Diane T. Holland

Reduced lopinavir exposure during pregnancy.

Background:Optimal antiretroviral exposure during pregnancy is critical for prevention of mother-to-child HIV transmission and for maternal health. Pregnancy can alter antiretroviral pharmacokinetics. Our objective was to describe lopinavir/ritonavir (LPV/r) pharmacokinetics during pregnancy. Methods:We performed intensive steady-state 12-h pharmacokinetic profiles of lopinavir and ritonavir (three capsules: LPV 400 mg/r 100 mg) at 30–36 weeks gestation and 6–12 weeks postpartum. Maternal and umbilical cord blood samples were obtained at delivery. We measured LPV and ritonavir by reverse-phase high-performance liquid chromatography. Target LPV area under concentration versus time curve (AUC) was ≥ 52 μg h/ml, the estimated 10th percentile LPV AUC in non-pregnant historical controls (mean AUC = 83 μg h/ml). Results:Seventeen women completed antepartum evaluations; average gestational age was 35 weeks. Geometric mean antepartum LPV AUC was 44.4 μg h/ml [90% confidence interval (CI), 38.7–50.9] and 12-h post-dose concentration (C12h) was 1.6 μg/ml (90% CI, 1.1–2.5). Twelve women completed postpartum evaluations; geometric mean LPV AUC was 65.2 μg h/ml (90% CI, 49.7–85.4) and C12h was 4.6 μg/ml (90% CI, 3.7–5.7). The geometric mean ratio of antepartum/postpartum LPV AUC was 0.72 (90% CI, 0.54–0.96). Fourteen of 17 (82%) pregnant and three of 12 (25%) postpartum women did not meet our target LPV AUC. The ratio of cord blood/maternal LPV concentration in ten paired detectable samples was 0.2 ± 0.13. Conclusions:LPV/r exposure during late pregnancy was lower compared to postpartum and compared to non-pregnant historical controls. Small amounts of lopinavir cross the placenta. The pharmacokinetics, safety, and effectiveness of increased LPV/r dosing during the third trimester of pregnancy should be investigated.

Diagnosis of enteroviral central nervous system infection by polymerase chain reaction during a large community outbreak.

Enteroviruses are common causes of localized and systemic infection in patients of all ages and are the most frequent cause of epidemic aseptic meningitis in the United States. We have developed a polymerase chain reaction (PCR) assay of cerebrospinal fluid (CSF) for rapid diagnosis of enteroviral meningitis. This assay was applied to 257 CSF specimens during a large community outbreak of enterovirus disease; 109 (97%) of 112 enterovirus culture-positive CSF samples contained enterovirus RNA. In addition 35 (66%) of 53 samples from patients with suspected central nervous system disease with negative or no CSF viral cultures were positive by enterovirus PCR. The enterovirus PCR detected 13 different enterovirus serotypes. PCR results are available within 24 hours compared with a mean of 6.8 days for enterovirus culture. The clinical characteristics of 141 patients with enterovirus central nervous system disease are presented. This study demonstrates the usefulness of enterovirus PCR for the rapid diagnosis of enterovirus central nervous system disease and the potential for PCR tests to shorten hospitalization.

Antiretroviral Concentrations in Breast-Feeding Infants of Women in Botswana Receiving Antiretroviral Treatment

BACKGROUND The magnitude of infant antiretroviral (ARV) exposure from breast milk is unknown. METHODS We measured concentrations of nevirapine, lamivudine, and zidovudine in serum and whole breast milk from human immunodeficiency virus type 1 (HIV-1)-infected women in Botswana receiving ARV treatment and serum from their uninfected, breast-feeding infants. RESULTS Twenty mother-infant pairs were enrolled. Maternal serum concentrations of nevirapine were high (median, 9534 ng/mL at a median of 4 h after nevirapine ingestion). Median breast-milk concentrations of nevirapine, lamivudine, and zidovudine were 0.67, 3.34, and 3.21 times, respectively, those in maternal serum. The median infant serum concentration of nevirapine was 971 ng/mL, at least 40 times the 50% inhibitory concentration and similar to peak concentrations after a single 2-mg/kg dose of nevirapine. The median infant serum concentration of lamivudine was 28 ng/mL, and the median infant serum concentration of zidovudine was 123 ng/mL, but infants were also receiving zidovudine prophylaxis. CONCLUSIONS HIV-1 inhibitory concentrations of nevirapine are achieved in breast-feeding infants of mothers receiving these ARVs, exposing infants to the potential for beneficial and adverse effects of nevirapine ingestion. Further study is needed to understand the impact of maternal ARV treatment on breast-feeding HIV-1 transmission, infant toxicity, and HIV-1 resistance mutations among infected infants.

Lopinavir concentrations in cerebrospinal fluid exceed the 50% inhibitory concentration for HIV.

Introduction:Lopinavir (LPV) is highly bound to plasma proteins and is a substrate for active drugs transporters, which may greatly limit the access of LPV to the central nervous system (CNS). However, even low lopinavir concentrations may be sufficient to inhibit HIV replication. Prior anecdotal reports indicated that lopinavir concentrations were below detection in cerebrospinal fluid (CSF). Methods:LPV was measured by liquid chromatography/mass spectrometry in 31 CSF–plasma pairs from 26 HIV-infected individuals who were taking LPV-containing antiretroviral regimens. The lower limit of quantification was 3.7 μg/l. Results:Seven of the sample pairs had very low plasma (and CSF) LPV concentrations, with a mean estimated plasma trough of 274 μg/l (range, < 3.7 to 608; typical trough values ∼4000 μg/l), suggesting poor recent adherence. In the remaining 24 sample pairs, the median LPV concentration was 5889 μg/l [interquartile range (IQR), 4805–9620] and all CSF samples had measurable LPV concentrations: median 17.0 μg/l (IQR, 12.1–22.7). The median CSF–plasma ratio was 0.23% (range, 0.12–0.75). All CSF concentrations in these samples were more than double the 50% inhibitory concentration for wild-type HIV virus. Conclusions:In patients with typical plasma levels of LPV, the drug is detectable in the CSF at concentrations that exceed those needed to inhibit HIV replication. Despite being > 98% bound to plasma proteins, LPV penetrates into the CNS and may contribute to the control of HIV in this potential reservoir.

Antiretroviral Concentrations in Breast-Feeding Infants of Mothers Receiving Highly Active Antiretroviral Therapy

ABSTRACT There are limited data describing the concentrations of zidovudine, lamivudine, and nevirapine in nursing infants as a result of transfer via breast milk. The Kisumu Breastfeeding Study is a phase IIb open-label trial of prenatal, intrapartum, and postpartum maternal treatment with zidovudine, lamivudine, and nevirapine from 34 weeks of gestation to 6 months postpartum. In a pharmacokinetic substudy, maternal plasma, breast milk, and infant dried blood spots were collected for drug assay on the day of delivery and at 2, 6, 14, and 24 weeks after delivery. Sixty-seven mother-infant pairs were enrolled. The median concentrations in breast milk of zidovudine, lamivudine, and nevirapine during the study period were 14 ng/ml, 1,214 ng/ml, and 4,546 ng/ml, respectively. Zidovudine was not detectable in any infant plasma samples obtained after the day of delivery, while the median concentrations in infant plasma samples from postpartum weeks 2, 6, and 14 were 67 ng/ml, 32 ng/ml, and 24 ng/ml for lamivudine and 987 ng/ml, 1,032 ng/ml, and 734 ng/ml for nevirapine, respectively. Therefore, lamivudine and nevirapine, but not zidovudine, are transferred to infants via breast milk in biologically significant concentrations. The extent and effect of infant drug exposure via breast milk must be well understood in order to evaluate the benefits and risks of maternal antiretroviral use during lactation.

Pharmacokinetics and Safety of Indinavir in Human Immunodeficiency Virus-Infected Pregnant Women

ABSTRACT Human immunodeficiency virus-infected women (n = 16) received indinavir (800 mg three times a day) plus zidovudine plus lamivudine from 14 to 28 weeks of gestation to 12 weeks postpartum. Two women and eight infants experienced grade 3 or 4 toxicities that were possibly treatment related. Indinavir area under the plasma concentration-time curve was 68% lower antepartum versus postpartum, suggesting increased intestinal and/or hepatic CYP3A activity during pregnancy.

Quality assurance program for Clinical measurement of antiretrovirals: AIDS Clinical Trials Group proficiency testing program for pediatric and adult pharmacology Laboratories

ABSTRACT Clinical trials designed to compare antiretroviral regimens, investigate therapeutic drug monitoring, or measure pharmacometrics often include protease inhibitors (PIs), nonnucleoside reverse transcriptase inhibitors (NNRTIs), and nucleoside reverse transcriptase inhibitors, requiring the measurement of these antiretrovirals in plasma. Within the adult and pediatric AIDS Clinical Trials Group (ACTG), a network of Pharmacology Support Laboratories (PSLs) is a component of the group laboratory infrastructure and conducts these types of pharmacologic assays. The adult ACTG has developed a comprehensive quality assurance program for the conduct of clinical pharmacology protocols, one component of which is the antiretroviral proficiency testing (PT) program that has been implemented between the adult and pediatric pharmacology laboratories of the ACTG. PT testing samples were prepared and distributed in July 2001, February 2002, and July 2002. High, medium, and low concentrations of PIs (indinavir, saquinavir, amprenavir, lopinavir, ritonavir, and nelfinavir) and NNRTIs (nevirapine and efavirenz) were added to drug-free EDTA plasma and distributed, on dry ice, to eight ACTG PSLs. One testing laboratory used liquid chromatography-tandem mass spectrometry, and seven used high-performance liquid chromatography-UV analysis. A result was considered acceptable if it was within 20% deviation of the assigned concentration. For all concentrations of PIs evaluated, 96% of samples tested (430 of 448 measurements) met the acceptance criteria. For both NNRTIs, 100% of samples tested (140 of 140 measurements) met the acceptance criteria. In conclusion, the PT program results presented demonstrate excellent interlaboratory agreement for all antiretrovirals tested and provide support for the merger of plasma concentration data among laboratories for large clinical trials.

Lopinavir exposure with an increased dose during pregnancy.

Background:Use of standard adult lopinavir/ritonavir (LPV/RTV) dosing (400/100 mg) during the third trimester of pregnancy results in reduced LPV exposure. The goal of this study was to determine LPV exposure during the third trimester of pregnancy and 2 weeks postpartum with a higher LPV/RTV dose. Methods:The Pediatric AIDS Clinical Trials Group Protocol 1026s is an ongoing, prospective, nonblinded study of antiretroviral pharmacokinetics in HIV-infected pregnant women that included a cohort receiving LPV/RTV 400/100 mg twice daily during the second trimester and 533/133 mg twice daily during the third trimester through 2 weeks postpartum. Intensive steady state 12-hour pharmacokinetic profiles were performed during the third trimester and at 2 weeks postpartum and were optional during the second trimester. LPV and RTV were measured by reverse-phase high-performance liquid chromatography with a detection limit of 0.09 μg/mL. Results:Twenty-six HIV-infected pregnant women were studied. Median LPV area under the plasma concentration-time curve (AUCs) for the second trimester, third trimester, and postpartum were 57, 88, and 152 μg·h−1·mL−1, respectively. Median minimum LPV concentrations were 1.9, 4.1, and 8.3 μg/mL. Conclusions:The higher LPV/RTV dose (533/133 mg) provided LPV exposure during the third trimester similar to the median AUC (80 μg·h−1·mL−1) in nonpregnant adults taking standard doses. However, the AUC on this increased dose at 2 weeks postpartum was considerably higher. These data suggest that the higher LPV/RTV dose should be used in third trimester pregnant women; that it should be considered in second trimester pregnant women, especially those who are protease inhibitor experienced; and that postpartum LPV/RTV dosing can be reduced to standard dosing by 2 weeks after delivery.

Simple high-performance liquid chromatography method for the simultaneous determination of serum caffeine and paraxanthine following rapid sample preparation

A simple reversed-phase high-performance liquid chromatography (HPLC) method for the simultaneous determination of caffeine and paraxanthine in human serum is described. Serum proteins are precipitated with perchloric acid and the resulting supernatant neutralized for direct injection onto an HPLC column. The method uses a phosphate-methanol mobile phase (85:15, v/v) at pH 4.9 with a flow-rate of 1.75 ml/min and quantitation is by UV absorbance at 274 nm. Elution times are approximately 18 min for caffeine and 8 min for paraxanthine. Theobromine and theophylline have elution times of 5.4 and 9.4 min and do not interfere in the assay. The intra-assay and between-assay means for precision and accuracy for both drugs are: 4.5% C.V. and 3.3% deviation. The sensitivity of the method is 50 ng/ml for each drug.

Quality assurance program for pharmacokinetic assay of antiretrovirals : ACTG proficiency testing for pediatric and adult pharmacology support laboratories, 2003 to 2004 A requirement for therapeutic drug monitoring

Proficiency testing (PT) is a mandated requirement for clinical laboratories doing therapeutic drug monitoring and is an invaluable tool to help laboratories identify and correct problems in analytical procedures. The AIDS Clinical Trial Group Pharmacology Quality Assurance Committee implemented a new antiretroviral PT program for all currently available antiretroviral drugs in 2001. The PT program was designed for the AIDS Clinical Trial Group Pharmacology Specialty Laboratories actively involved in assaying these drugs in clinical trial samples so as to comply with the Clinical Laboratory Improvement Amendments. Results from the first 3 rounds of PT have been analyzed and reported1 and provided support for formalizing the guidelines of the PT testing program. The PT program has expanded with the addition of nucleoside reverse transcription inhibitors (NRTIs) and atazanavir. This report includes results from rounds 4 to 7 over 2 additional years of standard operations. Additionally we include results from NRTIs for all rounds and atazanavir for a single round. There were 9 participating laboratories. Eight used high-performance liquid chromatography as the primary method of detection and 2/8 also reported LC-MS-MS results. One laboratory used LC-MS-MS as their primary detection method. All laboratories measured protease inhibitors, most measured at least 1 non-nucleoside reverse transcription inhibitor and 5 had NRTI capabilities. Results were normally distributed and the acceptance range of ±20% best corresponded to a 95% confidence interval. Overall score for 9 participating laboratories was 96% correct out of 1826 challenges over 4 rounds. Laboratories scored 95, 98 and 97% correct for protease inhibitors, non-nucleoside reverse transcription inhibitorss and NRTIs, respectively. Three laboratories reporting LC-MS-MS results had 92% correct (347/378) challenges for all drugs. The percentage of correct results is about the same as previously reported1. There is a continued need for a PT program to help participating laboratories maintain essential quality assurance and quality control.