 min, and the absorbance monitoredat 210 nm.

LC/MS Method for In Vitro MicrosomalHalf-Life Screen

An LC/MS method was developed to quantifyHIV-1 attachment inhibitors in the samples fromin vitro microsomal half-life screen. In addition,all the in vitro studies conducted with BMS-488043, including blood stability, blood-to-plasmaconcentration ratios, serum protein binding,microsomal incubations and human hepatocyteincubations, were analyzed using this method.

The in vitro samples were treated with threevolumes of acetonitrile containing a structurallyrelated analog as an IS at 0.1mg/mL. Aftercentrifugation at 10,000g for 5 min at 48C(Eppendorf Centrifuge 5417R, Brinkman Instru-ments, Westbury, NY), a 10-mL clear supernatantwas injected onto LC/MS for analysis. A ShimadzuHPLC system that consisted of two LC-10ADvppumps, a SIL-10ADvp autosampler, a CTO-10ASvpcolumn compartment, and an SCL-10Avp systemcontroller (Columbia, MA) was used. The columnwas an YMC ProC18 column (2.0� 50 mm, 5mmparticles, Waters Co.), maintained at 408C and aflow rate of 0.2 mL/min. The mobile phaseconsisted of 0.1% formic acid in 5 mM ammoniumacetate (A) and acetonitrile (B). The initial mobilephase composition was 100%A/0%B. After sampleinjection, the mobile phase was changed to 50%A/50%B over 1 min and held at that composition foran additional 2.5 min. The mobile phase was then

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changed to 10%A/90%B over 0.25 min and heldat that composition for an additional 2.75 minto clean up the column. Afterwards, the mobilephase returned to initial conditions and thecolumn reequilibrated for 1 min. The total analy-sis time was 7.5 min.

The HPLC was interfaced to a MicromassZMD2000 mass spectrometer (Waters Co.)equipped with an electrospray interface. Dataacquisition was via selected ion monitoring usingions representing the (MþH)þ species for boththe analyte and IS. For BMS-488043, the ionsmonitored were m/z 423.4. The retention times forHIV-1 attachment inhibitors ranged from 1.5 to3.5 min. In the case of BMS-488043, the retentiontime was 2.66 min. The lower limit of quantifica-tion (LLQ) was 10 ng/mL, with measured concen-trations of at least two-thirds of quality controlsamples within 20% of nominal values.

LC/MS/MS Method for In VivoPharmacokinetic Studies

Samples from in vivo pharmacokinetic studieswere analyzed by LC/MS/MS. They were treatedwith 2 volumes of acetonitrile containing an IS at0.5mg/mL. After centrifugation to remove pre-cipitated proteins, 10mL supernatant was injectedonto a column for analysis. After centrifugation toremove precipitated proteins, 10mL supernatantwas injected onto a column for analysis. Rat brainsamples were prepared as 25% brain homoge-nates. Urine and bile samples were diluted withwater and injected directly.

The HPLC system consisted of two ShimadzuLC10AD pumps (Columbia, MA), a Perkin ElmerSeries 200 autosampler (Norwalk, CT) and aHewlett Packard Series 1100 column compart-ment (Palo Alto, CA). The column used was aKeystone Hypersil C18 (2� 20 mm2, 3mm parti-cles, Bellefonte, PA) maintained at 608C and aflow rate of 0.3 mL/min. The mobile phaseconsisted of solvent A (10 mM ammonium acetateand 25% methanol in water, pH adjusted to 5.5with glacial acetic acid) and solvent B (10 mMammonium acetate in methanol) at a flow rate of0.2 mL/min. The initial mobile phase compositionwas 95%A/5%B. After sample injection, the mobilephase was changed to 15%A/85%B over 0.1 minand held at that composition for an additional1 min. The mobile phase was then returned toinitial conditions and the column reequilibratedfor 0.9 min. The total analysis time was 2 min.

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The HPLC system was interfaced to a Micro-mass Quattro LC tandem mass spectrometer(Beverly, MA) equipped with an electrosprayinterface. Ions representing the (MþH)þ speciesfor both the analyte and IS were selected in MS1and collisionally dissociated with argon at apressure of 2� 10�3 Torr to form specific productions, which were subsequently monitored by MS2.The transitions monitored were m/z 423.3! 205.0for BMS-488043 and m/z 362.6! 143.9 for the IS.The retention times for both BMS-488043 and ISwere approximately 0.8 min. The LLQ was 3.9 ng/mL, with measured concentrations of at least two-thirds of quality control samples within 20% ofnominal values.

Data Analysis

Data are expressed as mean� standard deviation(SD).

The hepatic intrinsic clearance (CLh,int) of BMS-488043 in various species was estimated fromliver microsome or hepatocyte data using themethod described by Obach et al.8 and Iwatsuboet al.9 Assuming linear kinetics and similarunbound intrinsic clearance in vitro and in vivo,the CLh,int was calculated based on the disap-pearance of the parent drug in liver microsome orhepatocyte incubations as follows:

Microsomes : CLh;int

¼ k

Cprotein

� �� 45 mg protein

1 g liver weight

� �

� g of liver weight

kg of body weight

� �

Hepatocytes : CLh;int

¼ k

Ccell

� �� 120 � 106 cells

1 g liver weight

� �

� g of liver weight

kg of body weight

� �

where k is the turnover rate constant in theincubation as described previously, Cprotein andCcell are microsomal protein and hepatocyteconcentrations in the incubation, respectively.The liver weight relative to body weight in rats,dogs, monkeys, and humans is 40, 32, 32, and 21 g/kg, respectively.10 Assuming the well-stirredmodel, similar protein binding in microsomes(or hepatocytes) and blood, and rapid equilibriumbetween blood and the liver, the hepaticblood clearance (CLh,blood) was estimated as


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follows:

CLh;blood ¼ Qh � CLint

Qh þ CLint

where Qh is the hepatic blood flow with a valueequal to 70, 35, 44, and 20 mL/min/kg in rats, dogs,monkeys, and humans, respectively.10,11

The pharmacokinetic parameters of BMS-488043were estimated from its plasma concentration–timedata using the noncompartmental analysis (Kine-ticaTM software v.3.0, InnaPhase Co., Philadel-phia, PA). The peak concentration (Cmax) and timefor Cmax (Tmax) were recorded directly fromexperimental observations. The total areas underthe concentration–time curve (AUC0–1) andunder the first moment curve were calculatedfrom time zero to infinity using a combination oflinear and log-linear trapezoidal summations. Thetotal body clearance from plasma (CLtot), steady-state volume of distribution (Vss), terminal half-life (T1/2), and mean residence time (MRT) werealso estimated from IV administration.12 The totalbody clearance from blood (CLtot,b) was calculatedas the CLtot divided by the blood-to-plasmaconcentration ratio. The oral bioavailability(expressed as percentage) was estimated as theratio of dose-normalized AUC values between POand IV doses. The percentage of the dose excretedunchanged in the urine and bile was calculatedas the cumulative amount of the unchanged drugin the respective fluid divided by the doseadministered.

The fraction of dose absorbed into theportal vein ( fafg) was calculated as the oralbioavailability divided by the fh, where fh is thefraction of the dose reaching the liver that escapesthe liver elimination. The fh was calculated as1 minus the hepatic extraction ratio, where theextraction ratio was estimated from CLtot,b

divided by the hepatic blood flow, assuming thatthe liver is a major organ of elimination. To assessthe rate of absorption, the absorption rateconstant (ka) was determined as the reciprocalof the mean absorption time (MAT), assuming afirst-order rate of absorption. MAT was calculatedas the difference in MRT between PO and IVroutes, assuming linear pharmacokinetics.

The human oral bioavailability was calculatedas fafgfh, where the fafg was averaged from animalsolution data and the fh was estimated fromhuman clearance predicted from human livermicrosomal results as described above. In addi-tion, simple allometry was also employed topredict human CLtot and Vss.

13 To predict human


plasma AUC after PO administration, the follow-ing equation was used

AUC ¼ FpoDose

CLtot

where Fpo is the predicted human oral bioavail-ability and CLtot is predicted human plasmaclearance.

RESULTS

Utility of the In Vitro Caco-2 Screen to PredictOral Absorption

In all the Caco-2 studies conducted herein, theTEER values were greater than 400Vcm2 and thePc values of mannitol were less than 24 nm/s,indicating that the tight junctions were wellmaintained during the studies. In addition, noapparent correlation was observed betweenthe CC50 determined using a 72-h XTT assay inHela 67 cells and the Caco-2 Pc values (data notshown), suggesting that a short 4-h exposure ofCaco-2 cells to HIV-1 attachment inhibitors didnot comprise the integrity of cell membranes.

At the early stage of the HIV-1 attachmentinhibitor program, the relevance of the in vitroCaco-2 screen to oral absorption was explored,with findings summarized in Table 1. When theCaco-2 apical-to-basolateral (A-to-B) Pc was low(15 nm/s or less), the oral bioavailability in ratswas found to be low (<10%), even though thesystemic clearance in a number of these cases waslow relative to the hepatic blood flow (70 mL/min/kg) in rats. With a modest Caco-2 A-to-B Pc value(51 nm/s), the oral absorption of BMS-378806 wasstill incomplete and variable in the animal speciestested (32–49% in rats and monkeys vs. 89% indogs).7 On the other hand, when the Caco-2 A-to-BPc value was greater than 100 nm/s, the oralbioavailability in rats was found to be complete fortwo compounds listed in Table 1. These observa-tions are consistent with the in-house calibrationdata set generated with marketed drugs, wherecompounds are well absorbed in humans when theCaco-2 A-to-B Pc value is greater than 100 nm/s(e.g., metoprolol) and are poorly absorbed whenthe Caco-2 A-to-B Pc value is below 30 nm/s (e.g.,mannitol, atenolol, etc.).14 Consequently, the in vitroCaco-2 permeability assay was utilized as aneffective screen in the HIV-1 attachment inhibitorprogram for assessing absorption potential.

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Table 1. Relationship between Caco-2 Permeability and Oral Absorption in the HIV-1 AttachmentInhibitor Program

BMS No.Caco-2 Pc at

pH 6.5a (nm/s)Rat Oral Bioavailability

at 5 mg/kg (%)CLtot (mL/min/kg)(Extraction Ratio)b

BMS-1 17 2.3 3.1 (0.04)BMS-2 <15 1.0 5.5 (0.08)BMS-3 <15 1.0 131 (>1.0)BMS-4 15 2.6 82 (>1.0)BMS-5 15 2.0 0.52 (0.007)BMS-6 <15 <1.0 26 (0.37)BMS-378806 51 19 32 (0.46)BMS-7 204 100 5.2 (0.07)BMS-8 >300 89 3.9 (0.06)

aThe Caco-2 Pc of metoprolol, a highly permeable marker, was 132 nm/s, whereas the Caco-2 Pc of mannitol, a poorly permeablemarker, was 15 nm/s.

bExtraction ratio calculated as the total body plasma clearance divided by rat hepatic blood flow of 70 mL/min/kg, assuming thehepatic blood clearance is equal to the total body plasma clearance.


Utility of the In Vitro Liver Microsomal Half-LifeScreen to Predict Systemic Clearance

The effectiveness of in vitro liver microsomal half-life studies as a way to predict in vivo clearancewas also evaluated in the early stage of the HIV-1attachment inhibitor program, and the results areshown in Figure 2. A good in vitro–in vivocorrelation in rat clearance was observed for 12out of 13 test compounds. More importantly, these

Figure 2. In vitro–in vivo correlation in rat clearanceof 13 HIV-1 attachment inhibitors. The elimination ofthese compounds was assumed to be via the liver. Inaddition, the blood-to-plasma concentration ratio wasassumed to be unity. Accordingly, the in vivo totalbody plasma clearance was assumed to be equal tothe hepatic blood clearance. Low-clearance category:CL<21 mL/min/kg; intermediate-clearance category:21 mL/min/kgCL49 mL/min/kg; high-clearancecategory: CL>49 mL/min/kg.

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12 compounds were well distributed among thecategories of low, intermediate, and high clear-ance classes in the rat, illustrating the utility ofthe in vitro liver microsomal half-life in predictingin vivo clearance and differentiating compoundsfrom low to high clearance. As a result, the in vitrohalf-life screen in human liver microsomes wasimplemented as a first-tier screen immediatelyafter potency determination for compoundtriaging. A half-life greater than 33 min underthe condition of 0.5mM CYP enzyme (�1 mg/mLmicrosomal proteins) in human liver microsomeswas used as a cut-off value for compound selection,which predicted a human clearance of less than9 mL/min/kg (45% of hepatic blood flow).

In addition, half-life results from different lotsof pooled human liver microsomes and fromseparate experiments were compared in orderto assess the reproducibility of the assay. Theaverage liver microsomal half-life of a programcompound serving as an internal control for theassay was 31� 4.3 min (n¼ 6) when tested in sixseparate experiments with four different lots ofhuman liver microsomes. Two additional programcompounds were tested in two separate experi-ments. The half-life for the 1st compound in the1st and 2nd experiment was 4.0 and 4.1 min,respectively; the half-life for the 2nd compoundwas 34 and 36 min, respectively. The coefficient ofdetermination (R2) of the nonlinear regressionline in all cases was greater than 0.98. Collec-tively, these results indicated the in vitro livermicrosomal half-life screen implemented in theHIV-1 attachment inhibitor program was reliableand reproducible.


2144 YANG ET AL.

Identification of BMS-488043 through In VitroCaco-2 and Liver Microsomal Half-Life Screens

In the course of the lead optimization campaignthat led to the identification of BMS-488043, 132and 89 compounds were evaluated in the humanliver microsomal half-life and Caco-2 permeabilityscreens, respectively. As shown in Figure 3a, itwas clearly a challenging task to balance humanliver microsomal half-life and antiviral potency.The majority of the 132 compounds were con-gregated in the lower left quadrant where theantiviral EC50 values in a pseudotype assay5 weregenerally less than 1 nM but with less optimalmicrosomal half-lives. The task of balancing theseproperties with Caco-2 permeability addedfurther to the challenge of identifying amolecule suitable for development, as shown inFigure 3b. Few compounds possessed a high Caco-2 A-to-B Pc value of greater than 100 nm/s and anoptimal microsomal half-life (i.e., upper rightquadrant). However, among these compounds,BMS-488043 clearly stood out with its Caco-2A-to-B Pc value of 178 nm/s and a human livermicrosomal half-life of greater than 100 min.

Figure 3. Relationship between human liver micro-somal half-life and antiviral potency for 132 HIV-1attachment inhibitors (a); and relationship betweenhuman liver microsomal half-life and Caco-2 perme-ability for 89 HIV-1 attachment inhibitors (b).


Evaluation of In Vitro and In Vivo Metabolism andPharmacokinetic Properties of BMS-488043

Bi-Directional Caco-2 Cell Permeability

BMS-488043 exhibited a high Caco-2 A-to-B Pc(178 nm/s) as indicated previously in the Caco-2screen tested at 200mM, suggesting that thecompound is likely to be well absorbed in vivo.

In the subsequent bi-directional Caco-2 perme-ability study, the A-to-B Pc of BMS-488043 at100mM was 113� 1.0 nm/s (n¼ 3), 1.6-fold lowerthan the B-to-A Pc (182� 4.9 nm/s, n¼ 3;p< 0.05). In the presence of 100mM verapamilconducted in the same experiment, the directionaldifference in permeability disappeared (A-to-BPc¼ 140� 14 nm/s; B-to-A Pc¼ 157� 5.5 nm/s;efflux ratio¼ 1.1), indicating that efflux transpor-ters such as P-gp are possibly involved, althoughtheir roles may not be prominent in limiting theoral absorption of BMS-488043.

Blood Stability and Blood-to-PlasmaConcentration Ratios

BMS-488043 was stable in human, rat, dog, andmonkey blood at 378C during a 2-h incubation.The blood-to-plasma concentration ratios (n¼ 6)in humans, rats, dogs, and monkeys were0.79� 0.06, 0.75� 0.09, 0.71� 0.06, 0.79� 0.07,respectively. These results suggest that BMS-488043 is less distributed to blood cells.

Serum Protein Binding

BMS-488043, at 1mg/mL, was 95.1� 0.3% boundto human serum proteins, and 97.0� 0.05%,75.9� 1.0%, and 92.9� 0.8% bound to rat, dog,and monkey serum proteins, respectively. Thenonspecific binding of BMS-488043 to ultrafiltra-tion devices was negligible.

Metabolic Stability in Human Liver Microsomesand Hepatocytes

BMS-488043 was metabolized at a slow rate in thepresence of either human liver microsomes orhepatocytes. The predicted human clearance(3.6 mL/min/kg) from liver microsomes was com-parable to that (4.5 mL/min/kg) from humanhepatocytes, suggesting that BMS-488043 wouldbe a low clearance drug in humans. Consistentwith what was observed in the liver microsomalhalf-life screen, a good in vitro–in vivo correlationexisted between the observed and predicted

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Table 2. In Vitro–In Vivo Correlation in the Clearance of BMS-488043

Parameter Rat LM Dog LM Monkey LM

Human

LM Hepatocyte

Predicted hepatic blood clearance (mL/min/kg) 13 2.3 10 3.6 4.5Observed hepatic blood clearance (mL/min/kg) 17 3.3a 5.5a — —

LM, liver microsomes.aTotal body blood clearance.


clearance values of BMS-488043 in animal species(Tab. 2).

Inhibition of Human Cytochrome P450

BMS-488043 exhibited a low potential to inhibitCYP enzymes. The IC50 for CYP1A2, 2C9, 2C19,2D6, 3A4 in a recombinant CYP system was >100,46, 99, >100, >100mM, respectively.

Reaction Phenotyping in Human Liver Microsomes

With the use of CYP isoform-selective chemicalinhibitors, BMS-488043 was found to be metabo-lized primarily by CYP3A4. The extent of inhibi-tion, measured as the disappearance of parentdrug at 1mM drug concentration, was 24� 21%by 20mM furafylline (CYP1A2), �14� 12% by30mM sulfaphenazole (CYP2C9), 7.2� 6.6% by1mM quinidine (CYP2D6), and 84� 15% by 3mMketoconazole (CYP3A4), respectively. Thus, vary-ing levels of the CYP3A4 expression may causeinter-subject variability in the pharmacokineticsof BMS-488043 and its disposition can also bealtered by a comedication that is a CYP3A4inhibitor.

Table 3. Pharmacokinetic Parameters of BMS-488043 in

Rat

BMS-488043 BMS-378806 BM

CLtot (mL/min/kg) 13� 4.0 32� 1.8Vss (L/kg) 1.1� 0.22 0.55� 0.023 0Terminal T1/2 (h) 2.4� 0.33 0.30� 0.031MRT (h) 1.5� 0.25 0.30� 0.014Oral bioavaiability (%) 90 19AUC at 5 mg/kg (mg h/mL) 6.3� 2.7 0.51� 0.24Cmax at 5 mg/kg (mg/mL) 1.9� 0.65 0.090� 0.035Tmax (h) 2.0� 0.0 2.7� 1.2

aLinearly extrapolated from 3.4 to 5 mg/kg.

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Pharmacokinetics in Rats

The pharmacokinetic parameters of BMS-488043following IV (1 mg/kg) and PO (5 mg/kg) admin-istration to rats are summarized in Table 3.Following IV administration, the plasma concen-trations of BMS-488043 displayed a bi-exponen-tial decline, with a terminal T1/2 of 2.4 h (Fig. 4a).The systemic clearance was low, with a CLtot,b

being 24% of the hepatic blood flow in rats. The Vss

value in rats (1.1 L/kg) was greater than thevolume of total body water (0.67 L/kg),10 demon-strating extravascular distribution. However, thebrain-to-plasma AUC ratio of BMS-488043 follow-ing an IV dose of 5 mg/kg was only 0.016. Sinceresidual blood generally contributes about 3–5%of drug concentrations measured in the brain, thepresent results indicate that BMS-488043 did notpenetrate into the brain to an appreciable extent.

The oral bioavailability of BMS-488043 in ratsat the 5 mg/kg dose was 90% (Tab. 3), with a Cmax

of 1.9mg/mL and a Tmax of 2 h, respectively.Consistent with the observed high Caco-2 Pcvalue, the oral plasma concentration–time profilewas parallel to the IV curve (Fig. 3a), suggestingno prolonged absorption. The fafg in rats wasestimated to be 100%, indicating complete oralabsorption. Furthermore, when dosed as a crystal-

Rats, Dogs, and Monkeys

Dog Monkey

S-488043 BMS-378806 BMS-488043 BMS-378806

2.4� 0.43 5.7� 0.48 4.3� 0.26 10� 1.1.45� 0.083 0.47� 0.065 0.63� 0.039 0.39� 0.132.6� 1.2 1.2� 0.063 4.7� 0.31 0.90� 0.223.3� 0.72 1.4� 0.11 2.4� 0.15 0.63� 0.1757� 17 77� 1.1 60� 4.0 24� 1120� 3.0 11� 0.84a 12� 1.2 1.9� 0.69a

2.7� 0.49 4.5� 0.64a 2.5� 0.50 0.31� 0.11a

2.9� 1.9 1.0� 0.50 2.7� 1.2 1.5� 0.50


Figure 4. Plasma concentration–time profiles ofBMS-488043 after IV and PO administration to rats(a), dogs (b), and monkeys (c). Plasma samples weretaken up to 24 h (data not shown are below LLQ).

2146 YANG ET AL.

line suspension at 5 mg/kg, the average AUC andCmax of BMS-488043 from the suspension was 62%and 75% of the solution formulation, respectively,suggesting that the high membrane permeability


of BMS-488043 compensated for its low aqueoussolubility (40mg/mL as a crystalline free base) andled to a relatively small reduction in oralexposure.

In BDC rats dosed orally at 25 mg/kg, about 12%and 1.3% of dose were excreted unchanged in theurine and bile, respectively. The renal clearance ofBMS-488043 in rats was estimated be 2.5 mL/min/kg, which was significantly higher than theproduct of the free fraction and glomerularfiltration rate in the rat (0.16 mL/min/kg), indicat-ing that the compound is actively secreted in thekidneys.

Pharmacokinetics in Dogs

Table 3 summarizes the pharmacokinetic para-meters of BMS-488043 following IV (1 mg/kg) andPO (5 mg/kg) administration to dogs. After IVadministration, the plasma concentration–timeprofile of BMS-488043 appeared to be convexfollowed by a bi-exponential decline (Fig. 3b),indicating some degree of saturation at the earlydistribution phase. The terminal T1/2 was 2.6 h.The CLtot,b was 10% of the hepatic blood flow,indicating a low clearance in dogs. The Vss (0.45 L/kg) in dogs was lower than the volume of totalbody water (0.60 L/kg) but greater than thevolume of the extracellular fluid (0.28 L/kg).10

The average oral bioavailability of BMS-488043in dogs was 57% (Tab. 3), with an average Cmax of2.7mg/mL and an average Tmax of 2.9 h. Similar torats, the oral plasma concentration–time profile indogs was parallel to that after IV dosing at theterminal phase, indicating no prolonged absorp-tion. The fafg in dogs was estimated to be 63%.

Pharmacokinetics in Monkeys

Table 3 also summarizes the pharmacokineticparameters of BMS-488043 following IV (1 mg/kg)and PO (5 mg/kg) administration to monkeys.After IV administration, the plasma concentra-tions of BMS-488043 displayed a multi-exponen-tial decline, with a terminal T1/2 of 4.7 h (Fig. 3c).The CLtot,b was low (12% of the hepatic blood flow).Similar to dogs, the Vss in monkeys (0.63 L/kg) wasgreater than the volume of extracellular fluid(0.21 L/kg) but less than the volume of total bodywater (0.69 L/kg).10

The average oral bioavailability of BMS-488043in monkeys was 60% (Tab. 3). The average Tmax

was 2.7 h, with an average Cmax of 2.5mg/mL.Similar to the other two species studied, theplasma concentration–time profiles in monkeys

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were parallel between the IV and PO route,indicating no prolonged oral absorption (Fig. 3c).Similar to dogs, the fafg in monkeys was estimatedto be 68%.

Prediction of Human Pharmacokinetics

Figure 5 displays the allometric scaling of CLtot

and Vss from animal data to humans using simpleallometry. The human CLtot and Vss werepredicted to be 1.2 mL/min/kg and 0.31 L/kg,respectively. The low CLtot predicted from simpleallometry is in agreement with the clearance (3.6–4.5 mL/min/kg) predicted from either human livermicrosomes or hepatocytes, suggesting that BMS-488043 would be a low clearance drug in humans.

To predict human oral bioavailability, the fafg

estimated from animal species was averaged(75%). The human fh (82%) was calculated from

Figure 5. Allometric scaling of pharmacokineticparameters of BMS-488043 from animal data tohumans. (a) CLtot; (b) Vss.

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human liver microsomal data as described above.The human liver microsomal data were used,because it offered a conservative estimate of thehuman clearance compared to that predicted bysimple allometry and because there also existed agood in vitro–in vivo correlation in the clearancefrom animal species using liver microsomal data.Together, the oral bioavailability of BMS-488043in humans, calculated as fafgfh (75%� 82%), waspredicted to be 60%.

The human oral plasma AUC of BMS-488043was estimated using the predicted human oralbioavailability and plasma clearance (after aconversion of the predicted blood clearance fromhuman liver microsomal data). The predictedplasma AUC of BMS-488043 at 100 mg was5.1mg h/mL, which was �10-fold higher than that(0.41mg h/mL) predicted for the prototype mole-cule BMS-378806 using the same predictionmethodology. This prospective analysis indicatesthat the human oral exposure of BMS-488043 islikely to be significantly improved over BMS-378806.

DISCUSSION

Optimizing pharmacokinetic properties to improveoral exposure is a common theme in modern drugdiscovery. This was of particular importance inthe efforts of discovering a novel anti-HIV agentfor which efficacy cannot be tested readily in thepreclinical setting. Furthermore, maintaining aconstant and sufficiently high drug level inplasma is required to prevent the productiveinteraction of virus with host. To this end,assessing the pharmacokinetic properties of dis-covery compounds following initial potency deter-minations became essential to lead optimizationefforts in the HIV-1 attachment inhibitor pro-gram. The challenges for discovery metabolismand pharmacokinetics scientists, however, were toestablish a rapid screen that had a sufficientthroughput to accommodate a large number ofcompounds while providing quantitative informa-tion on ADME attributes that enabled thedevelopment of SAR by medicinal chemists. Inthe present work, we described the utilization oftwo in vitro screens, Caco-2 permeability and livermicrosomal half-life, during the lead optimizationstage of the HIV-1 attachment inhibitor programthat led to the identification of BMS-488043, acompound with much improved pharmacokinetic


2148 YANG ET AL.

properties compared to the prototype moleculeBMS-378806.

Utilization of in vitro screens to optimizepharmacokinetic properties in a drug discoveryprogram is especially attractive in light of typicalconstraints on in vivo resources. The in vitroscreens could serve as an effective tool to triageand prioritize compounds for in vivo pharmaco-kinetic studies. More importantly, the in vitroscreens, such as Caco-2 permeability and livermicrosomal half-life studies, provide mechanisticinsights to the two key issues (absorption andfirst-pass metabolism) that potentially limit oralbioavailability. The quantitative informationgenerated is critical to the development of SARin optimizing ADME properties. In contrast, othertypes of pharmacokinetic screens such as cassettedosing15–17 and abbreviated pharmacokineticstudies18 generally require significant in vivoresources. In addition, they are more empirical innature, providing limited insight into the under-lying mechanism that is responsible for poor oralexposure.

In vitro Caco-2 studies have been widely used asa permeability screen in the pharmaceuticalindustry.19 However, as with any in vitro screen,the relevance of the in vitro Caco-2 permeabilityassay to assessing the in vivo absorption potentialneeds to be clearly demonstrated for programcompounds prior to implementing such a screen ina discovery program. This is because differencesin culturing conditions, passage numbers, andtesting conditions could greatly affect the perme-ability results.19 In addition, the varying expres-sion levels of known and unknown transporters inCaco-2 cells could further confound the correlationof permeability to in vivo absorption. In thepresent work, we examined the in vitro and in vivocorrelation between Caco-2 permeability andin vivo absorption at an early stage of a discoveryprogram in order to determine the relevance ofsuch a screen to in vivo absorption. As shown inTable 1, the Caco-2 permeability was quantita-tively predictive of in vivo oral absorption. Whenthe Caco-2 Pc value that was 15 nm/s or less, oralbioavailability was poor despite low systemicclearance. When the Caco-2 Pc value was in anintermediate range (e.g., 50 nm/s), the oralabsorption of BMS-378806 was still suboptimaland species dependent (Tab. 3).7 Therefore, it wasdeemed desirable to have a compound thatexhibits a good Caco-2 Pc of 100 nm/s or greater.These calibrations provided quantitative informa-tion to the medicinal chemistry team that guided


SAR development as part of effort to improve oralabsorption. For the purpose of screening, a cut-offvalue of 70 nm/sec was used to delineate com-pounds with greater absorption potential. Theguideline is consistent with an in-house data setestablished with marketed drugs.14

The current Caco-2 screen was conducted at acompound concentration of 200mM. The relevanceof such a concentration to oral absorption shouldbe considered. In our typical pharmacokineticstudies, a compound was dosed at 1 mg/mL (5 mg/kg with a dosing volume of 5 mL/kg). With a MW of400, this would translate to a gastrointestinal (GI)luminal concentration of 2.5 mM. It has beenreported that residual GI fluid volume is quitelimited in rats.20 Therefore, the use of 200mM toassess oral absorption potential is justified. Ahigher testing concentration may also minimizethe potential of nonspecific binding to Caco-2 cellstudy devices, as this issue often contributes to theproblem of a low recovery in Caco-2 permeabilitystudies.19 Obviously, the downside of a highcompound testing concentration is that solubilitycan often be a limiting factor even with 1% DMSOin the buffer. However, our experience with HIV-1attachment inhibitors (crystalline solubility in therange of 1–50mg/mL) is that this was not a majorissue in the screen.

In addition to poor permeability, solubility oftenplays a role in limiting oral absorption. In thecurrent work, all the in vivo pharmacokineticstudies were conducted with test compoundsdosed in a 90% PEG400/10% ethanol solution.The possibility that precipitation in the GI lumenoccurred to at least some extent after oral dosingof the solution formation cannot be completelyexcluded and crash-resistant formulations werenot explored at the time. However, for the HIV-1attachment inhibitor program, a number ofcompounds with a range of solubility showeddose-proportional or more than dose-proportionalincreases in systemic exposure when adminis-tered at doses 10- or 20-fold higher than theroutine pharmacokinetic screening dose of 5 mg/kg (data not shown). Since the propensity ofprecipitating in the GI tract after a high solutiondose is higher than that following a low dose, theseresults suggest that the HIV-1 attachmentinhibitors studied were unlikely to have precipi-tated to a significant extent in the GI lumen whendosed in a solution formulation at the 5 mg/kgdose. This line of evidence, together with a goodcorrelation observed between Caco-2 permeabilityand oral absorption, indicates that solubility

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unlikely played a major role in limiting the oralabsorption of HIV-1 attachment inhibitors whendosed in a solution formulation at a pharmacoki-netic screening dose of 5 mg/kg.

Similar to the approach taken with the in vitroCaco-2 permeability screen, the relevance ofin vitro liver microsomal half-life studies topredicting the in vivo clearance needed to beproven prior to employing such a screen as a triagemechanism immediately following the initialpotency evaluation. In addition, the reliabilityand reproducibility of the assay needed to bedemonstrated while testing with different lots ofpooled human liver microsomes and/or on differ-ent study occasions. In the current work, it wasdemonstrated early on in the HIV-1 attachmentinhibitor program that a strong correlationexisted between the clearance predicted fromrat liver microsomes and the clearance observedin vivo. The assay had a sufficient resolution todifferentiate three clearance classes among 12 outof 13 compounds tested. The only outlier in thecorrelation plot (Fig. 2) had a predicted clearancefrom liver microsomes 2.5-fold higher than thatobserved. Such a discrepancy was not unreason-able, given the fact that there were a number ofassumptions (see Data Analysis Section) involvedin the prediction of clearance from liver micro-somes and violating any of the assumptions couldaffect the accuracy of the prediction results.Furthermore, the data generated from the livermicrosomal half-life screen greatly aided theSAR development in a quantitative manner andeffectively guided chemistry efforts towards iden-tifying metabolically more stable compounds.

It is important to point out that the livermicrosomal half-life screen that utilizes NADPHas a cofactor only represents a portion ofbiotransformation reactions (i.e., oxidative meta-bolism). Cautions should be exercised not to over-interpret the microsomal stability data when afunctional moiety in a structure is capable ofundergoing Phase II metabolism. The tendencytowards Phase II metabolism could unknowinglyincrease if a metabolic stability screen weresolely geared towards NADPH-fortified livermicrosomal stability studies. Therefore, it isimportant to periodically evaluate metabolicstability in a more complete in vitro system suchas hepatocytes to ensure that a broad range ofpotential biotransformation reactions are covered.

Factoring microsomal binding in the predictionof clearance has been advocated in the litera-ture.21–23 In the present work, we assumed that

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in vitro microsomal binding was similar to that inblood, thus the two factors canceled out eachother. While this assumption may be scientificallydebated, it did result in a very good in vitro–in vivocorrelation in the clearance for HIV-1 attachmentinhibitors. The same approach (i.e., withoutconsidering microsomal protein binding in theclearance prediction) had been applied to theclearance prediction of BMS-378806 and BMS-488043 across species, which also resulted in areasonably good in vitro and in vivo correlation.This again illustrates the importance of under-standing the in vitro–in vivo correlation forclearance in individual discovery programs. It ispossible that other factors (e.g., high extentdistribution to the liver) may offset the bindingdifference between microsomal incubations andblood, leading to a reasonable prediction ofclearance from in vitro data without consideringbinding to microsomal proteins.

Implementation of two in vitro ADME screensin the HIV-1 attachment inhibitor programgreatly enhanced the throughput of compoundevaluation and the ability of triage efficientlyprior to conducting in vivo pharmacokineticstudies. Moreover, the quantitative data gener-ated from the two in vitro screens providedmeaningful guidance to the effort of improvingthe pharmacokinetic properties of newly synthe-sized HIV-1 attachment inhibitors. However, thecritical challenge was to balance high antiviralpotency with a liver microsomal half-life predic-tive of a low clearance in humans. As illustrated inFigure 3a, a majority of compounds that hadantiviral activity (EC50) of less than 1 nM fell intothe category of a short liver microsomal half-life.It is understood that increasing lipophilicitygenerally increases affinity to targets. However,it is often associated with a cost, since the higherthe lipophilicity of a molecule, the higher thepropensity to be metabolized. The opposite is oftentrue as well. When compounds become more polarand less lipophilic, they tend to be more stable inthe liver microsomal half-life screen. However, thepotency for the target is often compromised.Furthermore, increases in polarity decrease theCaco-2 permeability, as shown in Figure 3b, wheremetabolically stable compounds generally exhibita low Caco-2 Pc value. This kind of dilemmapresented a significant challenge to the HIV-1attachment inhibitor program. To address thisdilemma, it was critical to map out the chemicalspace for optimal combination of potency andADME properties early on in a program, taking a


2150 YANG ET AL.

holistic and balanced approach rather than simplyoptimizing potency alone into a narrow chemicalspace where adequate ADME properties would bedifficult to achieve. A balanced approach is clearlyimportant, because a biological target oftendefines the range of physicochemical propertiesof small molecules that it interacts with, whichfurther determines pharmacokinetic properties inthe body.

It is of interest to note from Figure 3b that,while there were a significant number of com-pounds that demonstrated high Caco-2 Pc butpoor metabolic stability, there existed an equalnumber of compounds that were poor in bothattributes. These results suggest that lipophili-city, as gauged from good metabolic stability, isnot the only factor that governs the Caco-2permeability. Clearly, other factors, such as effluxtransporters, also play an important role ingoverning the Caco-2 permeability.

The identification of BMS-488043 through thein vitro Caco-2 and liver microsomal half-lifescreens was a major step in the HIV-1 attachmentinhibitor program. Although BMS-488043 was notthe most potent molecule of the compoundsevaluated (Fig. 3a), its Caco-2 Pc value (178 nm/sat 200mM) and liver microsomal half-life(>100 min at 1mM and �1 mg/mL microsomalproteins) were clearly superior among the >100compounds tested. Compared to the programprototype, BMS-378806 (Caco-2 Pc of 51 nm/sand microsomal half-life of 37 min), theseimprovements were significant. The significantimprovements in the in vitro ADME attributesalso translated well into in vivo pharmacokineticproperties. Unlike BMS-378806 where oral bioa-vailability was modest and species dependent(Tab. 3), the oral bioavailability of BMS-488043was consistently high across animal species,largely owing to the improvement in membranepermeability. High membrane permeability alsotranslated into �2-fold increases in the Vss forBMS-488043 compared to BMS-378806, asobserved in rats and monkeys. The benefit ofhigh membrane permeability was also evident in amodest reduction in the oral exposure of BMS-488043 when dosed in a crystalline suspension.Compared to BMS-378806, the aqueous solubilityof BMS-488043 as a crystalline free base was4-fold lower at pH 4–8 (170mg/mL vs. 40mg/mL).However, the relative oral bioavailability fromthe suspension in rats was similar betweenBMS-488043 and 378806 (62% vs. 61%) whenevaluated at 5 mg/kg. Clearly, in this case, higher


membrane permeability of BMS-488043 com-pensated for its lower aqueous solubility thatmight otherwise have had a negative effect onbioavailability.

The improvement in liver microsomal half-lifealso led to a twofold improvement in the systemicclearance of BMS-488043 compared to BMS-378806. The systemic clearance of BMS-488043was low in all three animal species tested (Tab. 3),with a hepatic extraction ratio in the range of 10–20%. Consistent with findings in animal species,the predicted human clearance of BMS-488043from either human liver microsomes or hepato-cytes was low, whereas the predicted humanclearance of BMS-378806 was in the intermediaterange. The improvements in the primary phar-macokinetic parameters of BMS-488043 overBMS-378806 resulted in 6- to 12-fold increasesin the AUC observed in rats and monkeys wherethe pharmacokinetics of BMS-378806 was sub-optimal. Similarly, the terminal half-life and MRTwere increased by 4- to 8-fold in these two species.Collectively, these data indicate that the improve-ment in Caco-2 permeability and liver microsomalhalf-life had led to a consistent enhancement ofpharmacokinetic behaviors observed for BMS-488043 in the three animal species tested andshould lead to a more desirable pharmacokineticprofile in humans. Indeed, the oral exposure ofBMS-488043 observed in humans was at least15-fold higher than that of BMS-378806 whencompared at the same dose under a similardosing condition (e.g., fasted/fed and solution vs.capsule).24,25 More importantly, the significantimprovement in the human oral exposure of BMS-488043 led to a proof-of-concept in the clinic forthis new class of anti-HIV agents.26

In comparison with the human solution data,the plasma AUC prospectively predicted fromanimal solution data was 85% of the observedvalue.24,25 When factored in the relative oralbioavailability of 62% obtained from a suspensionstudy in rats, the predicted AUC were 1.6-foldhigher than that of the capsule data.24,25 Thebigger discrepancy in the capsule data betweenthe predicted and observed AUC is not unex-pected, as the final form used in clinical studieswas not available at the discovery stage and thedissolution step could not be readily assessed atthe time of the candidate nomination.

One desirable pharmacokinetic feature thatwas not achieved with BMS-488043 was to attaina high extent of brain penetration, since the brainis believed to be a sanctuary site for HIV-1 where

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the virus can elude drug treatment and possiblydevelop resistance.27 Despite significant improve-ments in the membrane permeability of BMS-488043, the molecule failed to penetrate into thebrain. A number of factors could contribute to thisobservation. First of all, BMS-488043 was highlyprotein bound (97% bound in rats and 95% boundin humans), which may limit its brain penetra-tion. Secondly, BMS-488043, as well as BMS-378806, is likely a substrate of efflux transporterssuch as P-gp and/or breast cancer resistanceprotein, which have been shown to limit brainpenetration to a significant extent.28,29 The bi-directional Caco-2 studies with BMS-488043,although conducted at a relatively high drugconcentration of 100mM, demonstrated that therewas a direction-dependent Caco-2 Pc and that thisdifference could be inhibited by 100mM verapa-mil. Furthermore, the compound was activelysecreted in rat kidneys, providing an additionalline of evidence that BMS-488043 is subject toefflux transporters. Clearly, a triple-knockoutmouse or P-gp knockout mouse study is warrantedin this case. Moreover, going forward, a consid-eration should be given to conducting bi-directional Caco-2 permeability studies at asubstantially lower substrate concentration(e.g., 3mM) on a routine basis in order to screenfor efflux transporter substrates and guidechemistry SAR to address this issue.

In summary, the in vitro Caco-2 permeabilityand liver microsomal half-life screens wereutilized to more rapidly and effectively guidechemistry SAR that led to the identification ofBMS-488043, a novel HIV-1 attachment inhibitorwith much improved pharmacokinetic propertiesover the prototype compound BMS-378806. Theseimproved pharmacokinetic properties in the pre-clinical setting had lead to a significant improve-ment in human pharmacokinetics that enabled aproof-of-concept in the clinic for this novel class ofanti-HIV agents.

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DOI 10.1002/jps

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