Hydrocodone is oxidized to a more potent mu-opioid agonist hydromorphone by cytochrome P450 2D6 (CYP2D6). CYP2D6 poor metabolizers (PMs) cannot convert hydrocodone to hydromorphone. It is believed that PMs will not gain meaningful analgesia from hydrocodone. Responses of PMs were compared with those of competent metabolizers (non-PMs) during hydrocodone/acetaminophen extended release (HC/APAP CR) treatment following bunionectomy surgery and in osteoarthritis patients, to learn whether CYP2D6 PMs might be effectively treated with HC/APAP CR. DNA samples collected from patients recruited into two multi-center placebo-controlled clinical trials were genotyped for major CYP2D6 PM alleles and assigned PM or non-PM status. In a study of acute pain relief after bunionectomy, efficacy variables were assessed descriptively. In a chronic pain study in osteoarthritis, efficacy of HC/APAP CR treatment was evaluated prospectively for the percentage change from baseline to week 12 of pain intensity score (VAS%), using analysis of covariance with a factor for PM status and baseline pain-intensity score as a covariate. Other efficacy endpoints were assessed to support the prospective analysis. Tolerability of HC/APAP CR in PMs was assessed descriptively in both studies. Among 130 bunionectomy subjects, four of six PMs dosed with HC/APAP CR experienced meaningful analgesia. Among 614 osteoarthritis subjects, eleven of nineteen PMs dosed with HC/APAP CR experienced meaningful analgesia. No difference was observed between PMs and non-PMs for VAS% (43.5% v –46.5%, p=0.770). PMs treated with placebo (–21.0%, n=19) did not respond as well as PMs treated with HC/APAP CR. Results for other key efficacy variables were consistent with those for VAS%. Safety-related study dropout and adverse event patterns were similar in PMs and non-PMs in both studies. These results indicate that PMs and non-PMs have similar analgesic responses to HC/APAP CR. This may distinguish HC/APAP CR from tramadol and possibly other opioid-based analgesics. Funded by Abbott Laboratories.

Cytochrome P450 2D7 (CYP2D7) has long been considered a pseudogene. A recent report described an indel polymorphism (CYP2D7 138delT) that causes a frameshift generating an open reading frame and functional protein. This polymorphism was observed in 6 of 12 samples from an Indian population. Individuals with the 138delT polymorphism expressed CYP2D7 protein from a brain-specific, alternatively spliced transcript (J Biol Chem 279:27383–27389, 2004). The unexpectedly high frequency of the variant allele and resulting CYP2D7 expression could have important implications for brain-specific metabolism of CYP2D substrates including many psychoactive drugs. However, the 138delT variant has not been detected in other studies (Pharmacogenetics 11:45–55, 2001; Biochem Biophys Res Commun 336:1241–1250, 2005). Our goal was to determine the frequency of this variant in a larger, ethnically diverse population. CYP2D7 138delT genotypes for 163 Caucasians, 95 East Asians, 50 Indians, 68 Hispanic Latinos, and 68 African Americans were determined by Pyrosequencing. The 138delT allele was observed at a frequency of 1.0% in East Asians and 0.74% in Hispanic Latinos. The deletion was not observed in Indians or the other ethnic populations. In addition, in each of the three samples with 138delT, the putative brain-specific transcript contains a premature stop codon that would preclude protein expression. The low frequency of the CYP2D7 138delT polymorphism in our ethnically diverse sample, and particularly the absence from 50 Indian samples, is in contrast to the high frequency previously reported. Our results suggest that CYP2D7 138delT is unlikely to be highly relevant for population variation of pharmacokinetics or drug response.

The 5′ untranslated regions (UTR) of chloroplast mRNAs often contain regulatory sequences that control RNA stability and/or translation. The petD chloroplast mRNA in Chlamydomonas reinhardtii has three such essential regulatory elements in its 362-nt long 5′ UTR. To further analyze these elements, we compared 5′ UTR sequences from four Chlamydomonas species (C. reinhardtii, C. incerta, C. moewusii and C. eugametos) and five independent strains of C. reinhardtii. Overall, these petD 5′ UTRs have relatively low sequence conservation across these species. In contrast, sequences of the three regulatory elements and their relative positions appear partially conserved. Functionality of the 5′ UTRs was tested in C. reinhardtii chloroplasts using β-glucuronidase reporter genes, and the nearly identical C. incerta petD functioned for mRNA stability and translation in C. reinhardtii chloroplasts while the more divergent C. eugametos petD did not. This identified what may be key features in these elements. We conclude that these petD regulatory elements, and possibly the corresponding trans-acting factors, function via mechanisms highly specific and surprisingly sensitive to minor sequence changes. This provides a new and broader perspective of these important regulatory sequences that affect photosynthesis in these algae.

Objective: Our objective was to learn whether genetic polymorphisms of metabolic enzymes or transport proteins provide a mechanistic understanding of the in vivo disposition of atrasentan, a selective endothelin A receptor antagonist.Methods. Atrasentan uptake was measured in HeLa cells transfected to express major alleles of organic anion transporting polypeptide 1B1 (OATP1B1). The results were used to classify individuals as extensive, intermediate, or poor OATP1B1 transporters according to their SLCO1B1 genotypes. Analysis of covariance including genotype, study, age, weight, sex, and ethnicity was used to identify factors influencing atrasentan single-dose (n = 44) and steady-state (n = 38) pharmacokinetic parameters. Genotypes for cytochrome P450 3A5, uridine diphosphate-glucuronosyltransferase (UGT) 1A1, UGT2B4, UGT2B15, adenosine triphosphate-binding cassette subfamily B (A-BCB) 1, solute carrier organic anion transporter (SLCO) 1B1, and solute carrier family 22 (SLC22) A2 were each assessed.Results. Single-dose atrasentan exposure (P = .0244), steady-state atrasentan exposure (P = .0108), and maximum postdose plasma concentration (P = .0002) were associated with OATP1B1 activity classified by SLCO1B1 genotype. No other tested genotypes were observed to be associated with both single-dose and steady-state atrasentan pharmacokinetics.Conclusions. OATP1B1 is a meaningful factor for atrasentan disposition. Individuals may be classified as having extensive, intermediate, or poor OATP1B1 transport phenotypes according to SLCO1B1 genotypes. Increased exposures of OATP1B1 substrates might be expected in individuals who have the poor transporter phenotype or are treated with an OATP1B1 inhibitor.

BACKGROUND Polymorphisms within cytochrome P450 2D6 (CYP2D6) result in different metabolizer phenotypes, but some discordance suggests the possibility of additional unknown alleles or factors contributing to metabolism. A recent study showed a high frequency frameshift-causing deletion, CYP2D7 138delT, which converted the CYP2D7 pseudogene to a functional gene within the brain.1 The high frequency of the deletion and the resulting CYP2D7 expression could have important implications for brain-specific metabolism of psychoactive substances. Our goal was to determine the frequency of this deletion in a larger ethnically diverse population. METHODS The CYP2D7 138delT genotypes for 163 Caucasians, 95 East Asians, 50 South Asians, 68 Hispanic Latinos, and 68 African Americans were determined by Pyrosequencing. RESULTS The 138delT allele was observed at a frequency of 1.0% in East Asians and 0.74% in Hispanic Latinos. The deletion was not observed in the other ethnic populations. CONCLUSIONS The very low frequency of the CYP2D7 138delT polymorphism in our panel is in contrast to the high frequency (50%) reported in the Indian population.1 Our results suggest that CYP2D7 138delT is unlikely to be highly relevant for population variation of pharmacokinetics or drug response. Clinical Pharmacology & Therapeutics (2005) 79, P42–P42; doi: 10.1016/j.clpt.2005.12.149

Purpose: ABT-751 is an oral antimitotic agent that binds to the colchicine site on beta-tubulin. A phase 1 study was conducted to determine the maximum tolerated dose and toxicities of ABT-751 in patients with advanced myelodysplastic syndrome and relapsed or refractory acute leukemias.Study Design: Thirty-two patients were treated: nine with 100 (n = 3),125 (n = 3), or 150 mg/m(2) (n = 3) of ABT-751 given orally once daily for 7 days every 3 weeks and 23 with. 75 2 (n = 3), 100 (n = 3),125 (n = 5),150 (n = 5),175 (n = 3), or 200 mg/m(2) (n = 4) of ABT-751 given orally once daily for 21 days every 4 weeks. Consenting patients had pharmacogenetic sampling and enumeration of circulating endothelial cells (CEC).Results: Dose-limiting toxicity consisted of ileus in one patient at 200 mg/m(2) with a subsequent patient developing grade 2 constipation at the same dose level. One patient with relapsed acute myelogenous leukemia achieved a complete remission that was sustained for 2 months. Four other patients had transient hematologic improvements, consisting of a decrease in peripheral blood blasts and improvements in platelet counts. CEC number was reduced in three patients with a concomitant reduction in peripheral blasts. A previously undescribed nonsynonymous single nucleoticle polymorphism, encoding Ala(185)Thr, was identified in exon 4 of the beta-tubulin gene, TUBB, in three other patients. The recommended phase 2 dose in hematologic malignancies is 175 mg/m(2) 75 daily orally for 21 days every 4 weeks.Conclusion: Further assessment of ABT-751, especially in combination with other agents, in patients with acute leukemias is warranted.

Purpose To assess whether genetic polymorphisms of drug metabolizing enzymes or drug transporters correlate with atrasentan pharmacokinetics. Methods Pharmacogenetic meta-analyses of single dose pharmacokinetic parameters from 3 clinical studies (n=44) and steady-state pharmacokinetic parameters from 2 clinical studies (n=38) were performed. ANCOVA including genotype, study, age, weight, gender & ethnicity as factors was the primary analysis. Genotypes in CYP3A5, UGT1A1, UGT2B4, UGT2B15, ABCB1, SLC21A6 & SLC22A2 were each assessed. Results Following a single dose of atrasentan, exposure (AUC0–∞) and apparent volume of distribution were correlated with organic anion transport protein C (OATP-C) activity predicted by SLC21A6 genotype (p=0.0034 and p=0.03, respectively). Analogous relationships of SLC21A6 genotype to steady-state atrasentan exposure (AUC0-24, p=0.03) and maximum post-dose plasma concentration (p=0.0003) confirmed the single dose results. No other tested genotypes were consistently associated with atrasentan pharmacokinetics. Conclusions Pharmacogenetic analysis suggests a testable hypothesis that atrasentan is an OATP-C substrate. In the current study, genotype-predicted OATP-C activity is the most influential covariate for atrasentan pharmacokinetics. Our work shows how pharmacogenetics can be used to gain insight into the pharmacokinetics of a new molecular entity. Clinical Pharmacology & Therapeutics (2004) 75, P94–P94; doi: 10.1016/j.clpt.2003.11.357

This study utilized cytochrome P450 2D6 (CYP2D6) genotypes to explain variability of desipramine pharmacokinetics in a cohort of non-poor metabolizer individuals. In an interaction study utilizing desipramine as a probe, genotyping for the CYP2D6*3, *4, *5 and *6 alleles was used to screen out CYP2D6 poor metabolizers. Individuals were categorized according to these and additional alleles (CYP2D6*2, *9, *10, *17, *41 and x2). Genotypes of individuals heterozygous for two or three of *2, *17 and *41 alleles were confirmed by molecular haplotyping. Pharmacokinetic parameters of desipramine were analysed according to CYP2D6 category. Molecular haplotyping was necessary to definitively categorize four of 16 individuals. A subject who had unusually high plasma elimination half-time, exposure and metabolic ratios carried an intermediate metabolizer (IM) *9 allele in combination with a non-functional allele. This combination has a population frequency of less than 1 : 200. Individuals with *1/*1, *1/*2 and *2/*2 genotypes had lower than average plasma elimination half-time, exposure and metabolic ratios. For desipramine, additional genotyping of CYP2D6 IM alleles helped define subgroups of the CYP2D6-positive cohort. This suggests that genotyping for IM alleles will aid in interpretation of clinical trials involving CYP2D6 substrates. Due to the diversity of IM alleles, molecular haplotyping may be necessary to fully characterize CYP2D6 genotype-phenotype relationships.

Understanding the mechanisms of hyperlipidemia in patients receiving highly active antiretroviral therapy (HAART) could assist in the treatment of current patients and the development of future ‘lipid neutral’ treatments. A pharmacogenetic approach can allow for such mechanistic insight; the correlation of a genetic variant with the degree of hyperlipidemia observed during treatment implies that the gene product may be part of a relevant pathway affected by a drug. Sterol response element binding protein 1c (SREBP-1c, gene SREBF1) is a transcription factor that activates genes of lipid metabolism [1]. Recently, a common variant in the 3’ untranslated region of SREBF1 (SREBF1 3′322C > G) was identified and associated with cholesterol and triglyceride changes in HAART patients [2]. We prospectively evaluated the association of the SREBF1 genotype with elevations of total cholesterol or triglycerides in a large clinical trial that compared the safety and efficacy of HAART regimens containing stavudine, lamivudine, and either lopinavir–ritonavir or nelfinavir for the initial treatment of HIV infection [3]. The pharmacogenetic analysis subgroup (n = 355) was representative of the entire study population (n = 653) according to demographic and lipid characteristics. SREBF1 genotypes were determined by pyrosequencing. Assay details are available on request. Genotypers were blind to the clinical results associated with the DNA samples. Data were analysed using SAS 6.12 (SAS Institute, Cary, NC, USA). The analysis plan was pre-specified. Analysis endpoints were the 48-week maximum change from baseline of total cholesterol and triglycerides. Sub-study subjects dosed with lopinavir–ritonavir and nelfinavir had similar baseline lipids, and a similar maximum change of total cholesterol distributions. The mean maximum change of triglycerides was significantly higher in lopinavir–ritonavir patients. Accordingly, data were analysed by two-way analysis of variance, with drug treatment as a block effect. This analysis had a greater than 80% power to detect a difference of means between two genotypes of 22 mg/dl for total cholesterol or 147 mg/dl for triglycerides. The maximum change of total cholesterol within 48 weeks of HAART treatment was not significantly dependent on either genotype (P = 0.12) or treatment regimen (P = 0.67). The mean changes observed in the six subgroups defined by genotype and treatment (e.g. CC genotype in ritonavir–lopinavir patients; CG genotype in nelfinavir patients) were similar, ranging from 63 ± 36 to 75 ± 40 mg/dl (see Fig. 1). Similarly, the maximum change of triglycerides was not significantly associated with the genotype (P = 0.80). Our results demonstrate that the SREBF1 3'322C > G genotype is not predictive of a risk of hyperlipidemia in the study patients. There was no statistically significant or clinically relevant difference between the three SREBF1 genotypes evaluated in the mean maximum change from the baseline of total cholesterol or triglycerides in patients treated with a HAART regimen containing stavudine, lamivudine and either lopinavir–ritonavir or nelfinavir, or in the combined population.Fig. 1.: Lack of dependence of total cholesterol changes in patients treated with highly active antiretroviral therapy on the SREBF1 genotype and treatment regimen. Box-and-whisker plots show the median, quartiles, and range (to 1.5 × interquartile distance beyond the quartile). Three high outliers (genotypes CC, CC, CG) and one low outlier (genotype CC) had cholesterol changes outside the scale of these plots. TC, Total cholesterol.Several factors may account for the difference between our results and those previously published [2]. First, the previous study was smaller (n = 67) with less power to resolve a difference. It is not uncommon to find that preliminary findings from small studies may not be confirmed by larger studies [4]. Second, we used the maximum change across multiple patient visits, providing a better chance of detecting a relationship, if one exists, than the percentage change between two visits separated by a variable length of time. Third, lipid measurements in our study were made without regard to fasting, which better reflects clinical practice. Fourth, we did not group CC and CG genotypes, as the prevrevious study did. In the absence of knowledge that the GG genotype has a recessive function, such grouping is unusual. We observed slight but consistent heterosis in our study; that is, the maximum lipid changes in CG heterozygotes were higher than those in either homozygous group. It would be of interest to learn whether a similar observation was made in the other study. Fifth, we did not exclude patients on the basis of abnormal baseline lipid levels or body mass index. Finally, our study included patients of greater racial diversity, more reflective of actual patient demographics. Our results provide no definitive evidence as to whether SREBP-1c is mechanistically involved in hyperlipidemia in HAART-treated patients. We studied a variant in the 3’ untranslated region of the gene to which no molecular phenotype has been ascribed. This may be non-functional, or the function may be sufficiently subtle that it does not substantially impact the complex phenotype of hyperlipidemia. Because our study included a limited variety of HAART regimens, we cannot formally exclude the possibility that the SREBF1 genotype is relevant for other drugs. Although no relationship was observed between the SREBF1 genotype and lipid elevations in this study, research to discover predictive factors for the risk of hyperlipidemia in HIV-positive patients should continue to be a priority.