John P. Donahue

Pharmacogenetics of Long-Term Responses to Antiretroviral Regimens Containing Efavirenz and/or Nelfinavir: An Adult AIDS Clinical Trials Group Study

BACKGROUND Efavirenz and nelfinavir are metabolized by cytochrome P-450 (CYP) 2B6 and CYP2C19, respectively, with some involvement by CYP3A. Nelfinavir is a substrate for P-glycoprotein, which is encoded by MDR1. The present study examined associations between genetic variants and long-term responses to treatment. METHODS Adult AIDS Clinical Trials Group study 384 randomized antiretroviral-naive subjects to receive efavirenz and/or nelfinavir plus 2 nucleoside analogues, with follow-up lasting up to 3 years. Population pharmacokinetics were estimated from a nonlinear mixed-effects model. Polymorphisms in CYP2B6, CYP2C19, CYP3A4, CYP3A5, and MDR1 were characterized. RESULTS The 504 participants in the genetic study included 340 efavirenz recipients and 348 nelfinavir recipients (184 of the 504 participants received both efavirenz and nelfinavir). Of the participants, 49% were white, 31% were black, and 19% were Hispanic. Plasma exposure to efavirenz and nelfinavir in each population was significantly associated with the polymorphisms CYP2B6 516G-->T and CYP2C19 681G-->A, respectively. Among efavirenz recipients, the MDR1 position 3435 TT genotype was associated with decreased likelihood of virologic failure and decreased emergence of efavirenz-resistant virus but not with plasma efavirenz exposure. Among nelfinavir recipients, a trend toward decreased virologic failure was associated with the polymorphism CYP2C19 681G-->A. CONCLUSIONS Genetic variants predict plasma exposure to efavirenz and nelfinavir, and they may predict virologic failure and/or emergence of drug-resistant virus. These associations with treatment responses must be validated in other studies.

Overcoming the restriction barrier to plasmid transformation of Helicobacter pylori

Helicobacter pylori strains demonstrate substantial variability in the efficiency of transformation by plasmids from Escherichia coli, and many strains are completely resistant to transformation. Among the barriers to transformation are numerous strain‐specific restriction‐modification systems in H. pylori. We have developed a method to protect plasmid DNA from restriction by in vitro site‐specific methylation using cell‐free extracts of H. pylori before transformation. In two cases, plasmid DNA treated with cell‐free extracts in vitro acquired the restriction pattern characteristic of genomic DNA from the source strain. Among three strains examined in detail, the transformation frequency by treated plasmid shuttle and suicide vectors was significantly increased compared with mock‐treated plasmid DNA. The results indicate that the restriction barrier in H. pylori can be largely overcome by specific DNA methylation in vitro. The approach described should significantly enhance the ability to manipulate gene function in H. pylori and other organisms that have substantial restriction barriers to transformation.

Drug Transporter and Metabolizing Enzyme Gene Variants and Nonnucleoside Reverse-Transcriptase Inhibitor Hepatotoxicity

This nested case-control study examined relationships between MDR1, CYP2B6, and CYP3A4 variants and hepatotoxicity during antiretroviral therapy with either efavirenz- or nevirapine-containing regimens. Decreased risk of hepatotoxicity was associated with MDR1 3435C-->T (odds ratio, 0.254; P=.021). An interaction between MDR1 and hepatitis B surface antigen status predicted risk with 82% accuracy (P<.001).

Genetic organization and heterogeneity of the iceA locus of Helicobacter pylori

The genetic organization and sequence heterogeneity of the iceA locus of Helicobacter pylori was studied, and the existence of two distinct gene families, iceA1 and iceA2, at this locus was confirmed. iceA1 has significant sequence homology to nlaIIIR, encoding an endonuclease in Neisseria lactamica, but the similarity at the protein level is limited, due to frameshift mutations of iceA1 in most H. pylori strains. In only five of the 19 iceA1 strains studied, a full-length open reading frame (ORF), capable of encoding a 228aa protein, with 52% homology to NlaIII was observed. The region upstream of iceA2 is highly variable in length, containing up to 15 copies of 8bp tandem repeats. iceA2 can encode proteins of 24, 59, 94, or 129 amino acids, consisting of 14 and 10aa domains, conserved in all iceA2 strains, flanking 0, 1, 2, or 3 copies of a 35aa cassette. This 35aa cassette consists of domains of 13, 16 and 6aa, respectively. The 13aa and 6aa domains are highly conserved, but the 16aa domain exists in two variants. In total, five distinct iceA2 subtypes were defined. Database searches did not reveal any homologous sequences. Recombinant IceA1 and IceA2 proteins were expressed in Escherichia coli, confirming the predicted ORFs. Genotype-specific PCR primers permitted iceA genotyping in 318 (99. 1%) of a worldwide collection of 321 H. pylori strains. The conserved sizes of the amplification products confirmed the worldwide distribution of discrete variants of iceA1 and iceA2.

The HIV-1 Vif PPLP motif is necessary for human APOBEC3G binding and degradation.

The HIV-1 virion infectivity factor (Vif) is required during viral replication to inactivate the host cell anti-viral factor, APOBEC3G (A3G). Vif binds A3G and a Cullin5-ElonginBC E3 ubiquitin ligase complex which results in the proteasomal degradation of A3G. The Vif PPLP motif (amino acids 161-164) is essential for normal Vif function because mutations in this motif reduce the infectivity of virions produced in T-cells. In this report, we demonstrate that mutation of the Vif PPLP motif reduces Vif binding to A3G without affecting its interaction with ElonginC and Cullin5. We demonstrate that the failure of the Vif mutant to bind A3G resulted in A3G incorporation into assembling virions with loss of viral infectivity.

Quantitative detection of Helicobacter pylori gene expression in vivo and relationship to gastric pathology.

ABSTRACT The iceA locus of Helicobacter pyloriincludes one of two mutually exclusive gene families, iceA1and iceA2. Colonization with iceA1 strains is associated with enhanced acute mucosal inflammation, and adherence to gastric epithelial cells in vitro induces expression oficeA1 but not iceA2 mRNA; however, both transcripts can be detected in vivo. The aim of this study was to determine whether differing levels of iceA transcription in vivo may contribute to disease pathogenesis. RNA from 41 H. pylori-positive gastric biopsy specimens was reverse transcribed to cDNA. Quantitative PCR was performed using biotinylated iceA1, iceA2, and 16S rRNA primers, and binding of biotinylated products to streptavidin-coated plates was detected by hybridization with a fluorescein-labeled probe. iceAgenotypes were determined by PCR and sequence analysis. All 41 samples contained detectable H. pylori 16S rRNA, with similar levels in iceA1- (n = 10) andiceA2 (n = 31)-colonized patients (P = 0.34). Biopsy specimens from four (40%) and 19 (61%) persons colonized with iceA1 or iceA2strains, respectively, had detectable iceA RNA. Acute inflammatory scores were significantly higher in iceA1RNA-positive patients than in iceA1 RNA-negative,iceA2 RNA-positive, or iceA2 RNA-negative subjects (P ≤ 0.05 for each). Within theiceA2 RNA-positive group, H. pylori strains with a single 35-amino-acid cassette were associated with significantly higher mucosal iceA2 transcript levels (P= 0.014 versus strains with two cassettes). These results indicate that the levels of transcription of H. pylori iceA1 andiceA2 and of 16S rRNA are independent and that particulariceA2 gene structures are associated with enhanced transcription. The finding that iceA1 transcription levels are significantly associated with the intensity of neutrophilic infiltration suggests that heterogeneity in inflammatory scores among persons colonized with H. pylori iceA1 strains reflects levels of iceA1 transcription in vivo.

Tariquidar, a selective P-glycoprotein inhibitor, does not potentiate loperamide's opioid brain effects in humans despite full inhibition of lymphocyte P-glycoprotein.

Background:Loperamide, a potent opioid, has been used as an in vivo probe to assess P-glycoprotein activity at the blood–brain barrier, because P-glycoprotein inhibition allows loperamide to cross the blood–brain barrier and exert its central opioid effects. In humans, studies with nonselective and moderately potent inhibitors resulted in mild opioid effects but were confounded by the concurrent inhibition of loperamide’s metabolism. The authors studied the effect of the highly selective, potent P-glycoprotein inhibitor tariquidar on loperamide’s central opioid effects. Methods:In a randomized, double-blind, crossover study, nine healthy subjects received on 2 study days oral loperamide (32 mg) followed by an intravenous infusion of either tariquidar (150 mg) or placebo. Central opioid effects (pupil diameter, sedation) were measured for 12 h, and blood samples were drawn up to 48 h after drug administration to determine plasma loperamide concentrations and ex vivo P-glycoprotein activity in T lymphocytes. Values for pupil diameter and loperamide concentrations were plotted over time, and the areas under the curves on the tariquidar and placebo study day were compared within each subject. Results:Tariquidar did not significantly affect loperamide’s central effects (median reduction in pupil diameter area under the curve, 6.9% [interquartile range, −1.4 to 12.1%]; P = 0.11) or plasma loperamide concentrations (P = 0.12) but profoundly inhibited P-glycoprotein in lymphocytes by 93.7% (95% confidence interval, 92.0–95.3%). Conclusion:These results suggest that despite full inhibition of lymphocyte P-glycoprotein, the selective P-glycoprotein inhibitor tariquidar does not potentiate loperamide’s opioid brain effects in humans.

Effects of nelfinavir and its M8 metabolite on lymphocyte P‐glycoprotein activity during antiretroviral therapy

The efflux pump P‐glycoprotein decreases drug penetration into cells and tissues. To determine whether nelfinavir or its metabolites inhibit P‐glycoprotein in lymphocytes from a healthy volunteer, whole blood cells from human immunodeficiency virus‐negative donors were incubated either in human plasma to which nelfinavir or its M8 metabolite were added ex vivo or in plasma from human immunodeficiency virus‐positive patients receiving nelfinavir. The 50% P‐glycoprotein inhibitory concentrations of purified nelfinavir and M8 were 10.9 μmol/L and 29.5 μmol/L, respectively, for CD4+ T cells and 19.3 μmol/L and >48 μmol/L, respectively, for CD8+ T cells. Significant inhibitory activity was present in plasma from 27 of 46 patients (59%) receiving nelfinavir. Plasma nelfinavir concentrations correlated with percent inhibition on CD4+ (ρ = 0.85, P < .0001) and CD8+ (ρ = 0.83, P < .0001) T cells. The M8 concentrations correlated weakly with both inhibition and nelfinavir concentrations. On the basis of our findings in lymphocytes from a healthy volunteer exposed to plasma from human immunodeficiency virus‐positive patients, we believe it is likely that CD4+ and CD8+ lymphocytes in patients receiving nelfinavir as therapy for human immunodeficiency virus may have P‐glycoprotein inhibited by plasma concentrations of nelfinavir.

Analysis of iceA1 transcription in Helicobacter pylori

Background. Transcription of the Helicobacter pylori iceA1 gene is induced following adherence of the bacterium to gastric epithelial cells in vitro, suggesting that this gene might be involved in H. pylori pathogenesis. Consequently, the current studies were undertaken to characterize iceA1 transcription and to define the structure of iceA1‐containing transcripts to evaluate the potential of this gene to encode functional proteins.

Implications of T-cell P-glycoprotein activity during HIV-1 infection and its therapy.

Objectives: P‐glycoprotein (P‐gp) may reduce antiretroviral efficacy by decreasing disposition of HIV‐1 protease inhibitors into tissues and cells. In contrast, P‐gp overexpression in vitro can inhibit HIV‐1 replication, and some drugs induce P‐gp expression. To explore which of these mechanisms predominate in vivo, this study characterized relationships between T‐cell P‐gp activity and clinical parameters in HIV‐infected adults. Methods: P‐gp activity was quantified in total and naive CD4+ and CD8+ T cells of HIV‐infected adults by flow cytometry using the substrate dye DiOC2(3). Demographic, virologic, immunologic, and treatment factors were obtained from medical records. Factors associated with P‐gp activity were identified using multivariate linear regression. Results: A total of 185 subjects (22% female; 34% African American) were studied, of whom 131 (71%) were receiving antiretroviral treatment. There was marked interindividual variability in P‐gp activity. By multivariate analysis, higher CD4+ T‐cell P‐gp activity was associated with lower log10 HIV‐1 RNA (P = 0.005), but not treatment or demographic factors. P‐gp activity was correlated across Tcell subsets. Conclusions: The inverse relationship between P‐gp activity and plasma HIV‐1 RNA is most consistent with an inhibitory effect on viral replication rather than drug disposition. Antiretroviral drug class did not independently predict P‐gp activity.