Johanna P. Daily

Protease Inhibitors as Inhibitors of Human Cytochromes P450: High Risk Associated with Ritonavir

Four protease inhibitor antiviral agents (ritonavir, indinavir, nelfinavir, saquinavir) were evaluated as in vitro inhibitors of the activity of six human cytochromes using an in vitro model based on human liver microsomes. Ritonavir was a highly potent inhibitor of P450‐3A activity (triazolam hydroxylation), having inhibitory potency slightly less than ketoconazole. Indinavir was also a potent 3A inhibitor, while nelfinavir and saquinavir were less potent. Ritonavir had high inhibition potency against cytochrome P450‐2C9 (tolbutamide hydroxylation), −2C19 (S‐mephenytoin hydroxylation), and −2D6 (dextromethorphan O‐demethylation and desipramine hydroxylation), while the other protease inhibitors had one or more orders of magnitude lower inhibitory activity against these reactions. None of the protease inhibitors had important inhibitory potency against P450‐1A2 (phenacetin O‐deethylation) or −2E1 (chlorzoxazone hydroxylation). Thus, among available protease inhibitors, ritonavir carries the highest risk of incurring drug interactions due to inhibition of cytochrome P450 activity.

Inhibition of Human Cytochrome P450 Isoforms by Nonnucleoside Reverse Transcriptase Inhibitors

The capacity of three clinically available nonnucleoside reverse transcriptase inhibitors (NNRTIs) to inhibit the activity of human cytochromes P450 (CYPs) was studied in vitro using human liver microsomes. Delavirdine, nevirapine, and efavirenz produced negligible inhibition of phenacetin O‐deethylation (CYP1A2) or dextromethorphan O‐demethylation (CYP2D6). Nevirapine did not inhibit hydroxylation of tolbutamide (CYP2C9) or S‐mephenytoin (CYP2C19), but these CYP isoforms were importantly inhibited by delavirdine and efavirenz. This indicates the likelihood of significantly impaired clearance of CYP2C substrate drugs (such as phenytoin, tolbutamide, and warfarin) upon initial exposure to these two NNRTIs. Delavirdine and efavirenz (but not nevirapine) also were strong inhibitors of CYP3A, consistent with clinical hazards of initial cotreatment with either of these drugs and substrates of CYP3A. The in vitro microsomal model provides relevant predictive data on probable drug interactions with NNRTIs when the mechanism is inhibition of CYP‐mediated drug biotransformation. However, the model does not incorporate interactions attributable to enzyme induction.

Alprazolam-ritonavir interaction: implications for product labeling.

Pharmacokinetic interactions involving antiretroviral therapies may critically influence the efficacy and toxicity of these drugs, as well as pharmacologic treatments of coincident or complicating diseases. The viral protease inhibitor ritonavir is of particular concern since it both inhibits and induces the activity of cytochrome P450 3A (CYP3A) isoforms.

Differential impairment of triazolam and zolpidem clearance by ritonavir.

Background: The viral protease inhibitor ritonavir has the capacity to inhibit and induce the activity of cytochrome P450‐3A (CYP3A) isoforms, leading to drug interactions that may influence the efficacy and toxicity of other antiretroviral therapies, as well as pharmacologic treatments of coincident or complicating diseases. Methods: The inhibitory effect of ritonavir on the biotransformation of the hypnotic agents triazolam and zolpidem was tested in vitro using human liver microsomes. In a double‐blind clinical study, volunteer study subjects received 0.125 mg triazolam or 5.0 mg zolpidem concurrent with low‐dose ritonavir (four doses of 200 mg), or with placebo. Results: Ritonavir was a potent in vitro inhibitor of triazolam hydroxylation but was less potent as an inhibitor of zolpidem hydroxylation. In the clinical study, ritonavir reduced triazolam clearance to <4% of control values (p < .005), prolonged elimination half‐life (41 versus 3 hours; p < .005), and magnified benzodiazepine agonist effects such as sedation and performance impairment. In contrast, ritonavir reduced zolpidem clearance to 78% of control values (p < .08), and slightly prolonged elimination half‐life (2.4 versus 2.0 hours; NS). Benzodiazepine agonist effects of zolpidem were not altered by ritonavir. Conclusion: Short‐term low‐dose administration of ritonavir produces a large and significant impairment of triazolam clearance and enhancement of clinical effects. In contrast, ritonavir produced small and clinically unimportant reductions in zolpidem clearance. The findings are consistent with the complete dependence of triazolam clearance on CYP3A activity, compared with the partial dependence of zolpidem clearance on CYP3A.

Short‐Term Exposure to Low‐Dose Ritonavir Impairs Clearance and Enhances Adverse Effects of Trazodone

Antiretroviral agents may participate in drug interactions that influence the efficacy and toxicity of other antiretrovirals, as well as pharmacologic treatments of coincident or complicating diseases. The viral protease inhibitor, ritonavir, may cause drug interactions by inhibiting the activity of cytochrome P450‐3A (CYP3A) isoforms. In a single‐dose, blinded, four‐way crossover study, 10 healthy volunteer subjects received 50 mg of trazodone hydrochloride or matching placebo concurrent with low‐dose ritonavir (four doses of 200 mg each) or with placebo. Compared to the control condition, ritonavir significantly reduced apparent oral clearance of trazodone (155 ± 23 vs. 75 ± 12 ml/min, p < 0.001), prolonged elimination half‐life (6.7 ± 0.7 vs. 14.9 ± 3.9 h, p < 0.05), and increased peak plasma concentrations (842 ± 64 vs. 1125 ± 111 ng/ml, p < 0.05) (mean ± SE). Coadministration of trazodone with ritonavir increased sedation, fatigue, and performance impairment compared to trazodone plus placebo; differences reached significance only for the digit‐symbol substitution test. Three subjects experienced nausea, dizziness, or hypotension when trazodone was given with ritonavir; 1 of these subjects also experienced syncope. Thus short‐term low‐dose administration of ritonavir impairs oral clearance of trazodone and increases the occurrence of adverse reactions. The findings are consistent with impairment of CYP3A‐mediated trazodone metabolism by ritonavir.

Reduced paediatric hospitalizations for malaria and febrile illness patterns following implementation of community-based malaria control programme in rural Rwanda.

BackgroundMalaria control is currently receiving significant international commitment. As part of this commitment, Rwanda has undertaken a two-pronged approach to combating malaria via mass distribution of long-lasting insecticidal-treated nets and distribution of antimalarial medications by community health workers. This study attempted to measure the impact of these interventions on paediatric hospitalizations for malaria and on laboratory markers of disease severity.MethodsA retrospective analysis of hospital records pre- and post-community-based malaria control interventions at a district hospital in rural Rwanda was performed. The interventions took place in August 2006 in the region served by the hospital and consisted of mass insecticide treated net distribution and community health workers antimalarial medication disbursement. The study periods consisted of the December–February high transmission seasons pre- and post-rollout. The record review examined a total of 551 paediatric admissions to identify 1) laboratory-confirmed malaria, defined by thick smear examination, 2) suspected malaria, defined as fever and symptoms consistent with malaria in the absence of an alternate cause, and 3) all-cause admissions. To define the impact of the intervention on clinical markers of malaria disease, trends in admission peripheral parasitaemia and haemoglobin were analyzed. To define accuracy of clinical diagnoses, trends in proportions of malaria admissions which were microscopy-confirmed before and after the intervention were examined. Finally, to assess overall management of febrile illnesses antibiotic use was described.ResultsOf the 551 total admissions, 268 (48.6%) and 437 (79.3%) were attributable to laboratory-confirmed and suspected malaria, respectively. The absolute number of admissions due to suspected malaria was smaller during the post-intervention period (N = 150) relative to the pre-intervention period (N = 287), in spite of an increase in the absolute number of hospitalizations due to other causes during the post-intervention period. The percentage of suspected malaria admissions that were laboratory-confirmed was greater during the pre-intervention period (80.4%) relative to the post-intervention period (48.1%, prevalence ratio [PR]: 1.67; 95% CI: 1.39 – 2.02; chi-squared p-value < 0.0001). Among children admitted with laboratory-confirmed malaria, the risk of high parasitaemia was higher during the pre-intervention period relative to the post-intervention period (age-adjusted PR: 1.62; 95% CI: 1.11 – 2.38; chi-squared p-value = 0.004), and the risk of severe anaemia was more than twofold greater during the pre-intervention period (age-adjusted PR: 2.47; 95% CI: 0.84 – 7.24; chi-squared p-value = 0.08). Antibiotic use was common, with 70.7% of all children with clinical malaria and 86.4% of children with slide-negative malaria receiving antibacterial therapy.ConclusionThis study suggests that both admissions for malaria and laboratory markers of clinical disease among children may be rapidly reduced following community-based malaria control efforts. Additionally, this study highlights the problem of over-diagnosis and over-treatment of malaria in malaria-endemic regions, especially as malaria prevalence falls. More accurate diagnosis and management of febrile illnesses is critically needed both now and as fever aetiologies change with further reductions in malaria.

Metabolomic analysis of patient plasma yields evidence of plant-like α-linolenic acid metabolism in Plasmodium falciparum

Metabolomics offers a powerful means to investigate human malaria parasite biology and host-parasite interactions at the biochemical level, and to discover novel therapeutic targets and biomarkers of infection. Here, we used an approach based on liquid chromatography and mass spectrometry to perform an untargeted metabolomic analysis of metabolite extracts from Plasmodium falciparum-infected and uninfected patient plasma samples, and from an enriched population of in vitro cultured P. falciparum-infected and uninfected erythrocytes. Statistical modeling robustly segregated infected and uninfected samples based on metabolite species with significantly different abundances. Metabolites of the α-linolenic acid (ALA) pathway, known to exist in plants but not known to exist in P. falciparum until now, were enriched in infected plasma and erythrocyte samples. In vitro labeling with (13)C-ALA showed evidence of plant-like ALA pathway intermediates in P. falciparum. Ortholog searches using ALA pathway enzyme sequences from 8 available plant genomes identified several genes in the P. falciparum genome that were predicted to potentially encode the corresponding enzymes in the hitherto unannotated P. falciparum pathway. These data suggest that our approach can be used to discover novel facets of host/malaria parasite biology in a high-throughput manner.

Cat scratch disease presenting as a breast mass.

A 39-year-old woman, gravida 3, para 3, with no significant medical or family history, presented with a 3-day report of a mass in her left breast that was preceded by 3 weeks of tenderness in the area. She was asymptomatic otherwise. On examination, she had a firm, mobile, tender, 2-cm left breast mass in the upper outer quadrant and two left axillary lymph nodes. Mammography showed an upper– outer-quadrant 2.5-cm density with irregular borders and numerous enlarged lymph nodes in the left axilla. On ultrasound, the mass was hypoechoic with septated, heterogenous echotexture and illdefined margins. A core biopsy of the mass was done and histology showed lymph node fragments with follicular and interfollicular hyperplasia, and a focus of necrotizing acute inflammation surrounded by histiocytes. Gram, silver, and Steiner stains for microorganisms were negative. Given the discordance between the pathologic findings and clinical impression of malignancy, full surgical excision of the mass was done and pathologic findings were similar. Stains and cultures including those for fungi and mycobacteria and immunohistochemical staining for toxoplasmosis were negative. Upon further questioning, the woman reported that she had received numerous scratches from her kitten and that before the development of breast tenderness, she had had an erythematous papule on her left chest wall below her breast. Four weeks after her initial presentation she had a Bartonella henselae indirect fluorescent antibody immunoglobulin (Ig)M titer of less than 1:20 and an IgG titer of 1:512; a titer greater than 1:64 is considered diagnostic of infection. Clarithromycin was prescribed at 250 mg twice a day for 3 weeks, with resolution of the lymphadenopathy. Comment

Iron Deficiency during Pregnancy: Blessing or Curse?

Iron deficiency is the most common micronutrient deficiency and the most common cause of anemia among pregnant women worldwide [1]. Pregnancy may require as much as 1000 mg of elemental iron to meet the demands for maternal red blood cell expansion and fetal erythropoiesis and to account for blood lost at delivery [2]. As part of its standards for maternal and neonatal care, the World Health Organization recommends routine iron supplementation of 60 mg daily for pregnant women living in areas where malnutrition is prevalent [3]. The rationale underlying this policy is the observed association of anemia with adverse pregnancy outcomes. Specifically, anemia has been linked with low birth weight, preterm birth, decreased infant iron stores, and consequent impaired cognition and growth of the child [4-7] . For the mother, severe anemia may increase the risks of cardiac failure or death from peripartum hemorrhage [7, 8]. In a recent review of 40 trials involving > 12,000 women, routine daily iron supplementation increased hemoglobin levels by -7.5 g/dL and was associated with a decreased risk for anemia at term (relative risk, 0.56; 95% con-

Correction: A Systematic Map of Genetic Variation in Plasmodium falciparum.

In PLoS Pathogens, volume 2, issue 6: DOI: 10.1371/journal.ppat.0020057 In the Materials and Methods section, the following Supporting Information files were mislabeled: Table S7 should be Table S10, Table S8 should be Table S11, and Table S9 should be Table S12. The corrected citations in text follow: To identify potential amplifications in 3D7, we compared the list of genes showing a 1.5-fold or greater change in 3D7 relative to each of the Senegal isolates (n = 5) as described above. This returned some genes that were likely to be highly polymorphic in individual Senegal strains, but such deletions were not held in common by all. Only two genes were shared by all the Senegal strains examined. These two genes were for GTP cyclohydrolase (PFL1155w) and P. falciparum 11–1 protein PF10_0374, the gene11–1 product, which is highly expressed during gametocytogenesis. Examination of the ratios for these genes (Table S10) is consistent with a 3D7 amplification (generally from 1.5- to 4-fold changes) rather than a Senegal deletion where ratios show a 20-fold difference in signal. Quantitative real-time PCR analysis further confirmed a probable amplification in 3D7 for GTP cyclohydrolase (Table S11). In contrast, gene deletions were identified as follows. The custom-designed Affymetrix malaria full-genome array consists of 2,397 probes for 100 viral genes that serve as background controls [14]. Intensities from these probes represent the level of cross-hybridization for a deleted gene. A new probe-to-gene map was generated to include both sense and antisense probes, and the MOID algorithm [59] was applied to assign “present” and “absent” calls to each gene. Based on all the background control data collected in this study, this analysis, similar to that described in Le Roch et al. [14], shows that a deleted gene has only a 2% chance to be misclassified as “present” if it is required to have both an intensity level of E > 10 and a Kolmogorov-Smirnov test of log10P of less than −0.5. Excluding genes with fewer than six probes and the highly variable var, rifin, and stevor genes, we found a total of 33 genes being called “absent” in at least two out of the three hybridizations for each strain (Table S12). Additionally, the Supporting Information file in the legend text for Table S7 was mislabeled. Table 5 should be Table S8. The corrected citation in text follows: Aside from those deleted genes (underlined), no distinct differences were observed between the classes of highly variable genes within the laboratory strains compared with the Senegal strains (Table S8). Indeed, no significant differences were observed in the variation within immunogens (p = 0.87) and protein biosynthesis (p = 0.45) genes between the two groups of isolates, but variation in multi-gene families was significant (p = 6.37E−58).