Disposition of radiolabeled BMS-204352 in rats and dogs

Received 11 September 2001Revised 12 November 2001

Copyright # 2002 John Wiley & Sons, Ltd. Accepted 13 November 2001

BIOPHARMACEUTICS & DRUG DISPOSITIONBiopharm. Drug Dispos. 23: 41–46 (2002)

Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/bdd.292

Disposition of Radiolabeled BMS-204352 in Rats and Dogs

Rajesh Krishna*, Ming Yao, Nuggehally R. Srinivas, Vinod Shah, Janice M. Pursley, Mark Arnold andNimish N. VachharajaniClinical Discovery, Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, NJ, USA

ABSTRACT: BMS-204352, a maxi-K channel opener, is currently under development for thetreatment of stroke. The objective of this study was to determine the pharmacokinetics, massbalance and absolute oral bioavailability of [14C]-BMS-204352 in rats and dogs. [14C]-BMS-204352was administered, to rats (n= 10/group; parallel design, 6mg/kg) and dogs (n= 4/group; crossoverdesign, 2mg/kg), as an oral (PO) or as a 3-min intraarterial (IA) infusion in rats and a 6-minintravenous (IV) infusion in dogs. Blood, urine, and feces samples were collected and analyzed forunchanged BMS-204352 (plasma) using a validated LC/MS assay and for total radioactivity(plasma, urine, feces) using liquid scintillation counting. The mean total body clearance (CLT) andsteady-state volume of distribution (VSS) values for the unchanged BMS-204352 were 2.58� 0.48 l/h/kg and 6.3� 1.14 l/kg, respectively, in rats and 0.21� 0.02 l/h/kg and 4.06� 0.47 l/kg,respectively, in dogs. In both species, the elimination half-life of total radioactivity was significantlylonger as compared to the unchanged drug. After IA administration of radiolabeled BMS-204352 torats, ca. 5.9 and 85% of radioactivity was recovered within 7 days in urine and feces, respectively;corresponding recoveries after PO dosing were 4.5 and 99.5%, respectively. The recoveries weresimilar in dogs, i.e., ca. 5.2 and 83% of administered radioactivity recovered in urine and feces,respectively, for IV dose and ca. 4 and 86%, respectively, for PO dose. These data indicate thatnonrenal (biliary) elimination in both species was predominant. Based on comparable urinaryrecovery of radioactivity and plasma AUCs of radioactivity, the extent of oral absorption of BMS-204352 appeared to be complete in both species. The absolute oral bioavailability was 55% in ratsand 79% in dogs. Bioavailability and extent of absorption data suggest evidence of first passmetabolism of BMS-204352 in the rat and dog. Copyright # 2002 John Wiley & Sons, Ltd.

Key words: BMS-204352; mass balance; absolute bioavailability; rats; dogs

Introduction

BMS-204352 (MaxiPostTM, [3S]-[+]-[5-chloro-2-methoxyphenyl]-1,3-dihydro-3-fluoro-6-[trifluor-omethyl]-2H-indol-2-one; Figure 1), a novelfluorooxindole maxi-K channel opener, is beingdeveloped for the treatment of stroke [1–3]. Thecompound is highly potent and specific for thetarget site of action and has been shown to beefficacious for therapy of acute forms of stroke

after intravenous dosing in animal stroke models[4]. In order to closely simulate clinical conditionof stroke, animal models included permanentMCA occlusion with delayed (2 h) drug applica-tion. Specifically, BMS-204352 significantly af-forded cortical neuroprotection 2 h after onsetof occlusion. In these models, BMS-204352produced remarkable decreases in corticalinfarct volume at relevant neuroprotective doses.This is notably important considering that com-parable doses of noncompetitive NMDA antago-nists MK-801 and CNS-1102 were ineffectiveupon drug application 2 h post-onset ofocclusion.

*Correspondence to: Dr. Rajesh Krishna, Mailstop E12-07, Bristol-Myers Squibb, Route 206 and Province Line Road, Princeton, NJ08540, USA. E-mail: [email protected]

The objective of this study was to assess thepharmacokinetics, absolute bioavailability, anddisposition of radiolabeled BMS-204352 in ratsand dogs. Both species have been used to assessthe toxicology profile of the compound. Proteinbinding studies with BMS-204352 have indicatedthat the compound is highly protein bound inboth species [5].

Experimental Section

Chemicals and formulations

BMS-204352 and [14C]-BMS-204352, as the freebase, with a specific activity of 28.4 mCi/mg(radiochemical purity 99.5%) were obtained fromBristol-Myers Squibb (BMS) Pharmaceutical Re-search Institute (Princeton, NJ). The dosingsolution for rats was prepared by dissolvingBMS-204352 in appropriate amounts of polyethy-lene glycol-300 (4%), polysorbate 80 (1.5%),ethanol (1%), and sterile water for injection(93.5%). For dogs, the dosing solution wascomposed of polyethylene glycol-300 (55%),ethanol (15%), and 5% dextrose in water (30%).Before administration, the dosing solution wassterilized by filtration through a 0.22 mm filter.

Animal studies

Approval for conducting the pharmacokineticstudies in rats and dogs were obtained from theBMS Animal Care and Use Committee.

Rats: Male Sprague-Dawley rats (ca. 250–300 g)were obtained from Hilltop Lab Animals, Inc(Scottdale, PA). Prior to the study initiation, ratswere acclimatized for a few days and fed astandard rodent diet (Purina rat chow andwater). A day prior to dosing, rats werecannulated with indwelling arterial (carotidartery) and venous (jugular vein) catheters andindividually housed in plastic metabolism cages.The animals were weighed prior to dosing. Thestudy was performed in two parts. In Part 1, 10rats were grouped into two groups of 5 rats each(A and B). Rats in group A received a nominaldose of 6mg/kg of [14C]-BMS-204352 (ca. 40 mCi/kg of radioactivity) by an intraarterial infusionover 3min through the carotid artery catheter.Serial urine and feces samples were collected at0–6, 6–24, 24–48, 48–72, 72–96, 96–120, 120–144,and 144–168 h post-dose. After each collectioninterval, the metabolism cages were rinsed withwater and cage rinses were counted for radio-activity. At the end of the study, the rats weresacrificed by an over dose of Metofane1 (meth-oxyflurane; Schering-Plough Animal Health, Un-ion, NJ). Aliquots of urine were transferred tolabeled polypropylene tubes and stored at orbelow �208C until analysis. Fecal samples werehomogenized for 10min in an ultrasonic proces-sor (Sonicator, Heat Systems-Ultrasonics, Inc.),and stored at or below �208C until analysis. InPart 2, the same procedure was followed fordosing and serial blood (0.4ml) samples werecollected from the jugular vein catheter at 3 (endof infusion), 6, 15, 45min, and 2, 3, 6, 8, 12, and24 h for IA; 15, 30, 45min, and 1, 2, 4, 6, 8, 12, and24 h for oral dosing. After the 24 h blood samplecollection, the rats were sacrificed by an overdose of Metofane1. Blood samples were collectedin tubes containing EDTA as the anticoagulant.Samples were analyzed for the determination oftotal radioactivity and for unchanged BMS-204352 by a validated LC/MS assay. All plasmasamples were stored at or below �208C untilanalysis.

Dogs: Male beagle dogs (ca. 11–14 kg) wereobtained from White Eagle Laboratories (Doyles-town, PA). Dogs, identified by tattoo numbers,were housed in individual cages and were fastedovernight and 2h post-dose. Water was provided

Figure 1. Chemical structure of BMS-204352

Copyright # 2002 John Wiley & Sons, Ltd. Biopharm. Drug Dispos. 23: 41–46 (2002)

R. KRISHNA ET AL.42

ad libitum. Dogs were surgically instrumentedwith indwelling venous access ports (femoralvein). In an open, 2-way crossover, randomizedmanner, four dogs received a single dose of2mg/kg [14C]-BMS-204352 (total radioactivity of50 mCi) either orally as a gavage or intravenouslyas a 6min infusion into the femoral vein in eachsession. There was a minimum of a 2-weekwashout period between sessions. Blood samples(2–5ml/time point) were collected at 0, 6 (end ofinfusion), 12, 20, 30, 45min, and 1, 2, 3, 6, 8, 12,24, 32, 48, and 72 h post-dose for IV administra-tion, and at 0, 15, 30, 45min, and 1, 1.5, 2, 3, 6, 8,12, 24, 32, 48, and 72 h post-dose for oraladministration. Blood samples were collected intubes containing EDTA to generate plasma uponcentrifugation. Samples were quantified for totalradioactivity and unchanged BMS-204352. Urineand feces from each dog were collected at 0–12,12–24, 24–48, 48–72, 72–96, 96–120, 120–144, and144–168 h post-dose. Plasma was analyzed forBMS-204352 using a validated LC/MS methodand total radioactivity in plasma, urine, and feceswas determined using liquid scintillation spec-trophotometry. Fecal samples were homogenizedafter addition of a 50:50 methanol:water mixture.All samples were stored at or below �208C untilanalysis.

Analytical methods

Determination of unchanged BMS-204352 inplasma: Plasma samples (0.1ml for rat and0.2ml for dog) were analyzed for unchangedBMS-204352 using a validated LC/MS methodwith a lower limit of quantitation of 2.5 ng/ml(rat) or 1 ng/ml (dog). Analyses were carried outusing a Hewlett–Packard HP 1090 Chromato-graphic System Series II and a Hypersil ODS2mm� 50mm� 3 mm column, operating with aVG Quattro I (Micromass) mass detector, mon-itoring m/z 358.1 for BMS-204352. The MS wasoperated in a negative electrospray mode usingnitrogen as a nebulizing gas at a flow rate of ca.20 bar and at a voltage of ca. 4000V. Data wereacquired and chromatographic peaks integratedusing the Masslynx1 software. BMS-204352 andan internal standard (stable-isotope labeled ana-log of BMS-204352) were extracted from bufferedplasma samples using toluene, evaporated to

dryness, and the residue reconstituted in amobile phase containing ammonium acetate inwater/methanol. The standard curve range was2.5–500 ng/ml in rat plasma and 1–500 ng/ml indog plasma; curves were linear with a coefficientof determination of � 0.998. Mean predictedconcentrations of quality control samples were5� 12.2% of nominal values and between- andwithin-day variabilities were 57.4% relativestandard deviation for both assay methods.

Determination of total radioactivity in plasma, urine,and feces: Blood samples were digested forapproximately 24–48 h at room temperature withToluene-350 (Packard Instrument Co., Meriden,CT), bleached with 20% benzoyl peroxide intoluene, neutralized with 0.1ml of a 4:3:1 mixtureof saturated sodium pyruvate in methanol:glacialacetic acid:methanol. This was followed byaddition of Hionic-Fluor scintillation cocktailand counted using liquid scintillation spectro-photometry. Plasma and urine samples weremixed with Hionic-Fluor liquid scintillationcocktail for radioactivity measurement. Homo-genized fecal samples were combusted in abiological oxidizer (Packard Sample Oxidizer,Packard Instrument Co., Meriden, CT), Perma-Fluor scintillation fluid added and radioactivitymeasured. Radioactivity in plasma, urine, andfecal samples was determined using a Tri-Carbliquid scintillation spectrophotometer (PackardInstrument Co., Meriden, CT) equipped with anautomatic data reduction system. Counts forradioactivity were corrected for backgroundand quenching.

Pharmacokinetic analysis

Plasma concentration vs time data for unchangedBMS-204352 and total radioactivity were ana-lyzed using a noncompartmental method usingPKMENU, an in-house pharmacokinetic analysissystem (Bristol-Myers Squibb, Princeton, NJ).The peak plasma concentration, Cmax, and thetime to reach peak concentration, Tmax, wererecorded directly from experimental observa-tions. The slope of the terminal log-linear phaseof the plasma concentration vs time curve (b) wasidentified by least-squares linear regression ofdata points that provided a minimum mean


BIOAVAILABILITY AND MASS BALANCE OF BMS-204352 43

square error. The T1/2 of the terminal log-linearphase was calculated as ln(2) divided by absolutevalue of b. The area under the plasma concentra-tion vs time curve from time 0 to infinity (AUC)was determined by a combination of trapezoidaland log-trapezoidal summations, with extrapola-tion to infinity. The extrapolated area wasdetermined by dividing the observed concentra-tion at the time of last nonzero plasma concen-tration by the slope of the terminal log-linearphase (b). The total body clearance (CLT), meanresidence time (MRT), and the steady-statevolume of distribution (VSS) of BMS-204352after intrarterial (rats) or intravenous (dogs)administration were calculated as follows:CLT=dose/AUC; MRT= [AUMC/AUC]�T0/2;and VSS=CLT�MRT, where AUMC is the areaunder the first moment curve and T0 is the infusiontime (i.e., 3min for rats and 6min for dogs). Theabsolute oral bioavailability (F%) of BMS-204352was calculated from the plasma AUC data ofunchanged BMS-204352 after IV or IA and oraltreatments. The total urinary recovery of BMS-

204352 or total radioactivity and fecal recovery oftotal radioactivity were calculated as the cumula-tive amount excreted in urine or feces andexpressed as a percentage of administered dose.

Results

The mean plasma concentration–time profiles ofunchanged BMS-204352 and total radioactivityfollowing drug administration in rats and dogsare shown in Figure 2, panels a–d. Pharmacoki-netic parameters are summarized in Table 1.

Pharmacokinetics in rats: After a 3-min IA infusionof 6mg/kg [14C]-BMS-204352 dose, mean Cmax

values were 1430 ng/ml and 2621 ng eq/ml forunchanged BMS-204352 and total radioactivity.Following termination of infusion, plasma con-centrations of unchanged BMS-204352 declinedmore rapidly than total radioactivity (Figure 2).In the terminal log-linear phase, plasma concen-tration–time profiles of unchanged BMS-204352and total radioactivity declined with mean T1/2

Figure 2. Mean (S.D.) plasma concentration–time profiles of unchanged BMS-204352 (closed circles; ng/ml) and totalradioactivity (TRA, closed triangles; ng eq/ml) in rats and dogs following intravenous (dogs) or intraarterial (rats) and oraladministration in rats and dogs following administration of [14C]-BMS-204352


R. KRISHNA ET AL.44

values of 2.3 and 21.6 h, respectively (Table 1).Comparison of the plasma AUC values forunchanged BMS-204352 and total radioactivityindicated that 53% of the plasma radioactivitywas accounted by the unchanged drug. Themean CLT and VSS of unchanged BMS-204352were 2.58 and 6.3 l/kg (Table 1). Radioactivitywas excreted primarily in the feces; from 0 to168 h post-drug administration, approximately85% of the dose was recovered in the feces (Table1). Thus, the total radioactivity in urine and fecesaccounted for ca. 91% of the administered dose(Table 1).

After oral administration of a 6mg/kg solutiondose of [14C]-BMS-204352, mean Cmax values forunchanged BMS-204352 and total radioactivitywere 215 ng/ml and 2506 ng eq/ml, respectively(Table 1). The mean plasma concentrations ofunchanged BMS-204352 and total radioactivitypeaked at a median Tmax value of 0.75 and 4h,respectively. Mean T1/2 values of unchangedBMS-204352 and total radioactivity were 2.69and 21 h, respectively. Over a 168 h period, theurinary excretion of total radioactivity amounted

to 4.5% of the orally administered dose. Radio-activity was excreted primarily in the feces; on anaverage, a total of 99.5% of the oral administereddose was recovered in the feces. The absolutebioavailability of BMS-204352 after oral adminis-tration of a 6mg/kg solution dose of [14C]-BMS-204352 was 55% (Table 1).

Pharmacokinetics in dogs: After a 6min IV infusionof a 2mg/kg dose of [14C]-BMS-204352, meanCmax values were 2155 ng/ml and 7026 ng eq/mlfor unchanged BMS-204352 and total radioactiv-ity, respectively (Table 1). Following terminationof infusion, plasma concentrations of BMS-204352 appeared to decline more rapidly thanthe total radioactivity (Figure 2). In the terminallog-linear phase, plasma concentration–time pro-files of unchanged BMS-204352 and total radio-activity declined with mean T1/2 values of 16 and158 h, respectively (Table 1, Figure 2). Compar-ison of the plasma AUC values for unchangedBMS-204352 and total radioactivity indicated that53% of the plasma radioactivity was accountedby the unchanged drug. The mean CLT and VSS

Table 1. Summary of the absorption, distribution, metabolism, and excretion parameters of unchanged BMS-204352 and totalradioactivity (TRA) in rats and dogs after IV/IA and oral administration

Species Analyte Dose(mg/kg),route

Cmaxa Tmax

(h)bAUC(INF)a T1/2

(h)CLT(l/h/kg)

VSS(l/kg)

F (%) UR(%)c

FR(%)c

Rat BMS-204352 6, IA 1430 0.05 1066 2.3 2.58 6.3 nad na na(498) (0.05, 0.05) (206) (0.3) (0.48) (1.14)

6, PO 215 0.75 585 2.7 na na 55 na na(52) (0.50, 0.75) (127) (0.6)

TRA 6, IA 2621 3.0 48804 22 na na na 5.9 85.0(245) (3.0, 6.0) (4447)e (5.5) (1.0) (2.1)

6, PO 2506 4.0 46781 21 na na na 4.5 99.5(134) (2.0, 6.0) (3133) (7.7) (0.8) (24)

Dog BMS-204352 2, IV 2155 0.1 9811 16 0.21 4.06 na na na(411) (0.1, 0.1) (881) (0.7) (0.02) (0.47)

2, PO 487 1.25 7714 15 na na 79 na na(153) (0.5, 2.0) (717) (2.7) (9)

TRA 2, IV 7026 32 444812 158 na na na 4.0 85.8(933) (24, 32) (54764)e (15) (0.6) (6.2)

2, PO 6194 32 569356 158 na na na 5.2 83.1(877) (24, 32) (238873)e (10) (1.1) (2.7)

a Units for Cmax: BMS-204352 (ng/ml) and TRA (ng eq/ml); units for AUC: BMS-204352 (ngh/ml) and TRA (ng eq h/ml).b Median (minimum, maximum).c Cumulative amount excreted in urine (UR%) and feces (FR%) over 7 days.d Not applicable.e AUC(0-T).


BIOAVAILABILITY AND MASS BALANCE OF BMS-204352 45

of BMS-204352 were 0.21 l/h/kg and 4.06 l/kg,respectively (Table 1). Radioactivity was excretedprimarily in the feces. Over a 7-day period post-drug administration, ca. 5 and 83% of the dosewas recovered in the urine and feces, respectively(Table 1). The overall total radioactivity in urineand feces accounted for approximately 88% ofthe administered dose.

After oral administration of a 2mg/kg solutiondose of [14C]-BMS-204352, the mean Cmax valuesfor unchanged BMS-204352 and total radioactiv-ity were 487 ng/ml and 6194 ng eq/ml, respec-tively (Table 1). Mean T1/2 values for unchangedBMS-204352 and total radioactivity were 15 and168 h, respectively. Comparison of the plasmaAUC values for unchanged BMS-204352 and totalradioactivity indicated that 52% of the plasmaradioactivity was accounted by the unchangeddrug. Radioactivity was excreted primarily in thefeces. Over a 7-day period post-drug adminis-tration, a total of ca. 4 and 86% of the dose wererecovered in the urine and feces, respectively(Table 1). The overall radioactivity in feces andurine accounted for nearly 90% of the adminis-tered dose. The absolute bioavailability of BMS-204352 after oral administration of a 2mg/kgsolution dose of [14C]-BMS-204352 was 79%(Table 1).

Discussion

Following intraarterial (rats) or intravenous(dogs) administration, plasma concentration vstime profiles of unchanged drug and total radio-activity were markedly different, with un-changed BMS-204352 levels declining morerapidly than total radioactivity. The more rapiddecline of BMS-204352 levels in plasma relativeto total radioactivity suggested that the T1/2 oftotal metabolites ([14C]-derived material) is muchlonger than that of the unchanged drug.

Radioactivity was primarily excreted in thefeces (ca. 85% of administered dose) in both ratsand dogs. Radioactivity in urine accounted forapproximately 5% of the administered dose,suggesting that nonrenal elimination plays apredominant role in the disposition of BMS-204352 in rats and dogs.

Following oral administration in rats and dogs,the absorption of [14C]-BMS-204352 from thesolution dose was rapid. The percentage of theadministered radioactive dose that was recov-ered from the urine was comparable following IA(rats) or IV (dogs) and oral dosing, suggestingthat oral absorption of BMS-204352 was essen-tially complete. The extent of oral absorption wasestimated based on plasma AUC of total radio-activity following IA (rats) or IV (dogs) and oraladministration. Based on comparable AUCs ofradioactivity, the extent of oral absorption ap-peared to be complete in rats and dogs. However,the absolute oral bioavailability was only 55% inrats and 79% in dogs, suggesting some evidenceof first pass metabolism of BMS-204352 in ratsand dogs.

In summary, BMS-204352 was rapidly andcompletely absorbed after oral administration inboth species, with absolute bioavailability ran-ging from 55 to 79%. The compound undergoesextensive distribution, as indicated by the VSSvalues in both species. Some evidence of first-pass metabolism of BMS-204352 is suggestedwith nonrenal (biliary) elimination appearing toplay a predominant role in the elimination of thecompound.

References

1. Gribkoff VK, Starrett Jr JE, Dworetzky SI, et al. Targetingacute ischemic stroke with a calcium-sensitive opener ofmaxi-K potassium channels. Nat Med 2001; 7: 471–477.

2. Cheney JA, Weisser JD, Bareyre FM, et al. The maxi-Kchannel opener BMS-204352 attenuates regional cerebraledema and neurologic motor impairment after experi-mental brain injury. J Cereb Blood Flow Metab 2001; 21: 396–403.

3. Starrett JE, Dworetzky SI, Gribkoff VK. Modulators oflarge conductance calcium-activated potassium (BK)channels as potential therapeutic targets. Curr Pharm Des1996; 2: 413–428.

4. Starrett JE, Hewawasam P, Oritz AA, et al. Synthesis,pharmacokinetic analysis and MCAO stroke activity ofthe maxi-K opener BMS-204352. Proceedings of the KeystoneSymposium: Potassium Channels, Keystone, CO, 2000, 57.

5. Krishna R, Yao M, Kaczor D, Vachharajani N, Srinivas NR.In-vitro protein binding studies with BMS-204352: lack ofin-vitro protein binding displacement interaction of BMS-204352 in human serum. Biopharm Drug Dispos 2001; 22:41–44.


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Disposition of radiolabeled BMS-204352 in rats and dogs