Solid-phase extraction combined with dispersive liquid-liquid microextraction/HPLC-UV as a sensitive and efficient method for extraction, pre-concentration and simultaneous determination of antiretroviral drugs nevirapine, efavirenz and nelfinavir in pharmaceutical formulations and biological samples.

Therapeutic drug monitoring (TDM) of antiretroviral drugs requires accurate and precise analysis of their trace amounts in plasma samples. Solid-phase extraction (SPE) coupled with dispersive liquid-liquid microextraction based on solidification of floating organic drop (DLLME-SFO) was introduced as a simple and sensitive method for extraction, pre-concentration and simultaneous determination of efavirenz (EFV), nelfinavir (NFV) and nevirapine (NVP) in human plasma and pharmaceutical formulations by using high performance liquid chromatography (HPLC) with UV detection. A response surface methodology (RSM) based on central composite design (CCD) was used for optimizing the main variables in the extraction procedure. Under the optimum experimental conditions, the calibration curves were linear in the range of 0.1-400 ng mL-1 with correlation coefficients from 0.9982 to 0.9997 and limits of detection (at S/N = 3) of 0.03 to 0.07 ng mL-1. Moreover the intra-day and inter-day precision (RSD%) were in the range of 2.2-4.2% and 3.1-5.2%, respectively. The enrichment factors and relative recoveries of the anti-HIV drugs were 459-1507 and 93.7-105.4%. The method was successfully applied to the simultaneous determination of the trace amounts of the antiretroviral drugs in blood plasma and pharmaceutical formulations.

Pharmacokinetics of Increased Nelfinavir Plasma Concentrations in Women During Pregnancy and Postpartum.

This study aims to evaluate the safety, acceptability, and pharmacokinetics (PK) of an increased dose of nelfinavir (NFV) during the third trimester of pregnancy. The study was registered as part of the International Maternal Pediatric Adolescent AIDS Clinical Trials network (IMPAACT-P1026s), an ongoing multicenter prospective cohort study of antiretroviral PK during pregnancy (NCT00042289). NFV intensive PK evaluations were performed at steady state during the third trimester of pregnancy and 2-3 weeks postpartum. Plasma concentrations of NFV and its active metabolite, hydroxyl-tert-butylamide (M8) were measured using high-performance liquid chromatography with ultraviolet detection. A total of 18 women are included in the analysis. NFV area under the concentration-time curve (AUC) with the increased dose during the third trimester was nearly identical to the standard dose postpartum, with a geometric mean ratio for third trimester to postpartum AUC of 0.98 (90%CI 0.71-1.35). Despite the increased dose, M8 AUC was lower during the third trimester compared to postpartum (0.53, IQR [0.38-0.75]), as was the M8/NFV AUC ratio (0.51, IQR [0.42-0.63]). NFV AUC0-12 was above target in 15 of 18 (83%) of participants during the third trimester compared to 14 of 16 (88%) postpartum. No major safety concerns were noted. Increasing the NFV dose to 1875 mg twice daily during the third trimester achieved similar concentrations postpartum compared to standard dosing (1250 mg twice daily). Increased NFV dose regimens may still have some benefit to human immunodeficiency virus (HIV)-positive pregnant women living in countries where novel protease inhibitors are currently unavailable or in individuals who are intolerant to ritonavir-boosted HIV medications.

Quality-by-design based development of a self-microemulsifying drug delivery system to reduce the effect of food on Nelfinavir mesylate.

Poor aqueous solubility and moderate permeability of Nelfinavir mesylate (NFM) leads to high variability in absorption after oral administration. To improve the solubility and bioavailability of NFM, the self microemulsifying drug delivery system (SMEDDS) was developed. For this purpose, Quality by design (QbD) approach employing D-optimal mixture design was used to prepare SMEDDS of NFM. Further, the software generated numerically optimized SMEDDS were developed by utilizing desirability function. Maisine 35-1, Tween 80, and Transcutol HP were identified as oil, surfactant, and co-surfactant that had best solubility for NFM. Ternary phase diagrams were plotted to identify the self-emulsification region. Dissolution of putative NFM in simulated fasted or fed small intestinal conditions, respectively, predicted that there is a positive food effect. However, NFM loaded SMEDDS showed absence of food effect with no significant difference in dissolution performance either in Fasted or fed state simulated intestinal fluid (FaSSIF or FeSSIF) biorelevent dissolution media. The prepared SMEDDS were thermodynamically stable with droplet size (121 nm), poly dispersity index (PDI) (0.198) and emulsification time (<1 min). Transmission electron microscopy (TEM) analysis confirmed the spherical shape of the reconstituted SMEDDS droplets. The ex vivo performance revealed 4.57 fold enhancement in the apparent permeability of NFM as compared to NFM suspension. The animal pharmacokinetic analysis in New Zealand strain rabbits indicated food effect on pure NFM suspension. However, absence of food effect and 3.5-3.6 fold enhancement in the oral bioavailability was observed when NFM was formulated into SMEDDS. Thus, it could be envisaged that development of SMEDDS formulation of NFM could be one of the best alternative to enhance oral bioavailability of NFM.

Impact of CYP2C19 polymorphism on the pharmacokinetics of nelfinavir in patients with pancreatic cancer.

AIM: This study evaluated the influence of CYP2C19 polymorphisms on the pharmacokinetics of nelfinavir and its metabolite M8 in patients with pancreatic cancer. METHODS: Nelfinavir was administered orally to patients for over 10 days. The plasma concentrations of nelfinavir and M8 were measured by HPLC. The genotypes of CYP2C19*1, CYP2C19*2 and CYP2C19*3 were determined by the polymerase chain reaction-restriction fragment length polymorphism method. RESULTS: Pharmacokinetic profiles of nelfinavir and M8 were characterized by wide interindividual variability. The mean Cmax of nelfinavir in CYP2C19*1/*1 patients was 3.89 ± 0.40 (n = 3) and 5.12 ± 0.41 (n = 30) µg ml(-1) , while that of CYP2C19*1/*2 patients was 3.60 (n = 1) and 6.14 ± 0.31 (n = 5) µg ml(-1) at the doses of 625 and 1250 mg nelfinavir twice daily, respectively. For the M8 metabolite, the mean Cmax of CYP2C19*1/*1 patients was 1.06 ± 0.06 (n = 3) and 1.58 ± 0.27 (n = 30) µg ml(-1) , while those of CYP2C19*1/*2 patients were 1.01 (n = 1) and 1.23 ± 0.15 (n = 5) µg ml(-1) at the doses of 625 and 1250 mg nelfinavir twice daily, respectively. The area under the plasma concentration-time curve (AUC(0,12 h)) values of nelfinavir for CYP2C19*1/*1 patients were 28.90 ± 1.27 and 38.90 ± 4.99 µg ml(-1) ·h and for CYP2C19*1/*2 patients, AUC(0,12 h) was 28.20 (n = 1) and 40.22 ± 3.17 (n = 5) µg ml(-1) ·h at the doses of 625 and 1250 mg nelfinavir twice daily, respectively. The Cmax of nelfinavir was significantly higher (P <0.05) in CYP2C19*1/*2 patients but there was no statistical difference in AUC(0,12 h). CONCLUSION: CYP2C19*1/*2 genotype modestly affected the pharmacokinetic profiles of nelfinavir and M8 in patients with locally advanced pancreatic cancer.

Predicting the outer boundaries of P-glycoprotein (P-gp)-based drug interactions at the human blood-brain barrier based on rat studies.

Using positron emission tomography (PET), (11)C-verapamil as the P-gp substrate, and cyclosporine A (CsA) as the P-gp inhibitor, we showed that the magnitude of P-gp-based drug interactions at the human blood-brain barrier (BBB) is modest. However, such interactions at clinically relevant CsA blood concentrations may be greater for substrates where P-gp plays an even larger role (fractional contribution of P-gp, ft > 0.97) in preventing the CNS entry of the drug (e.g., nelfinavir). Since we have shown that the rat is an excellent predictor of the verapamil-CsA interaction at the human BBB, we determined the magnitude of drug interaction at the rat BBB between nelfinavir and CsA. Under isoflurane anesthesia, male Sprague-Dawley rats were coadministered IV infusions of nelfinavir and escalating doses of CsA to achieve pseudo steady-state plasma/blood and brain concentrations of both drugs (blood CsA ranged 0-264.9 µM, n = 3-6/group). The percent increase in the brain:blood nelfinavir concentration ratio (determined by LC/MS) was described by the Hill equation with Emax = 6481%, EC50 = 12.3 µM, and γ = 1.6. Then, using these data, as well as in vitro data in LLCPK1 cells expressing the human P-gp, we predicted that CsA (at clinically relevant blood concentration of 1.5 µM) will increase the distribution of nelfinavir into the human brain by 236%. Collectively, our data suggest that clinically significant P-gp based drug interactions at the human BBB are possible for P-gp substrates highly excluded from the brain (ft > 0.97) and should be investigated using noninvasive approaches (e.g., PET).

Phase I trial of the combination of the Akt inhibitor nelfinavir and chemoradiation for locally advanced rectal cancer.

PURPOSE: To investigate the toxicity of nelfinavir, administered during preoperative chemoradiotherapy (CRT) in patients with locally advanced rectal cancer. MATERIAL AND METHODS: Twelve patients were treated with chemoradiotherapy to 50.4 Gy combined with capecitabine 825 mg/m(2) BID. Three dose levels (DL) of nelfinavir were tested: 750 mg BID (DL1), 1250 mg BID (DL2) and an intermediate level of 1000 mg BID (DL3). Surgery was performed between 8 and 10 weeks after completion of CRT. Primary endpoint was dose-limiting toxicity (DLT), defined as any grade 3 or higher non-hematological or grade 4 or higher hematological toxicity. RESULTS: Eleven patients could be analyzed: 5 were treated in DL1, 3 in DL2 and 3 in DL3. The first 3 patients in DL1 did not develop a DLT. In DL2 one patient developed gr 3 diarrhea, 1 patient had gr 3 transaminase elevation and 1 patient had a gr 3 cholangitis with unknown cause. An intermediate dose level was tested in DL3. In this group 2 patients developed gr 3 diarrhea and 1 patient gr 3 transaminase elevation and gr 4 post-operative wound complication. Three patients achieved a pathological complete response (pCR). CONCLUSIONS: Nelfinavir 750 mg BID was defined as the recommended phase II dose in combination with capecitabine and 50.4 Gy pre-operative radiotherapy in rectal cancer. First tumor response evaluations are promising, but a further phase II study is needed to get more information about efficacy of this treatment regimen.

Phase I study of nelfinavir in liposarcoma.

PURPOSE: HIV protease inhibitors are associated with HIV protease inhibitor-related lipodystrophy syndrome. We hypothesized that liposarcomas would be similarly susceptible to the apoptotic effects of an HIV protease inhibitor, nelfinavir. METHODS: We conducted a phase I trial of nelfinavir for liposarcomas. There was no limit to prior chemotherapy. The starting dose was 1,250 mg twice daily (Level 1). Doses were escalated in cohorts of three to a maximally evaluated dose of 4,250 mg (Level 5). One cycle was 28 days. Steady-state pharmacokinetics (PKs) for nelfinavir and its primary active metabolite, M8, were determined at Levels 4 (3,000 mg) and 5. RESULTS: Twenty subjects (13 males) were enrolled. Median (range) age was 64 years (37-81). One subject at Level 1 experienced reversible, grade 3 pancreatitis after 1 week and was replaced. No other dose-limiting toxicities were observed. Median (range) number of cycles was 3 (0.6-13.5). Overall best responses observed were 1 partial response, 1 minor response, 4 stable disease, and 13 progressive disease. Mean peak plasma levels and AUCs for nelfinavir were higher at Level 4 (7.3 mg/L; 60.9 mg/L × h) than 5 (6.3 mg/L; 37.7 mg/L × h). The mean ratio of M8:nelfinavir AUCs for both levels was ~1:3. CONCLUSIONS: PKs demonstrate auto-induction of nelfinavir clearance at the doses studied, although the mechanism remains unclear. Peak plasma concentrations were within range where anticancer activity was demonstrated in vitro. M8 metabolite is present at ~1/3 the level of nelfinavir and may also contribute to the anticancer activity observed.

Nelfinavir and its active metabolite, hydroxy-t-butylamidenelfinavir (M8), are transferred in small quantities to breast milk and do not reach biologically significant concentrations in breast-feeding infants whose mothers are taking nelfinavir.

Antiretroviral drugs cross from maternal plasma to breast milk and from breast milk to the infant in different concentrations. We measured concentrations of nelfinavir and its active metabolite (M8) in maternal plasma and breast milk from women and in dried blood spots collected from their infants at delivery and postnatal weeks 2, 6, 14, and 24 in the Kisumu Breastfeeding Study, Kisumu, Kenya. Nelfinavir-based antiretroviral regimens given to mothers as prevention of mother-to-child HIV transmission (PMTCT) do not expose the breast-feeding infant to biologically significant concentrations of nelfinavir or M8.

Transplacental transfer of antiretroviral drugs and newborn birth weight in HIV-infected pregnant women.

Although it is well known that antiretroviral drugs (ARVs) across the placenta in different extents, few data are available concerning the impact of the transplacental passage of ARVs on newborn outcome. The aim of this study is to evaluate the transplacental diffusion of ARVs and the clinical assessment of the newborn. Mother and cord lopinavir, nelfinavir, atazanavir and nevirapine plasma levels were determined by high-performance liquid chromatography. Newborn gestational age, weight, and Apgar score were recorded. Cord-to-mother ratio (C:M) was calculated to estimate the placental passage of ARVs. Preterm birth was defined as delivery at <37 weeks of gestation and low birth weight was defined as a birth weight of <2500g. Twenty-six HIV-infected pregnant women were enrolled. Nevirapine presented the highest C:M ratio (0.60 +/- 0.19), the C:M ratio of nelfinavir and atazanavir was 0.37 +/- 0.38 and 0.20 +/- 0.14, respectively. The lopinavir level in the cord was undetectable. The observed prevalence rate of neonatal low birth weight and preterm delivery was 19,2% (n = 5) and 15.4% (n = 4), respectively. A significant linear regression analysis was reported between the C:M ratio and newborn birth weight (p = 0.01). Although the role of highly active antiretroviral therapy (HAART) in preventing mother-to-child transmission is indisputable, these data indicate a pharmacological rationale to the association between birth weight and highly active antiretroviral therapy during pregnancy.

Pregnancy does not increase CYP3A or P-glycoprotein activity in the non-human primate, Macaca nemestrina.

Plasma concentrations of protease inhibitors are lower in pregnant women than in nonpregnant women or men. Using nelfinavir as a model protease inhibitor, we have shown that this phenomenon can be reproduced in a representative non-human primate model, Macaca nemestrina (J Pharmacol Exp Ther 329:1016-1022, 2009). Nelfinavir is cleared from the body predominantly by CYP3A metabolism and P-glycoprotein (P-gp) efflux. Therefore, using midazolam (MDZ) as a CYP3A probe and digoxin (DIG) as a P-gp probe, we determined the antepartum (73-118 days) and postpartum (61-130 days) in vivo intestinal and hepatic CYP3A or P-gp activity in the macaque. Although the systemic clearance of MDZ was significantly increased ( approximately 70%) during pregnancy after intra-arterial (IA) administration of the drug ((15)N-labeled MDZ; 40 microg/kg), pregnancy did not affect the oral clearance of the drug administered simultaneously (1 mg/kg p.o.) with the IA dose. In vitro studies in hepatic and intestinal S-9 fractions indicated no effect of pregnancy on CYP3A activity or protein expression in the small intestine or liver. In contrast, neither the oral (100 microg/kg) nor the IA (10 microg/kg) clearance of DIG was significantly altered by pregnancy, indicating no effect of pregnancy on P-gp activity. Assuming that MDZ and DIG are selective substrates of the macaque CYP3A enzymes and P-gp, respectively, these results suggest that factors other than increased CYP3A or P-gp activity contribute to the increased clearance of protease inhibitors during M. nemestrina pregnancy.

Zidovudine, lamivudine, and nelfinavir concentrations in amniotic fluid and maternal serum.

PURPOSE: The objective of this study was to examine lamivudine (3TC), zidovudine (ZDV), nelfinavir (NFV), and its active nelfinavir metabolite (M8) concentrations in paired maternal plasma and amniotic fluid samples to determine antiretroviral penetration or accumulation in the fetal compartment. METHOD: Ten paired amniotic fluid and maternal plasma samples were obtained during caesarian section for pharmacokinetic analysis. Antiretroviral concentrations were measured in both matrices using high-performance liquid chromatography (HPLC) and mass spectrometry (LC/MS) methodologies. RESULTS: Median maternal plasma concentrations for NFV, M8, 3TC, and ZDV were 456, 244, 176, and 794 ng/mL, respectively, while median amniotic fluid concentrations were 118, 21, 2537, and 1483 ng/mL, respectively. The median NFV amniotic fluid to maternal plasma ratio was 0.44; the median M8 ratio was 0.11. Median 3TC and ZDV amniotic fluid to plasma ratios were 11.9 and 1.5, respectively. CONCLUSIONS: NFV and M8 exhibited partial drug transfer and/or accumulation in the amniotic compartment, whereas ZDV and 3TC concentrations mostly exceeded that in maternal plasma. Overall, all drugs achieved exposures in the amniotic fluid in excess of their wild-type viral susceptibilities. Amniotic fluid is an important compartment in the prevention of mother-to-child transmission; a further understanding of protease inhibitor and other antiretroviral drug penetration into amniotic fluid is warranted.

As in humans, pregnancy increases the clearance of the protease inhibitor nelfinavir in the nonhuman primate Macaca nemestrina.

The apparent oral clearance of protease inhibitors (PIs) is increased in pregnant women. Although this phenomenon is reproduced in the mouse, because of the multiplicity of mouse cytochrome P450 isoforms, lack of information on their substrate and inhibitor selectivity, and lack of reagents (e.g., antibodies, purified protein), it is difficult to study the mechanistic basis of this phenomenon in this animal model. To investigate the mechanistic basis of this phenomenon in a more representative model, the nonhuman primate, we first determined whether this phenomenon could be reproduced in Macaca nemestrina, using nelfinavir as a model PI. Consistent with the human and mouse studies, we found that the apparent oral clearance of nelfinavir (NFV) in the macaques was significantly increased (3.14-fold) antepartum (n = 3) versus postpartum (n = 4). This increased apparent oral clearance was a result of an increased systemic clearance (1.9-fold) and a decreased bioavailability (approximately 45%) during pregnancy. In vitro, pregnancy significantly enhanced the rate of NFV depletion in hepatic, but not intestinal S-9 fractions. Human CYP3A inhibitors erythromycin (0.5 mM), ketoconazole (0.5 microM), and troleandomycin (0.01-1 mM), but not the CYP2C inhibitor, sulfaphenazole (3 microM), significantly inhibited the depletion of NFV in hepatic S-9 fractions and expressed rhesus CYP3A64 enzyme. Based on these data, we conclude that increased hepatic activity of NFV-metabolizing enzymes (perhaps CYP3A enzymes) results in increased clearance of PIs during pregnancy in the macaques. The M. nemestrina should be further investigated as a model to study the mechanisms by which the clearance of PIs is increased during pregnancy.

Quantification of 8 HIV-protease inhibitors and 2 nonnucleoside reverse transcriptase inhibitors by ultra-performance liquid chromatography with diode array detection.

BACKGROUND: Most HPLC-UV methods for therapeutic drug monitoring of anti-HIV drugs have long run times, which reduce their applicability for high-throughput analysis. We developed an ultra-performance liquid chromatography (UPLC)-diode array detection method for the simultaneous quantification of the HIV-protease inhibitors (PIs) amprenavir, atazanavir, indinavir, lopinavir, nelfinavir, ritonavir, saquinavir, and tipranavir (TPV), and the nonnucleoside reverse transcriptase inhibitors (NNRTIs) efavirenz and nevirapine. METHODS: Solid-phase extraction of 1 mL plasma was performed with Waters HLB cartridges. After 3 wash steps, we eluted the drugs with methanol, evaporated the alcohol, and reconstituted the residue with 50 microL methanol. We injected a 4-microL volume into the UPLC system (Waters ACQUITY UPLC BEH C8 column maintained at 60 degrees C) and used a linear gradient of 50 mmol/L ammonium acetate and 50 mmol/L formic acid in water versus acetonitrile to achieve chromatographic separation of the drugs and internal standard (A-86093). Three wavelengths (215, 240, and 260 nm) were monitored. RESULTS: All drugs were eluted within 15 min. Calibration curves with concentrations of 0.025-10 mg/L (1.875-75 mg/L for TPV) showed coefficients of determination (r(2)) between 0.993 and 0.999. The lower limits of quantification were well below the trough concentrations reported in the literature. Inter- and intraassay CVs and the deviations between the nominal and measured concentrations were <15%. The method was validated by successful participation in an international interlaboratory QC program. CONCLUSIONS: This method allows fast and simultaneous quantification of all commercially available PIs and NNRTIs for therapeutic drug monitoring.

Trough concentrations of lopinavir, nelfinavir, and nevirapine with standard dosing in human immunodeficiency virus-infected pregnant women receiving 3-drug combination regimens.

The objective of this study was to evaluate the plasma drug concentrations in human immunodeficiency virus (HIV)-infected pregnant women receiving highly active antiretroviral therapy (HAART) and to define the rate of occurrence of subtherapeutic concentrations for some commonly used antiretroviral drugs during pregnancy. We evaluated HIV-infected women (n = 68) in the third trimester of pregnancy in steady-state treatment with an HAART regimen administrated on a twice a day basis, which included 2 nucleoside reverse transcriptase inhibitors plus nelfinavir (NFV), lopinavir/ritonavir (LPV/r), or nevirapine (NVP). Blood samples were collected at predose (C(trough)). The following thresholds were used to define therapeutic drug concentrations-NFV: 0.8 microg/mL; LPV: 4.0 microg/mL/1.0 microg/mL (experienced/naive); and NVP: 3.1 microg/mL. At predose sampling, adequate drug concentrations were found in a higher proportion of women receiving NFV (70.8%) and LPV (75.0%) than NVP (55.6%). Median C(trough) plasma concentrations were 1.2 microg/mL for NFV, 5.5 microg/mL for LPV, and 3.1 microg/mL for NVP. Women receiving lopinavir/ritonavir had the lowest rates of detectable (>50 copies/mL) HIV RNA (15.4%) compared with rates of 22.2% and 41.7% among women receiving NVP and NFV, respectively. Genotypic resistance was detected in 50% of women with detectable HIV RNA for whom samples were available for testing. Subtherapeutic predose concentrations among HIV-infected pregnant women were more commonly found with NVP than with protease inhibitors. LPV administration was associated with the best viral load suppression.

Pharmacokinetic monitoring of HIV-1 protease inhibitors in the antiretroviral therapy.

Combination therapy with protease inhibitors (PIs) is used for the treatment of patients infected with the human immunodeficiency virus (HIV). To achieve optimal drug concentrations for viral suppression and avoidance of drug toxicity, therapeutic drug monitoring of PIs levels has been considered essential. In this study an analytical procedure for simultaneous monitoring the plasma concentrations of a total four protease inhibitors: indinavir, lopinavir, nelfinavir and saquinavir was presented. The plasma samples were liquid/liquid extracted and subjected to high performance liquid chromatography (HPLC) analysis. The lopinavir and saquinavir in the patient plasma samples were monitored by ultraviolet detection at 215 nm. Extraction procedure using methyl tert-butyl ether and the mobile phase consisted from acetonitrile with phosphate buffer on a Symmetry C18 column were used to monitor these two compounds. Linearity of the method was obtained in the concentration range of 0.1 - 15.0 mg/mL for all four protease inhibitors. Under steady state conditions, the plasma concentrations of protein inhibitors in 23 patients were determined and area under the plasma concentration-time curve was estimated to maintain optimal viral suppression. We developed a simple and specific method for the simultaneous determination of lopinavir and saquinavir.

Promoter polymorphisms and allelic imbalance in ABCB1 expression.

OBJECTIVE: The ABCB1 (MDR1) gene, encoding the transporter P-glycoprotein, is known to act on a broad range of prescription medicines. For this reason a large number of studies have assessed the functional consequences of variation in this gene. Particular attention has focused on the ABCB1_3435C>T polymorphism, an exonic variant resulting in a synonymous change. This variant has been associated with mRNA, protein and serum levels, and with responses to a number of medicines. The results of association studies have, however, been variable and it is not currently clear whether this polymorphism is functional or is in linkage disequilibrium with functionally distinct alleles. RESULTS: To identify functional variation in the ABCB1 gene we assessed allelic imbalance by pyrosequencing cDNA from 80 lymphoblastoid B cell lines from the Centre d'Etude du Polymorphisme Humain (CEPH) collection, including 74 individuals heterozygous for 3435C>T. We found that the degree of ABCB1 allelic imbalance differed among B-cell lines. In an effort to fine-map variants that influence the proportion of the two allelic mRNA species we genotyped representative common variations near the 3435C>T polymorphism by using a tagging single nucleotide polymorphism (SNP) approach. In one approach, we assessed in segregating families the impact of cis-acting variants on mRNA levels by using allelic imbalance as the phenotype in a regression analysis that distinguishes the coupling arrangements (phase) among alleles. In a second approach, we assessed allelic imbalance levels in lymphoblastoid B-cell lines from unrelated HapMap individuals, and performed an association using tagSNPs in a 5-Mb region surrounding the gene. Two potential cis-acting variants, a promoter rs28656907/rs28373093 dinucleotide polymorphism (P=0.007) and the rs10245483 SNP (P=0.0003) located 2 Mb upstream from the gene, were predictors of ABCB1 expression. CONCLUSIONS: The study outlines a general experimental approach for fine mapping gene variants that influence mRNA expression by using cultured cell lines.

Effect of highly active antiretroviral therapy on tacrolimus pharmacokinetics in hepatitis C virus and HIV co-infected liver transplant recipients in the ANRS HC-08 study.

OBJECTIVE: To characterise the interactions between tacrolimus and antiretroviral drug combinations in hepatitis C virus-HIV co-infected patients who had received a liver transplant. DESIGN: An observational, open-label, multiple-dose, two-period, one-sequence design clinical trial in which patients received tacrolimus as an immunosuppressive therapy during the postoperative period and then had an antiretroviral drug regimen added. Tacrolimus pharmacokinetics were evaluated at steady state during these two periods. METHODS: Fourteen patients participated in the study and seven participated in the intensified pharmacokinetic protocol. Patients were included if they had undergone liver transplantation for end-stage chronic hepatitis C, absence of opportunistic infection, a CD4 cell count of >150 cells/microL and an undetectable HIV plasma viral load (<50 copies/mL) under highly active antiretroviral therapy. During the posttransplantation period, the tacrolimus dose was adjusted according to blood concentrations. When liver function and the tacrolimus dose were stable, antiretroviral therapy was reintroduced. RESULTS: When lopinavir/ritonavir were added to the tacrolimus regimen (seven patients), the tacrolimus dose was reduced by 99% to maintain the tacrolimus concentration within the therapeutic range. Only two patients were treated with nelfinavir, which led to a wide variation in inhibition of tacrolimus metabolism. When efavirenz (four patients) or a nucleoside analogue combination (one patient) was added, very little change in tacrolimus dosing was required. CONCLUSION: The lopinavir/ritonavir combination markedly inhibited tacrolimus metabolism, whereas the effect of efavirenz was small. Tacrolimus dosing must be optimised according to therapeutic drug monitoring and the antiretroviral drug combination.

Evaluation of Elisa test for therapeutic monitoring of Nelfinavir in HIV-positive patients.

Therapeutic drug monitoring (TDM) is an important tool in the management of antiretroviral (ARV) therapy. The gold standard for measuring drugs plasma levels is High-Performance Liquid Chromatographic Assay (HPLC) however it is technically-demanding and time-consuming. We evaluated a new immunoenzymatic test (TDM-ELISA, Biostrands, Trieste, Italy) for nelfinavir and its active metabolite M8 in comparison with HPLC. A statistically significant difference in Ctrough between the two different tests was demonstrated but this difference was no longer significant when a value of 29% due to M8 aliquot was deleted. This faster TDM-ELISA may have an important role for TDM in HIV patients taking ARVs.

Pharmacokinetic modelling of the placental transfer of nelfinavir and its M8 metabolite: a population study using 75 maternal-cord plasma samples.

AIMS: A population pharmacokinetic model was developed to characterize the transfer of nelfinavir and its active metabolite M8 from maternal to cord plasma and amniotic fluid. METHODS: Concentration data were obtained from 75 women on the day of delivery and for whom maternal, umbilical plasma and amniotic fluid samples were collected. Data from 53 pregnant, 61 nonpregnant and seven consecutively pregnant and non pregnant women were then added to the database, the contents of which were analyzed using NONMEM. RESULTS: Nelfinavir and M8 concentrations in maternal plasma, umbilical plasma and amniotic fluid were described by six connected compartments. Mean (% intersubject variability) population estimates were: absorption rate 00.67 h(-1), lag time 00.87 h, oral clearance and volume of distribution: 39.5 l h(-1) (53%), and 557 l for non pregnant and pregnant women, respectively, and 115 l h(-1) (132%) and 1626 l, respectively, on the day of delivery, M8 formation clearance 0.77 l h(-1) and M8 elimination rate constant 03.41 h(-1) (74%). For nelfinavir and M8, respectively, the mother-to-cord parameters were 0.058 l h(-1) (34%), and 00.35 h(-1) (76%), the cord-to-amniotic fluid rate constants were 0.23 and 00.59 h(-1), and the elimination rate constants from amniotic fluid were 0.36 and 00.49 h(-1). The nelfinavir fetus : maternal concentration ratio was 25% for maternal concentrations between 0.1 and 2.5 mg l(-1), between the 31 and 41st week of gestation. CONCLUSIONS: The low transfer of nelfinavir from the placenta is unlikely to protect the fetus from vertical HIV-1 transmission.