Simple and rapid high-performance liquid chromatographic method for nelfinavir,M8 nelfinavir metabolite,ritonavir and saquinavir assay in plasma

A simple reversed-phase liquid chromatographic method has been developed to determine protease inhibitors concentrations in plasma. Plasma samples (250 micro l) containing protease inhibitors were prepared by a simple deproteinization (recovery: 92, 91, 91 and 90.5% for ritonavir, saquinavir, nelfinavir and M8 nelfinavir metabolite, respectively). Chromatography was accomplished using a Hypersil octadecylsilyl column (100 x 4.6 mm I.D.) and a mobile phase composed of acetonitrile, tetrahydrofuran and dihydrogenophosphate buffer (pH 4) (32:10:58, v/v). Ultraviolet detection at 210 nm was used. The limit of detection was 200 ng/ml for ritonavir, saquinavir, nelfinavir and M8 nelfinavir metabolite. Calibration curves were linear up to 20000 ng/ml, with correlation coefficients better than 0.997 for all compounds. Intra- and inter-day coefficients of variation of the assay were 相似文献   

High-performance liquid chromatographic method for the determination of nelfinavir, a novel HIV-1 protease inhibitor, in human plasma

Nelfinavir mesylate, a potent and orally bioavailable inhibitor of HIV-1 protease (K_i=2 nM), has undergone Phase III clinical evaluation in a large population of HIV-positive patients. A high-performance liquid chromatography analytical method was developed to determine the pharmacokinetic parameters of the free base, nelfinavir, in these human subjects. The method involved the extraction of nelfinavir and an internal standard, 6,7-dimethyl-2,3-di-(2-pyridyl)quinoxaline, from 250 μl of human plasma with a mixture of ethyl acetate–acetonitrile (90:10, v/v). The analysis was via ultraviolet detection at 220 nm using a reversed-phase C₁₈ analytical column and a mobile phase consisting of 25 mM monobasic sodium phosphate buffer (adjusted to pH 3.4 with phosphoric acid)–acetonitrile (58:42, v/v) that resolved the drug and internal standard peaks from non-specific substances in human plasma. The method was validated under Good Laboratory Practice (GLP) conditions for specificity, inter- and intra-assay precision and accuracy, absolute recovery and stability. The mean recovery ranged from 92.4 to 83.0% for nelfinavir and was 95.7% for the internal standard. The method was linear over a concentration range of 0.0300 μg/ml to 10 μg/ml, with a minimum quantifiable level of 0.0500 μg/ml for nelfinavir.     

Simultaneous quantitative determination of the HIV protease inhibitors indinavir,amprenavir, ritonavir,lopinavir, saquinavir,nelfinavir and the nelfinavir active metabolite M8 in plasma by liquid chromatography

A simple HPLC method that quantitates all six currently available protease inhibitors and the nelfinavir active metabolite M8 in one assay is presented. A 500-microliter plasma sample was treated by liquid-liquid extraction with a mixture of heptane and ethyl acetate. After evaporation, the residue was redissolved in sodium dihydrogenphosphate and acetonitrile and washed twice with heptane. Chromatography was performed with an analytical C(18) column. Ultraviolet detection at 210 and 239 nm was used. The present method is associated with high accuracy and low imprecision in the concentration range of 25-5000 ng/ml of all six protease inhibitors and M8. This makes it suitable for monitoring purposes.     

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.     

Simultaneous determination of 8 HIV protease inhibitors in human plasma by isocratic high-performance liquid chromatography with combined use of UV and fluorescence detection: amprenavir, indinavir, atazanavir, ritonavir, lopinavir, saquinavir, nelfinavir and M8-nelfinavir metabolite

A simple, accurate and fast method was developed for determination of the commonly used HIV protease inhibitors (PIs) amprenavir, indinavir, atazanavir, ritonavir, lopinavir, nelfinavir, M8-nelfinavir metabolite and saquinavir in human plasma. Liquid-liquid extraction was used with hexane/ethylacetate from buffered plasma samples with a borate buffer pH 9.0. Isocratic chromatographic separation of all components was performed on an Allsphere hexyl HPLC column with combined UV and fluorescence detection. Calibration curves were constructed in the range of 0.025-10 mg/l. Accuracy and precision of the standards were all below 15% and the lowest limit of quantitation was 0.025 mg/l. Stability of quality control samples at different temperature conditions was found to be below 20% of nominal values. The advantages of this method are: (1) inclusion and determination of the newly approved atazanavir, (2) simultaneous isocratic HPLC separation of all compounds and (3) increased specificity and sensitivity for amprenavir by using fluorescence detection. This method can be used for therapeutic drug monitoring of all PIs currently commercialised and is now part of current clinical practice.     

Nelfinavir induces TRAIL receptor upregulation in ovarian cancer cells

HIV protease inhibitors are currently being discussed to be useful as new and alternative anti-cancer agents, especially as second line treatments for chemo-resistant human cancer types. Among three clinically applied HIV protease inhibitors tested, we found a high efficacy of nelfinavir on ovarian cancer cells, accompanied by apoptosis (annexin binding) and necrosis (propidium iodide permeability). In vitro, at concentrations used to induce cell death in ovarian cancer cells, nelfinavir had no effect on the cellular viability of fibroblasts or peripheral blood mononuclear leukocytes. Nelfinavir sensitized ovarian cancer cells to treatment with an apoptosis-inducing TRAIL receptor antibody due to upregulation of the TRAIL receptor DR5 as shown by RT-PCR and FACScan analysis. We conclude that nelfinavir, an already approved drug, is a highly efficient agent against ovarian cancer cells and could sensitize ovarian cancer cells to TRAIL treatment, either therapeutically applied or endogenously produced by cells of the immune system.     

HPLC-MS method for the simultaneous quantification of the new HIV protease inhibitor darunavir, and 11 other antiretroviral agents in plasma of HIV-infected patients

A new method using high performance liquid chromatography coupled with electrospray mass spectrometry (HPLC-MS) was developed and validated, for the quantification of plasma concentration of the new protease inhibitors darunavir (DRV) and other 11 antiretroviral agents (ritonavir, amprenavir, atazanavir, lopinavir, saquinavir, indinavir, nelfinavir and its metabolite M-8, nevirapine, efavirenz and tipranavir). A simple protein precipitation extraction procedure was applied on 50 microl of plasma aliquots and chromatographic separation of drugs and Internal Standard (quinoxaline) was achieved with a gradient (acetonitrile and water with formic acid 0.05%) on an C-18 reverse phase analytical column with 25 min of analytical run. Calibration curves were optimised according to expected ranges of drug concentrations in patients, and correlation coefficient (r2) was higher than 0.998 for all analytes. Mean intra- and inter-day precision (relative standard deviation %) for all compounds were 8.4 and 8.3%, respectively, and mean accuracy (% of deviation from nominal level) was 3.9%. Extraction recovery ranged within 93 and 105% for all drugs analysed. This novel HPLC-MS methodology allows a specific, sensitive and reliable determination of DRV and 11 other antiretrovirals. In our hand, it was used to measure DRV and ritonavir plasma concentration in HIV-positive patients, and it is now successfully applied for routine therapeutic drug monitoring and pharmacokinetics studies.     

Subnanogram on-line column-switching liquid chromatographic-tandem mass spectrometric quantification method for nelfinavir and methoxyphenol metabolite M1 in rat plasma

A new on-line, rapid and sensitive column-switch LC/MS/MS method to measure nelfinavir (NFV), an HIV-1 protease inhibitor, and its major metabolite (M1) in rat plasma was developed. Rat plasma containing the analytes and the internal standard was treated with acetonitrile and the supernatant was processed through an on-line extraction and an analytical columns, with a column-switch device. ESI-LC/MS with multiple reaction monitors for appropriate analytes was performed. This assay gave a limit of quantitation (LOQ) of <1 ng/mL for the analytes with 5 min run time. The within-run and between-run precisions were <12 and <10%, respectively. This analytical method was successfully applied to a study to correlate changes in maternal and placental NFV plasma concentrations in rats following NFV exposure in utero.     

Synthesis and anti-HIV activity of glucose-containing prodrugs derived from saquinavir, indinavir and nelfinavir.

With the aim at improving the transport of the current HIV protease inhibitors across the intestinal and blood brain barriers and their penetration into the central nervous system, the synthesis of various acyl and carbamatoyl glucose-containing prodrugs derived from saquinavir, indinavir and nelfinavir, their in vitro stability with respect to hydrolysis, and their anti-HIV activity have been investigated. D-Glucose, which is actively transported across these barriers, was connected through its 3-hydroxyl to these antiproteases via a linker. The liberation of the active free drug during the incubation time of the prodrugs with the cells was found to be crucial for HIV inhibition. The labile ester linking of the glucose-containing moiety to the peptidomimetic hydroxyl of saquinavir or to the indinavir C-8 hydroxyl, which is not part of the transition state isostere, is not an obstacle for anti-HIV activity. This is not the case for its stable carbamate linking to the peptidomimetic hydroxyl of saquinavir, indinavir and nelfinavir. The chemical stability with respect to hydrolysis of some of the saquinavir and indinavir prodrugs reported here, the liberation rate of the active free drug and the HIV inhibitory potency are acceptable for an in vivo use of these prodrugs. These glucose-linked ester and carbamate prodrugs display a promising therapeutic potential provided that their bioavailability, penetration into the HIV sanctuaries, and/or the liberation of the active free drug from the carbamate prodrugs are improved. Furthermore, no cytotoxicity was detected for the prodrugs for concentrations as high as 10 or even 100 microM, thus indicating an encouraging therapeutic index.     

Ion-pair liquid chromatography of steady-state plasma levels of chlorimipramine and demethylchlorimipramine

A method for the determination of chlorimipramine and its metabolite demethylchlorimipramine in the plasma of depressed patients during treatment is described. The method involves extraction of the parent drug, its metabolite and the internal standard from plasma, back-extraction into an acidic aqueous phase and re-extraction into a small volume of organic phase. Separation and quantitation are carried out by ion-pair partition chromatography with UV detection. Accurate determination is possible down to levels of 30 and 60 nmole per liter of plasma for chlorimipramine and the metabolite, respectively, when 1 ml of plasma is used.The coefficient of variation is 7.3% or less at different levels for chlorimipramine and demethylchlorimipramine. Plasma levels of the parent drug and the metabolite measured by this liquid chromatographic method and by a gas chromatographic procedure with electron-capture detection were in good agreement (r = 0.98).The steady-state plasma level of the metabolite was always higher than that of the parent drug in the 34 depressed patients investigated. The mean ratio between the metabolite and the parent drug was 2.7 ± 1.1 (S.D.) Large inter-individual differences in the levels of the two compounds in patients receiving similar doses were found.     

Simultaneous determination of HIV protease inhibitors amprenavir, atazanavir, indinavir, lopinavir, nelfinavir, ritonavir and saquinavir in human plasma by high-performance liquid chromatography-tandem mass spectrometry

We report a precise and accurate method for simultaneous quantification of protease inhibitors (PIs) amprenavir, atazanavir, indinavir, lopinavir, nelfinavir, ritonavir and saquinavir in plasma. An internal standard was added to samples prior to protein precipitation with acetonitrile followed by addition of ammonium formate buffer. Analysis was by HPLC-MS/MS. Calibration curves were validated over concentration ranges encompassing both subtherapeutic and potentially 'toxic' drug concentrations. Inter- and intra-assay variation were below 11% and PI recovery was above 87%. The bioanalytical method described is successfully applied to measure PI concentrations obtained from clinical pharmacokinetic studies and routine therapeutic drug monitoring (TDM).     

Effect of ethanol on spectral binding, inhibition, and activity of CYP3A4 with an antiretroviral drug nelfinavir

Cytochrome P450 3A4 (CYP3A4) is the most abundant CYP enzyme in the liver and metabolizes approximately 50% of the drugs, including antiretrovirals. Although CYP3A4 induction by ethanol and impact of CYP3A4 on drug metabolism and toxicity is known, CYP3A4-ethanol physical interaction and its impact on drug binding, inhibition, or metabolism is not known. Therefore, we studied the effect of ethanol on binding and inhibition of CYP3A4 with a representative protease inhibitor, nelfinavir, followed by the effect of alcohol on nelfinavir metabolism. Our initial results showed that methanol, ethanol, isopropanol, isobutanol, and isoamyl alcohol bind in the active site of CYP3A4 and exhibit type I spectra. Among these alcohol compounds, ethanol showed the lowest K_D (5.9 ± 0.34 mM), suggesting its strong binding affinity with CYP3A4. Ethanol (20 mM) decreased the K_D of nelfinavir by >5-fold (0.041 ± 0.007 vs. 0.227 ± 0.038 μM). Similarly, 20 mM ethanol decreased the IC₅₀ of nelfinavir by >3-fold (2.6 ± 0.5 vs. 8.3 ± 3.1 μM). These results suggest that ethanol facilitates binding of nelfinavir with CYP3A4. Furthermore, we performed nelfinavir metabolism using LCMS. Although ethanol did not alter k_cat, it decreased the K_m of nelfinavir, suggesting a decrease in catalytic efficiency (k_cat/K_m). This is an important finding because alcoholism is prevalent in HIV-1-infected persons and alcohol is shown to decrease the response to antiretroviral therapy.     

Identification of a pyrovalerone metabolite in the rat by gas chromatography-mass spectrometry and determination of pyrovalerone by gas chromatography-nitrogen-phosphorus detection

Pyrovalerone and its hydrolated metabolite have been identified by gas chromatography-mass spectrometry in rat urine and plasma. A sensitive gas chromatographic method for the quantitative analysis of pyrovalerone in rat urine and plasma is described. The method also permits the quantitative monitoring of the urinary excretion of the drug and its metabolite. Pyrovalerone and its hydroxylated metabolite are detected up to 18 h after a single oral administration to the rat at a dose of 20 mg/kg.     

The contribution of naturally occurring polymorphisms in altering the biochemical and structural characteristics of HIV-1 subtype C protease

Fourteen subtype B and C protease variants have been engineered in an effort to study whether the preexistent baseline polymorphisms, by themselves or in combination with drug resistance mutations, differentially alter the biochemical and structural features of the subtype C protease when compared with those of subtype B protease. The kinetic studies performed in this work showed that the preexistent polymorphisms in subtype C protease, by themselves, do not provide for a greater level of resistance. Inhibition analysis with eight clinically used protease inhibitors revealed that the natural polymorphisms found in subtype C protease, in combination with drug resistance mutations, can influence enzymatic catalytic efficiency and inhibitor resistance. Structural analyses of the subtype C protease bound to nelfinavir and indinavir showed that these inhibitors form similar interactions with the residues in the active site of subtype B and C proteases. It also revealed that the naturally occurring polymorphisms could alter the position of the outer loops of the subtype C protease, especially the 60's loop.