Received 16 November 2001Revised 19 December 2001

Copyright # 2002 John Wiley & Sons, Ltd. Accepted 20 December 2001

BIOPHARMACEUTICS & DRUG DISPOSITIONBiopharm. Drug Dispos. 23: 83–86 (2002)

Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/bdd.294

SHORT COMMUNICATION

Pharmacokinetics and Dose Proportionality of BMS-204352after Intravenous Administration to Dogs

Rajesh Krishna*, Vinod R. Shah, Subbaro Mantha, Nimish N. Vachharajani 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 dogs. In an open, three-way crossover study, three beagle dogsreceived a single intravenous dose of BMS-204352 as a 6-min infusion into the femoral vein at 0.4,0.9, and 2.0mg/kg dose levels. There was at least a 1-week washout period between treatments.Serial blood samples were collected for up to 32 h post dose and plasma samples were analyzed forthe concentrations of intact BMS-204352 using a validated liquid chromatographic mass spectro-metric (LC/MS) method. Pharmacokinetic analysis was performed using a non-compartmentalmethod. Results indicated that peak BMS-204352 concentrations (Cmax) and area under the plasmaconcentration–time curves (AUC) values increased in a dose proportional manner. Mean residencetime (MRT, 18.2–21.9 h) and elimination half-life (Thalf, 13.5–17 h) did not change with dose.There was no dose dependency in the mean BMS-204352 total body clearance (CLT, 134–158ml/h/kg) and mean steady state volume of distribution (VSS, 2839-3291ml/kg). The high VSS valueindicated that BMS-204352 was distributed extensively in the extravascular tissues. In conclusion,BMS-204352 exhibits linear pharmacokinetics over the dose range tested (0.4–2mg/kg). Copyright# 2002 John Wiley & Sons, Ltd.

Key words: BMS-204352; dogs; dose proportionality; pharmacokinetics

Introduction

BMS-204352, ([3S]-[+]-[5-chloro-2-methoxyphe-nyl]-1,3-dihydro-3-fluoro-6-[trifluoromethyl]-2H-indol-2-one), a novel fluorooxindole maxi-Kchannel opener, is being developed for thetreatment of stroke [1–3]. The compound isefficacious for therapy of acute forms of strokeafter intravenous dosing in various animal strokemodels [4]. The beagle dog has been used as oneof the primary species for the toxicologic evalua-tion of BMS-204352. In addition, the protein

binding in dog plasma and the disposition ofradiolabeled BMS-204352 in dogs have beencharacterized [5,6]. The objective of the currentinvestigation was to determine the pharmacoki-netics and dose proportionality of BMS-204352 indogs receiving single intravenous doses of BMS-204352.

Materials and Methods

Animals

Approval for animal investigations was obtainedfrom the in-house animal care and use commit-tee. Healthy adult male beagle dogs (weighing ca.

* Correspondence to: Mailstop E12-07, Bristol-Myers Squibb,Route 206 and Province Line Road, Princeton, NJ 08540, USA.E-mail: rajesh.krishna@bms.com

10–12 kg) were used in this study. The animalswere identified by ear tattoos and individuallyhoused in stainless steel metabolic cages. Duringthe washout period of the study, food wasprovided daily between 7:00 and 10:00 a.m. andfresh drinking water was provided ad libitum. Alldogs had an indwelling venous (femoral vein)access port. Animals were weighed prior to drugadministration.

Chemicals and formulations

BMS-204352 was obtained from Bristol-MyersSquibb Pharmaceutical Research Institute (Prin-ceton, NJ). A stock solution of BMS-204352 wasprepared by dissolving the compound in asolvent mixture composed of polyethylene gly-col-400, ethanol, and 5% dextrose in water. Thedosing solution was sterile filtered using a SterileAcrodisc1 filter.

Study design

This study was an open, three-way crossoverexperimental design in which three male beagledogs received a single intravenous dose of BMS-204352 as a 6min constant rate infusion via thefemoral vein, at either 0.4, 0.9, or 2mg/kg doselevels. There was at least a 1week washoutperiod between successive treatment periods. Fordrug administration, an appropriate volume(1ml/kg) of the dosing solution was infusedover 6min via a femoral vein using an infusionpump that employed a 22-gauge huber pointneedle attached to a syringe.

Sample collection and preparation

Blood samples (approximately 2ml) were col-lected at predose, 6 (end of infusion), 10, 15, 20,30, 45min, and 1, 2, 4, 6, 8, 10, 12, 24, and 32 hpost-dosing. Blood samples were collected inEDTA containing Vacutainers and within 30minof blood collection, plasma was harvested bycentrifuging at 1000 g 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 that

was accurate, precise, specific, sensitive andreproducible. Analyses were carried out using aWaters Chromatoraphic 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 0.5ml volume of plasma, a structurally similarinternal standard, BMS-223110 and 5mM ammo-nium acetate buffer were added. The mixturewas gently vortex-mixed and extracted withtoluene. The organic phase was separated andevaporated to dryness under a gentle stream ofnitrogen at 408C. The residue was reconstitutedin the mobile phase (bi-phasic mixture of twosolvent mixtures, A composed of 5mM ammo-nium acetate and 0.1% triethylamine in 75:25 v/vwater:methanol, adjusted to pH 5, and B, 5mMammonium acetate in methanol. Standard curveswere linear (R250.998) over the concentrationrange 0.5–1000 ng/ml. The mean predicted qual-ity control concentrations deviated 55.1% fromnominal values; the intra- and inter-assay preci-sion values were 55.5% relative standard devia-tion. On the basis of the performance of thestandard curves and quality control samples, theplasma assay for BMS-204352 was sensitive,selective, accurate, precise, and reproducible.

Pharmacokinetic analyses

Plasma data were subjected to non-compartmen-tal pharmacokinetic analysis [7]. 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-

Copyright # 2002 John Wiley & Sons, Ltd. Biopharm. Drug Dispos. 23: 83–86 (2002)

R. KRISHNA ET AL.84

zero plasma concentration by the slope of theterminal log-linear phase. Other pharmacokineticparameters described include peak plasma con-centration (Cmax), elimination half-life (Thalf),mean residence time in the body (MRT), totalbody clearance (CLT), and steady-state volume ofdistribution (VSS).

Statistical methods

The pharmacokinetic parameters, MRT, Thalf,CLT, and VSS were analyzed in the context of athree-way crossover design. The effect of se-quence (carryover) and period were not testeddue to the small sample size. The reduced modelfor this analysis of variance (ANOVA) was:

PKðijÞ¼ meanþ doseðijÞ þ eðijÞ

where, i =A, B, and C (A= 0.4mg/kg; B= 0.9mg/kg; C= 2mg/kg), j=dog 1, 2, and 3; PK is theparameter of interest; dose(ij) denotes the effectof each dose treatment level; e(ij) is the randomerror term used for testing all effects. Tukey’sunweighted pairwise comparisons, at the p= 0.05level, was used to make comparisons amongdose means if significant dose effect was found.Weighted linear regression was used to deter-mine whether Cmax and AUC values were doselinear or dose proportional. The test for doselinearity was based on the t-test for a linear trendacross doses. The test for non-linearity wasperformed using a lack of fit t-statistic [8]. Inthe absence of significant non-linearity, the

parameter was concluded to be dose propor-tional if the intercept was not statisticallysignificantly different from zero.

Results and Discussion

Mean (SD) plasma BMS-204352 concentration–time curves for the three dose levels arepresented in Figure 1. Within 4 min after thecessation of the intravenous administration, theplasma levels declined quickly (ca. 40% declinefrom Cmax), after which the decline appearedslower. The mean (SD) pharmacokinetic para-meters of BMS-204352 are presented in Table 1.

Time (h)0 5 10 15 20 25 30 35

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sma

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on

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Figure 1. Mean (SD) plasma concentration–time curves ofBMS-204352 in dogs receiving single 0.4, 0.9, and 2mg/kgintravenous doses of BMS-204352

Table 1. Mean (SD) pharmacokinetic parameters for BMS-204352 in dogs (n=3) after a single intravenous dose of BMS-204352at 0.4, 0.9, and 2mg/kg dose levels

Dose (mg/kg) Cmax (ng/ml) AUC (ng.h/ml) MRT (h) Thalf (h) CLT (ml/h/kg) VSS (ml/kg)

0.4 600 3212 20.3 15.6 134 2839(132) (1140) (4.8) (4.0) (39) (1326)

0.9 1465 7081 21.9 17.0 145 3291(574) (3337) (3.3) (4.5) (58) (1722)

2.0 2710** 14030 18.2 13.5 158 2848(}) (4979) (2.2) (1.4) (67) (1127)

*Statistical Comparison NA NA NS NS NS NSp=0.500 p=0.519 p=0.869 P=0.906

*Performed at p= 0.05.

**n= 2.

NS= not significantly different.

NA=not applicable.

Copyright # 2002 John Wiley & Sons, Ltd. Biopharm. Drug Dispos. 23: 83–86 (2002)

DOSE PROPORTIONALITY OF BMS-204352 IN DOGS 85

Mean BMS-204352 Cmax values at 0.4, 0.9, and2mg/kg doses were 600, 1465, and 2710 ng/ml,respectively. Mean BMS-204352 Thalf values ran-ged from 13.5–17h over the three dose levels.Figure 2 depicts the plot of Cmax and AUC as afunction of dose. Weighted linear regressionanalysis of Cmax versus dose suggested a doseproportional relationship (r2 = 0.821). As dose ofBMS-204352 increased in the ratio 1:2.3:5, Cmax

increased in the ratio of 1:2.4:4.5. Similarly,the weighted regression analysis of AUC alsosuggested a dose proportional relationship(r2 = 0.734), with BMS-204352 AUC increasing inthe ratio of 1:2.2:4.4.

BMS-204352 CLT values were not statisticallydifferent at the three dose levels. Mean CLTvalues ranged from 134–158ml/h/kg (Table 1).Similarly, mean VSS values for BMS-204352 weredose independent and ranged from 2839 to3291ml/kg. These VSS values were almost 4–5fold higher than the total body water in dogs, i.e.604ml/kg [9], suggesting that BMS-204352 isextensively distributed into extravascular tissues.MRT (18.2–21.9h, over the three doses) valueswere not significantly different among the threedose levels. In summary, BMS-204352 exhibited

linear pharmacokinetics after intravenous ad-ministration to dogs over the dose range of 0.4–2mg/kg. Mean CLT, VSS, MRT, and Thalf valueswere dose independent. BMS-204352 is alsorapidly distributed into extravascular tissues, asindicated by a large VSS value.

Acknowledgements

The authors acknowledge the expert assistance ofthe staff of the Technical Services Unit for animalexperimentation and C.-W. Soong for assistancewith statistical analyses.

References

1. Gribkoff VK, Starrett JE Jr, 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 of largeconductance calcium-activated potassium (BK) channels aspotential therapeutic targets. Current Pharm Design 1996; 2:413–428.

4. Starrett JE, Hewawasam P, Oritz AA, et al. Pharmacokineticanalysis and MCAO stroke activity of the maxi-K openerBMS-204352. Proc Keystone Symp: potassium channels, Key-stone, 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.

6. Krishna R, Yao M, Srinivas NR, et al. Disposition ofradiolabeled BMS-204352 in rats and dogs. Biopharm DrugDispos 2002; 23: 41–46.

7. Riegelman S, Collier P. An application of statistical momenttheory to the evaluation of in vivo dissolution time andabsorption time. J Pharmacokinet Biopharm 1980; 8: 509–534.

8. Myers RH. Classical, Modern Regression with Applications,2nd Edn. PWS-Kent Publishing Co: Boston, MA, 1990.

9. Davis B, Morris T. Physiological parameters in laboratoryanimals and humans. Pharm Res 1993; 10: 1093.

Dose (mg/kg)0 1 2 3

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Figure 2. Relationship between Cmax and AUC versus dose indogs receiving single 0.4, 0.9, and 2mg/kg intravenous dosesof BMS-204352. Regression equations: Y=1380*X+89 (Cmax)and Y=5780*X+1129 (AUC)

Copyright # 2002 John Wiley & Sons, Ltd. Biopharm. Drug Dispos. 23: 83–86 (2002)

R. KRISHNA ET AL.86