CLINICAL I’I-IARIVIACOLOGY Sc 'I‘l"Il:LRAPEUTICS
`VOLUME 73, NUMBER2
`
`American Societyfor Clinical I’/{mu/macology m/wl Therapeuticr P77
`
`This article is protected by copyright and is provided by the University of Wisconsin-
`Madison under license from John Wiley & Sons. All rights reserved.
`
`PIII—55
` C¥P"7D6—B¥ EARQX
`
`ETINE.
`I{_.l,\/l_._Bertelsen K. Venkatakrishnan, L.L. von Moltke, R.
`Scott Obach, and D.J. Greenblatt. Tufts University, Boston, MA and
`Pfizer Inc., Groton, CT.
`The selective serotonin reuptake inhibitor paroxetine (PX) is a
`potent CYP2D6 inhibitor, but the mechanism of inhibition is not
`established. Inhibition of CYP2D6 in vitro by PX, fluoxetinc (FLU)
`and quinidine (Q) was studied in human liver mierosomes (HLM)
`using dextromethorphan O—demethylati0n (25 uM) as the index re-
`action. Without preincubation of HLM with inhibitor, mean IC50
`values were: 2.5 i.LM for PX, 0.2 p.M for FLU, and 0.23 i.tM for Q.
`Preincubation of inhibitor with l-ILM significantly reduced the PX
`IC5n by eight-fold to 0.3 p.M; in contrast, the IC50 for FLU and Q
`were actually increased (to 0.42 uM and 0.36 p.M, respectively). PX
`produced time—dependent inactivation, with an apparent rate constant
`of 0.17/min based on a Kitz—Wilson plot. This was not true for FLU
`or Q. Incubation of PX with recombinant CYP2D6 yielded a differ-
`ence spectrum with maximum absorbance at 456 nm, and a time
`dependent increase in absorbance, consistent with inactivation via
`formation of a carbene-heme complex. Thus PX, unlike FLU and Q,
`appears to inhibit CYP2D6 at least in part through a mechanism-
`based process, probably attributable to the methylenedioxy substitu-
`ent of PX.
`
`PIII-56
`PHARMACOGENOMIC STUDIES USING PARAFFIN EM—
`
`BEDDED TUMOR SAMPLES,
`J. M. Rae PhD 1. Scheys, BS,
`K. E. Cordero, BS, M. D. Johnson, PhD, University of Michigan,
`Georgetown University, Ann Arbor, MI.
`Paraffin embedded tumor samples are valuable in the study of
`cancer. Besides use in routine staging and marker analysis, they are
`used to test new prognostic and predictive markers. However, their
`use in retrospective pharrnacogenomic analysis of clinical trials has
`yet to be evaluated. We set out to establish genotyping methods for
`polymorphic CYP450s in forma1in—fixed archival tumor samples and
`determine if fixation, processing or somatic changes in the tumor
`affect our ability to determine germ—line mutations. To establish the
`assay, paraffin blocks were made from 8 cell lines. DNA from cell
`cultures and paraffin sections was purified using the Qiagen system.
`These samples were genotyped for CYP2C8, CYP2C9, CYP2Cl9
`and CYP2D6 alleles using PCR—Rl-‘LP. "his allowed optimization of
`DNA extraction and genotyping for these materials and showed that
`fixation and processing did not significantly alter the genotypes
`‘
`ll, .......'
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`.. s_
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`sections were cut from archival tumor blocks from l0 patients for
`whom gDNA from peripheral blood was available. Again, concor-
`dance was very high with the same genotype being obtained for 97%
`of the alleles tested. Conclusions: accurate genetic testing for poly-
`morphisms on CYP2Cs and CYP2D6 can be obtained from archival
`paraffin embedded tumor samples. Thus, pharmacogenetie analysis
`can be applied to existing cancer therapy trials to test associations
`between these polymorphisms and treatment response.
`
`PIII-57
`sm
`FUNCTIONAL 5 ’—FLANKING REGION (5’-FR) POLYMOR—
`
`PHISMS.
`J. Prondzinski B. Thomae, L. Wang, B. Eckloff, E.
`Wieben, PhD, R. Weinshilboum, MD, Mayo Clinic, Mayo Graduate
`School—Mayo Clinic, Rochester, MN.
`SULTlAl catalyzes the sulfate conjugation of many drugs. xeno-
`biotics and hormones. Human platelet SULTlAl activity and thermal
`stability vary widely among individuals and are regulated by inher-
`itance. A single nucleotide polymorphism (SNP) in the SULTIAI
`open reading frame (ORF) (Arg2l3His) is associated with low levels
`of enzyme activity and thermal stability. However, not all SULTlAl
`phenotypic variation is explained by this SNP. We set out to deter-
`mine whether polymorphisms in the 5’—FR also influence SULTlAl
`expression. We resequeneed 2 kb of the SULT1A1 5’—FR using 32
`DNA samples selected for extreme platelet activity and thermal
`stability. Three common SNPs were observed (—l985 C/G, -624 C/G,
`-397 G/A — nuinbered relative to the “A” in the ATG codon). The
`—624 and -397 SNPs were associated with SULTIAI ORF genotype
`and with platelet SULTlAl phenotype. Luciferase reporter gene
`constructs containing these SNPs were used to transfect HepG2 cells.
`There was a 59% average decrease in activity (N=9) for the construct
`containing (-624, -397) GA as compared with CG, and a 90.5%
`average decrease in activity (N=9) for a shorter construct that con-
`tained only (-397) A vs. G. These results indicate that 5'—FR genetic
`polymorphisms may contribute to SULTlAl pharmacogenetic vari~
`ation and that inherited variation in the regulation of transcription
`could influence sulfate conjugation catalyzed by this isoform.
`
`PIII-58
`INFLUENCE OF CYP2D6 GENOTYPE ON THE QTc INTER-
`VAL AND PLASMA CONCENTRATIONS OF THIORIDAZINE
`AND ITS METABOLITES IN PSYCHIATRIC PATIENTS TAK-
`ING CHRONIC THERAPY. R. Thanacoody, MRCP, A. K. Daly,
`PhD, S. H. Thomas, MD, University of Newcastle, Newcastle, United
`Kingdom (Great Britain).
`Purpose. The antipsychotic agent thioridazine is an important
`cause of QTC interval prolongation and torsade de pointes and
`CYP2D6 is important in its metabolism. This study was performed to
`determine whether CYP2D6 genotype influences the QTc interval in
`long—term recipients of thioridazine.
`Methods: Patients receiving stable doses of thioridazine were
`recruited from psychiatric hospitals and clinics. A I2—lead ECG was
`recorded and the QTe interval measured using a digitiser. CYP2D6
`
`
`
`with absence of activity (*3, *4, *5 and *6). When available, plasma
`was analysed for concentrations of thioridazine and its metabolites,
`thioridazine—2—sulfoxide (T—2SO),
`thioridazine—2—sulfone (T—SO2),
`and thioridazine—5-sulfoxide (T-5SO) using high performance liquid
`chromatography.
`the 93 patients studied in-
`Results: Genotyping indicated that
`cluded 54 extensive metabolizers (EM), 30 intermediate metabolizers
`(IM. heterozygotes with 1 mutant allele) and 9 poor metabolizers
`(PM, 2 mutant alleles). The mean doses of thioridazine talcen in the
`previous 24 h (104 i122, ll3 i 86, and ll5:t I85 mg respectively)
`and mean Q'l‘e intervals (425 i 29 427 i 22 41 l i 4] ms
`respectively) were similar for the 3 groups. For the 30 subjects for
`whom data were available, QTc intervals were significantly corrc~
`lated with plasma concentrations of thioridazine, T—2SO and T«SO2
`but not T—5SO. Mean thioridazine and metabolite concentrations did
`not differ significantly between EM, IM and PM subjects, although
`the numbers involved were small (l5, l2 and 3 respectively).
`Conclusions: This study does not suggest that CYPZD6 genotype
`substantially affects risk of thioridazine induced QTC prolongation.
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`Vanda Exhibit 2028 - Page 1
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`VNDA 02699850
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`Vanda Exhibit 2028 - Page 1