Received 14 February 2002Revised 28 March 2002

Copyright # 2002 John Wiley & Sons, Ltd. Accepted 5 April 2002

BIOPHARMACEUTICS & DRUG DISPOSITIONBiopharm. Drug Dispos. 23: 233–237 (2002)

Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/bdd.318

Pharmacokinetics and Dose Proportionality of BMS-204352after Intraarterial Administration to Rats

Rajesh Krishna*, Vinod R. Shah and Nuggehally SrinivasClinical Discovery, Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, NJ 08543-4000, USA

ABSTRACT: BMS-204352 is a novel maxi-K channel opener that is being developed for thetreatment for stroke. The current study was designed to evaluate the dose proportionality andpharmacokinetics of BMS-204352 in rats. In an open, parallel fashion, sixteen rats per genderreceived a single intraarterial dose of BMS-204352 as a 3-min infusion into the carotid artery at 0.4,2.0, 5.0 and 10.0mg/kg dose levels. Serial blood samples were collected for up to 24 h post-doseand plasma samples were analyzed for the concentrations of intact BMS-204352 using a validatedliquid chromatographic mass spectrometric (LC/MS) method. Pharmacokinetic analysis wasperformed using a non-compartmental method. Results revealed a gender difference in thepharmacokinetics of BMS-204352 in rats at all doses excluding the first (i.e., 0.4mg/kg) dosepanel. BMS-204352 peak plasma concentration (Cmax) and area under the plasma concentration–time curve (AUC) values increased in a proportion greater than the increment in dose.Specifically, as dose increased in the ratio 1:5:12.5:25, Cmax increased in the ratio 1:7:18:31 in malerats and 1:7:22:51 in female rats. The respective AUC ratios were 1:6:20:42 in male rats and1:12:29:77 in female rats. Mean total body clearance (CLT) values for BMS-204352 ranged from 879–3242ml/h/kg over the four dose levels and generally decreased with increase in dose. Similarly,steady state volume of distribution (VSS) values ranged from 3621–8933ml/kg over the four doselevels and generally decreased with increase in dose. However, mean residence time (MRT)and elimination half-life (T1/2) values for BMS-204352 were independent of dose and rangedfrom 2.42–4.54 to 2.08–4.70 h, respectively. In conclusion, BMS-204352 appears to exhibitdose-dependent pharmacokinetics in rats. In addition, there appeared to be some evidenceof gender related differences in the pharmacokinetics of BMS-204352. Copyright# 2002 John Wiley& Sons, Ltd.

Key words: BMS-204352, rats, dose proportionality, pharmacokinetics

Introduction

BMS-204352 is being developed for the treatmentof stroke [1–3]. Chemically, ([3S]-[+]-[5-chloro-2-methoxyphenyl]-1,3-dihydro-3-fluoro-6-[trifluor-omethyl]-2H-indol-2-one) BMS-204352 is a novelfluorooxindole maxi-K channel opener that has

been shown to be efficacious in acute forms ofstroke in experimental animal stroke models [4].The rat has been used as one of the primaryspecies for the toxicologic evaluation of BMS-204352.

The objectives of the current investigationwere two-fold: (1) to delineate the pharmacoki-netics of BMS-204352 in rats receiving singleintraarterial doses of BMS-204352, and (2) toevaluate the relationship of the pharmacokineticcharacteristics of BMS-204352 and the adminis-tered dose.

*Correspondence to: Dr. Rajesh Krishna, Clinical Discovery,Bristol-Myers Squibb, Route 206 and Province Line Road,Princeton, NJ 08540, USA. E-mail: rajesh.krishna@bms.com

Experimental

Animals

Approval for animal investigations was obtainedfrom the in-house animal care committee. Sixteenadult Sprague–Dawley rats per gender (weighingca 180–220 g, females and 250–300 g, males) wereused for experimentation. The rats were acclima-tized for a minimum of 3 days with appropriatetemperature and humidity control. Each rat wastagged with a unique identification number(metal ear tag). Rats were housed in stainless-steel cages and were provided free access towater and Purina rat chow (Ralston Purina).Animals were weighed prior to drug adminis-tration. Prior to experimentation, rats weresurgically prepared for placement of arterialand venous catheters. Indwelling catheters wereplaced in the carotid artery and jugular vein,after which rats were individually housed in steelmetabolic cages. After each bleed, the patency ofthe catheter was assured by flushing the catheterwith 0.2ml heparinized-saline solution.

Chemicals and formulations

BMS-204352 was obtained from Bristol-MyersSquibb Pharmaceutical Research Institute (Prince-ton, NJ). A stock solution of BMS-204352 wasprepared by dissolving the compound in asolvent mixture composed of polyethylene gly-col-400, polysorbate 80, ethanol, and 5% dextrosein water. The dosing solution was sterile filteredusing a 0.22 mm filter prior to drug administration.

Study design

This was an open, parallel group experimentaldesign. Sixteen Sprague–Dawley rats per genderreceived a single intraarterial dose of BMS-204352 as a 3min constant rate infusion via thecarotid artery, at either 0.4, 2, 5, or 10mg/kg doselevels. For drug administration, an appropriatevolume of the dosing solution was infused over3min via the carotid artery using an infusionpump that employed a 20-gauge Luer stubadapter attached to a syringe.

Sample collection and preparation

Blood samples (approximately 0.3ml) were col-lected from the jugular vein catheter at 3 (end ofinfusion), 5, 10, 20, 30min, and 1, 2, 4, 6, 8, 10, 12,and 24 h post-dosing. Blood samples were col-lected in potassium EDTA containing Vacutai-ners and processed for plasma as follows. Within30min of blood collection, plasma was harvestedby centrifuging at 1000g for 10min at 48C. Plasmasamples were stored at or below �208C untilanalysis.

Analysis of BMS-204352 in plasma

Plasma concentrations of BMS-204352 were de-termined using a validated LC/MS method thatwas accurate, precise, specific, sensitive andreproducible. Analyses were carried out using aWaters Chromatographic System Alliance modelno. 2690 (Waters Corporation, Milford, MA) anda Hypersil ODS 2mm� 50mm� 3 mm column(Phenominex Inc., Wilmington, NC), operatingwith a LC/MS API 100 mass detector (Perkin-Elmer, Foster City, CA), monitoring m/z 358.1 forBMS-204352. The MS was operated in a negativeion spray mode using nitrogen as a nebulizinggas at a flow rate of ca 40 psi and at a voltage ofca �2690mV. Data were acquired and chromato-graphic peaks integrated using the Sciex1 soft-ware program MacQuan, version 1.4b. Briefly, toa 0.1ml volume of plasma, internal standard,BMS-223110 and 5mM ammonium acetate bufferwere added. The mixture was gently vortex-mixed and extracted with toluene. The organicphase was separated and evaporated to drynessunder a gentle stream of nitrogen at 408C. Theresidue was reconstituted in the mobile phase(bi-phasic mixture of two solvent mixtures, Acomposed of 5mM ammonium acetate and 0.1%triethylamine in 75:25 v/v water:methanol, ad-justed to pH 5, and B, 5mM ammonium acetatein methanol). Standard curves were linear (R2 50.996) over the concentration range 2.5–1000 ng/ml, with a lower limit of quantitation of 2.5 ng/ml. The mean predicted quality control concen-trations deviated 47.6% from nominal values.The intra- and inter-assay precision values were45.6% relative standard deviation. Prior to theanalysis of samples, three concentrations (nom-inal concentrations of 15, 300, and 600 ng/ml) of

Copyright # 2002 John Wiley & Sons, Ltd. Biopharm. Drug Dispos. 23: 233–237 (2002)

R. KRISHNA ET AL.234

quality control (QC) samples were preparedin rat plasma. The QC samples were analyzedtogether with study samples to determineaccuracy, precision, and reproducibility of theassay.

Pharmacokinetic analyses

Plasma data were subjected to non-compartmen-tal pharmacokinetic analysis [5]. The terminallog-linear phase of the plasma concentration–time curve was identified by least-squares linearregression of data points which yielded a mini-mum mean square error. The area under theplasma concentration–time curve from time zeroto time infinity (AUC) was determined using acombination of trapezoidal and log-trapezoidalmethods plus the extrapolated area. The extra-polated area was determined by dividing thepredicted concentration at the time of last non-zero plasma concentration by the slope of theterminal log-linear phase. Other pharmacokineticparameters reported included elimination half-life (T1/2), mean residence time in the body (MRT,estimated as equal to [AUMC/AUC]-T/2, whereAUMC is the area under the first moment curveand T, the infusion time, 3min), total bodyclearance (CLT, estimated as dose/AUC), andsteady-state volume of distribution (VSS, esti-mated as a product of MRT and CLT). The peakplasma concentration (Cmax) was obtained fromobserved data.

Results and Discussion

Analytical results

The R2 of the standard curves, ranging from 2.5to 1000 ng/ml were 50.996. The mean predictedQC concentrations deviated 42.07% of thenominal values. The values for the between-run, and within-run precision estimates for theQC samples were within 6.23% relative standarddeviation. Based on the performance of standardcurves and QC samples, the plasma assayfor BMS-204352 was accurate, precise andreproducible.

Pharmacokinetic results

Mean (S.D.) plasma BMS-204352 concentration–time profiles in male and female rats areillustrated in Fig. 1A and B, respectively. Table1A and B summarizes the mean pharmacokineticparameters for BMS-204352 in male (A) andfemale (B) rats receiving 0.4–10mg/kg doses.

Plasma concentration–time curves indicate thatBMS-204352 is rapidly distributed upon dosing(Fig. 1A and B). Within 7min after cessation ofintraarterial administration, plasma levels ofBMS-204352 declined ca 30–40% from Cmax.Consistent with this result, VSS values for BMS-204352 were approximately 5–13 fold greaterthan the total body water in rats [6], indicatingthat the compound is extensively distributed intoextravascular tissues.

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Figure 1. Mean (S.D.) plasma concentration-time curves of BMS-204352 in male rats (A) and female rats (B) receiving single 0.4, 2,5, and 10mg/kg intraarterial doses of BMS-204352

Copyright # 2002 John Wiley & Sons, Ltd. Biopharm. Drug Dispos. 23: 233–237 (2002)

DOSE PROPORTIONALITY OF BMS-204352 IN RATS 235

Exposure of BMS-204352 was approximately2–3 fold higher in female rats as compared tomale rats at doses 2mg/kg and higher (Table 1Aand B), indicating gender differences in pharma-cokinetics of BMS-204352 with respect to AUCvalues.

Both Cmax and AUC values of BMS-204352appeared to increase in a proportion greater thanthe increment in dose. Specifically, as doseincreased in the ratio 1:5:12.5:25, Cmax increasedin the ratio 1:7:18:31 in male rats and 1:7:22:51 infemale rats. The respective AUC ratios were1:6:20:42 in male rats and 1:12:29:77 in femalerats.

Mean total body clearance (CLT) values forBMS-204352 ranged from 879–3242ml/h/kg overthe four dose levels and generally decreased withincrease in dose. As dose increased from 0.4 to10mg/kg, CLT values for BMS-204352 decreasedby approximately 41% in male rats and by 69% infemale rats (Table 1A and B). The reduced CLTwith increasing doses implies a possible satura-tion in the metabolism of BMS-204352.

Like CLT values, steady-state volume of dis-tribution (VSS) values (3621–8933ml/kg over thefour dose levels) generally decreased withincrease in dose. VSS values decreased byapproximately 23% in male rats and by approxi-

mately 54% in female rats as dose increased from0.4 to 10mg/kg. The reasons for the apparentdifferences in pharmacokinetics of BMS-204352in male and female rats are not known, however,sexual dimorphism in drug metabolism has beenknown to occur in rats [7].

However, mean residence time (MRT) andelimination half-life (T1/2) values for BMS-204352 were consistent across dose levels in bothmale (MRT: 2.42–3.76 h; T1/2: 2.08–4.70 h) andfemale (MRT: 3.08–4.54 h; T1/2: 2.94–4.32 h) rats.

In conclusion, there appeared to be someevidence of gender differences in the pharmaco-kinetics of BMS-204352 in rats. BMS-204352 Cmax

and AUC values appeared to increase in aproportion greater than the increment in dose,as dose increased from 0.4 to 10mg/kg. BMS-204352 is extensively distributed into extravas-cular tissues given a high VSS value. MRT andT1/2 values were independent of dose andgender.

Acknowledgements

The authors acknowledge the expert assistance ofthe staff of the Technical Services Unit for animalexperimentation.

Table 1. Mean (S.D.) pharmacokinetic parameters for BMS-204352 in rats (n=4/dose/gender) after a singleintraarterial dose of BMS-204352 at 0.4, 2, 5, and 10mg/kg dose levels

Dose (mg/kg) Cmax (ng/ml) AUC (ngh/ml) MRT (h) T1/2 (h) CLT (ml/h/kg) VSS (ml/kg)

A. Male rats0.4 171 125 2.42 2.08 3242 7749

(46) (17) (0.92) (0.68) (395) (2877)2.0 1218 692 3.12 3.98 2940 8933

(235) (100) (1.51) (3.03) (461) (3708)5.0 3060 2539 3.76 2.95 1969 7401

(708) (33) (0.80) (1.88) (26) (1550)10.0 5374 5271 3.10 4.70 1911 5975

(963) (553) (0.95) (2.15) (182) (2161)B. Female rats0.4 122 155 3.08 2.94 2763 8005

(21) (46) (0.84) (1.10) (863) (736)2.0 906 1883 4.54 4.32 1154 4911

(239) (590) (1.60) (1.56) (406) (1091)5.0 2706 4424 4.36 3.40 1172 4993

(1030) (1071) (0.74) (0.48) (230) (465)10.0 6155 11978 4.31 3.47 879 3621

(228) (3131) (1.01) (0.75) (230) (105)

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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 experimen-tal 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 of themaxi-K opener BMS-204352. Proc Keystone SymposiumPotassium Channels, Keystone CO, 2000; 57.

5. Gibaldi M, Perrier D. Pharmacokinetics (2nd edn). MarcelDekker: New York, 1982.

6. Davies B, Morris T. Physiological parameters inlaboratory animals and humans. Pharm Res 1993; 10:1093–1095.

7. Waxman DJ, Dannan GA, Guengerich FP. Regulation of rathepatic cytochrome P-450: age-dependent expression,hormonal imprinting, and xenobiotic inducibility of sex-specific isozymes. Biochem 1985; 24: 4409–4417.

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