CLINICAL pHARMACOLOGY & THERAPEUTICS P 6 8 American Society fbr Clinical Pharmacology and ~Iherapeutics ~EBRUARY2003

PIII-22 GENOTYPIC AND PHENOTYPIC EFFECTS OF CHRONIC

ETHIDIUM BROMIDE (EtBr) EXPOSURE IN MOLT-4 (WT) CELLS. R. Armand, MD, J. Kintner, MS, W. Kristy, BS, C. Y. Jacqueline, PhD, P. P. Raymond, MD, L. D. Lewis, MB, BCh, MD, Dartmouth Hitchcock Medical Center, Dartmouth Medical School, Lebanon, NH.

Purpose: Ethidium Bromide (EtBr) inhibits mitochondrial DNA (mtDNA) biogenesis. Our objective was to develop a MOLT-4 lym- phoblastic cell line lacking mtDNA (pO) and study their properties.

Methods: MOLT-4 cells (wt) were exposed for 26 days to EtBr (50 ng/ml) plus pyruvate and uridine. DNA content was determined by southern blotting and quantitative polymerase chain reaction (PCR). Lactate production, cytochrome c oxidase and citrate synthase activities were measured by spectrophotometry; cellular oxygen con- sumption was measured by electron paramagnetic resonance oxime- try. A~&1 was measured by JC-1 flow cytometry. Transmission elec- tron microscopy was performed.

Results: po cells were generated. PCR revealed a mean (_+SD) mtDNA copy number in wt vs. po cells of 833 (_+433) vs 2 # (_+2) copies/cell (n=6; # p < 0.001) respectively. For wt vs pO cells the mean (±SD) doubling time was 24.1h (SD_+I.6) vs 42.3h" (SD ± 2.7) In=5; * p < 0.000002]; mean (±SD) lactate production was 4.3 (SD ±1) vs 16.3"* (SD± 5.4) fmol/min/cell (n=6; **p < 0.00t); mean 02 consumption was 0.73 (SD -+. 02) vs 0,09* (SD -+ 0.02) pmol/min/cell (n= 3; *p <0.001) respectively. Mean (+SD) cyto- chrome c oxidase/citrate synthase ratio in wt vs 9°cells was 0.31 (SD±. 02) vs 0.001"* ( S D - + .0005 (n=6; **p < 0.00000001) respectively. By JC-1 flow cytometry pO cells had a lower Atb m than wt cells, which 20 mM sodium fluoride reduced further, pO cells had vacuolated mitochondria, with cristae that were absent or had irreg- ular patterns.

Conclusion: Compared to wt cells, pO grew more slowly, con- sumed less oxygen and produced more lactate, with reduced activity of mitochondrially encoded enzymes, and had a lower A0m which was dependent upon glycolysis.

PIII-23 L-TYPE CALCIUM CHANNELS MEDIATE THE VASCULAR

EFFECTS OF POTASSIUM CHLORIDE IN HUMAN HAND VEINS IN VIVO. C. Schindler, M. Grossmann, MD, PhD, D. Dobrev, MD, PhD, E. Hultsch, W. Kirch, MD, PhD, Institute of Clinical Pharmacology Dresden University of Technology, Institute of Clinical Pharmacology Bobenheim, Institute of Pharmacology and Toxicology, Dresden University of Technol, Dresden, Germany.

Potassium chloride (KC1) is a frequently used venoconstrictor for investigation of vascular function in vitro. Recent studies indicate that low mM concentrations of KC1 dilate, whereas higher concen- trations constrict the vessels. Experimental animal studies in arteries suggest that K + ions might act as an endothelium derived hyperpo- larization factor.

We studied the effects of increasing concentrations of potassium (0.7-43 raM) in native (n=6) and in constricted dorsal hmnan hand veins (n=7). In addition, we tested whether L-type calcium channels mediate the vascular effects of KCI by administering the selective L-type calcium channel blocker nifedipine, in native veins infusion of KC1 (0.7-43 mM) resulted in dose-dependent venoconstriction (max- imum at 43 raM: 5.6_ + 15.2% basal vein size [BVS]). Coinfusion of nifedipine (0.3 ~g/min) strongly inhibited KCl-induced venoconstric- tion (43 mM KCl+nifedipine: 88.2_+4.8% BVS). To test for potential venodilatory effects of low miltimolar concentrations of KC1 (up to 15 mM), hand veins were constricted to 20% BVS by infusing the selective (x 1-adrenoceptor-agouist phenylephrine (dose range: 94-750 ng/min). Infusion of KC1 in phenylephrine preconstricted veins did not change vein diameter.

In conclusion, human hand veins in vivo did not respond to KC1 in a biphasic manner showing dilation followed by constriction. Admin- istration of KC1 caused venoconstriction mediated mainly by opening of L-type calcium channels. Thus, KCt may be a useful tool for venous preconstriction in order to investigate venodilatory effects of drugs in vivo.

PIII-24 ASSOCIATION OF ALPHA 2B-ADRENERGIC RECEPTOR

INSERTION/DELETION POLYMORPHISM WITH HYPERTEN- SION AND OBESITY. R.P. Rantinella, T. Y. Langaee, PhD, C. L. Aquilante, PharmD, J. A. Johnson, PharmD, University of Florida College of Pharmacy, Gainesville, FL.

The OL2b-adrenoceptor (oL2bAR) is involved in vasoconstriction and energy regulation in humans. A nine nucleotide deletion (D) variant has been identified in the e~2bAR gene that results in a deletion of three glutamic acids at codons 301 to 303. We tested whether this polymorphism was associated with hypertension or obesity. Three hundred nine men and women (Caucasian and African-American) aged 35 to 65 were studied. Hypertensives had sitting DBP > 90 mmHg or had previously diagnosed hypertension and were on anti- hypertensive drug therapy. Normotensives had no family history of hypertension. Obesity was defined as body mass index (BMI) ~> 30. C~2bAR genotypes were determined by PCR and RFLP. Genotype frequencies were not different between hypertensives and normoten- sives (shown in Table) and were in Hardy-Weinberg equilibrium. The D allele frequencies were also not significantly different between obese and nonobese subjects (whites: 42.2% vs. 32.8%; blacks: 20% vs. 21.4%). As previously documented, there were significant differ- ences in allele frequency by race.

Conclusion: Our data suggest that the (x2bAR I/D polymorphism is not associated with hypertension or obesity.

C a u c a s i a n African-American

GENOTYPE HT(n=100) NT(n=85) HT(n=86) NT(n=38)

I t 37% 40% 60.5% 68.4% I/D 53% 46% 34.8% 28.9% D/D 10% 14% 4.6% 2.6% D allele* 36.5 % 37% 22% 17. 1%

HT=hypertensive; NT=nolTnotensive; *p=NS within racial groups

PIII-25 DOSE-RESPONSE RELATIONSHIP CHARACTERIZATION

IN CURRENT ANTI-HYPERTENSIVE DRUG DEVELOPMENT. J. V. Gobburu, PhD, R. J. Lipicky, MD, OCPB/OPS/CDER/FDA, Private Consultant, Rockville, MD, Rockville, MD.

Purpose: Historically, dose (concentratiou)-response relationships have occupied a prominent role in antihypertensive drug develop- ment. Data from such trials (which some might classically classify as Phase 2) have been the primary basis of approval of all recently approved antihypertensive drugs. The purpose of such trials, methods of analysis, frameworks of reference that should surround the choice of methods of analysis, and inferences that one takes from results, have not been the subject of much rigorous discussion. The aim of the current report is to investigate the adequacy of the dose-response characterization of anti-hypertensives. Methods: We have abstracted clinical trials data from 10 NDAs that were approved for an antihy- pertensive indication for two classes of antihypertensive drugs. Namely angiotensin converting enzyme inhibitors (ACE inhibitors) and angiotensin II blocking agents (A2 blockers). Dose, mean arterial blood pressure (MAP) in rats, and dose, diastolic blood pressure (DBP), and systolic blood pressure (SBP) in man were the data analyzed using linear, log-linear and Emax models. All blood pres- sure data were group means (placebo subtracted in man, control subtracted in rats). Results: By parsimony, a linear model was adequate to describe the dose - response data for the antihyperten- sives analyzed. No clear separation of the 3 models tested could be made based on routine goodness-of-fit diagnostics. Conclusions: It is important to not only establish exposure-response relationship, but determine the maximal effect for antihypertensives. The results from these analyses will be employed to put forth a discussion on the need for innovative clinical trial designs.