Association between social psychological status and efavirenz and nevirapine plasma concentration among HIV patients in Kenya.

HIV-related stigma, lack of disclosure and social support are still hindrances to HIV testing, care, and prevention. We assessed the association of these social-psychological statuses with nevirapine (NVP) and efavirenz (EFV) plasma concentrations among HIV patients in Kenya. Blood samples were obtained from 254 and 312 consenting HIV patients on NVP- and EFV-based first-line antiretroviral therapy (ART), respectively, and a detailed structured questionnaire was administered. The ARV plasma concentration was measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS). There were 68.1% and 65.4% of the patients on NVP and EFV, respectively, who did not feel guilty for being HIV positive. The disclosure rates were approximately 96.1% and 94.6% of patients on NVP and EFV, respectively. Approximately 85% and 78.2% of patients on NVP and EFV, respectively, received social support as much as needed. There were 54.3% and 14.2% compared to 31.7% and 4.5% patients on NVP and EFV, respectively, with supratherapeutic and suboptimal plasma concentrations. Multivariate quantile regression analysis showed that feeling guilty for being HIV positive was associated with increased 954 ng/mL NVP plasma concentrations (95% CI 192.7 to 2156.6; p = 0.014) but not associated with EFV plasma concentrations (adjusted ß = 347.7, 95% CI = - 153.4 to 848.7; p = 0.173). Feeling worthless for being HIV positive was associated with increased NVP plasma concentrations (adjusted ß = 852, 95% CI = 64.3 to 1639.7; p = 0.034) and not with EFV plasma concentrations (adjusted ß = - 143.3, 95% CI = - 759.2 to 472.5; p = 0.647). Being certain of telling the primary sexual partner about HIV-positive status was associated with increased EFV plasma concentrations (adjusted ß 363, 95% CI, 97.9 to 628.1; p = 0.007) but not with NVP plasma concentrations (adjusted ß = 341.5, 95% CI = - 1357 to 2040; p = 0.692). Disclosing HIV status to neighbors was associated with increased NVP plasma concentrations (adjusted ß = 1731, 95% CI = 376 to 3086; p = 0.012) but not with EFV plasma concentrations (adjusted ß = - 251, 95% CI = - 1714.1 to 1212.1; p = 0.736). Obtaining transportation to the hospital whenever needed was associated with a reduction in NVP plasma concentrations (adjusted ß = - 1143.3, 95% CI = - 1914.3 to - 372.4; p = 0.004) but not with EFV plasma concentrations (adjusted ß = - 6.6, 95% CI = - 377.8 to 364.7; p = 0.972). HIV stigma, lack disclosure and inadequate social support are still experienced by HIV-infected patients in Kenya. A significant proportion of patients receiving the NVP-based regimen had supra- and subtherapeutic plasma concentrations compared to EFV. Social-psychological factors negatively impact adherence and are associated with increased NVP plasma concentration compared to EFV.

Vitamin D as Modulator of Drug Concentrations: A Study on Two Italian Cohorts of People Living with HIV Administered with Efavirenz.

To date, vitamin D seems to have a significant role in affecting the prevention and immunomodulation in COVID-19 disease. Nevertheless, it is important to highlight that this pro-hormone has other several activities, such as affecting drug concentrations, since it regulates the expression of cytochrome P450 (CYP) genes. Efavirenz (EFV) pharmacokinetics is influenced by CYPs, but no data are available in the literature concerning the association among vitamin D levels, seasonality (which affects vitamin D concentrations) and EFV plasma levels. For this reason, the aim of this study was to evaluate the effect of 25-hydroxy vitamin D (25(OH)D3) levels on EFV plasma concentrations in different seasons. We quantified 25(OH)D3 by using chemiluminescence immunoassay, whereas EFV plasma concentrations were quantified with the HPLC-PDA method. A total of 316 patients were enrolled in Turin and Rome. Overall, 25(OH)D3levels resulted in being inversely correlated with EFV concentrations. Some patients with EFV levels higher than 4000 ng/mL showed a deficient 25(OH)D3 concentration in Turin and Rome cohorts and together. EFV concentrations were different in patients without vitamin D supplementation, whereas, for vitamin D-administered individuals, no difference in EFV exposure was present. Concerning seasonality, EFV concentrations were associated with 25(OH)D3 deficiency only in winter and in spring, whereas a significant influence was highlighted for 25(OH)D3 stratification for deficient, insufficient and sufficient values in winter, spring and summer. A strong and inverse association between 25(OH)D3and EFV plasma concentrations was suggested. These data suggest that vitamin D is able to affect drug exposure in different seasons; thus, the achievement of the clinical outcome could be improved by also considering this pro-hormone.

A fast-screening dispersive liquid-liquid microextraction-gas chromatography-mass spectrometry method applied to the determination of efavirenz in human plasma samples.

We demonstrate the suitability of a fast, green, easy-to-perform, and modified sample extraction procedure, i.e., dispersive liquid-liquid microextraction (DLLME) for the determination of efavirenz (EFV) in human plasma. Data acquisition was done by gas chromatography-mass spectrometry (GC-MS) in the selected ion monitoring (SIM) mode. The simplicity of the method lies in, among others, the avoidance of the use of large organic solvent volumes as mobile phases and non-volatile buffers that tend to block the plumbing in high-performance liquid chromatography (HPLC). Chromatographic and mass spectral parameters were optimized using bovine whole blood for matrix matching due to insufficient human plasma. Method validation was accomplished using the United States Food and Drug Administration (USFDA) 2018 guidelines. The calibration curve was linear with a dynamic range of 0.10-2.0 µg/mL and an R2 value of 0.9998. The within-run accuracy and precision were both less than 20% at the lower limit of quantification (LLOQ) spike level. The LLOQ was 0.027 µg/mL which compared well with some values but was also orders of magnitude better than others reported in the literature. The percent recovery was 91.5% at the LLOQ spike level. The DLLME technique was applied in human plasma samples from patients who were on treatment with EFV. The human plasma samples gave concentrations of EFV ranging between 0.14-1.00 µg/mL with three samples out of seven showing concentrations that fell within or close to the recommended therapeutic range.

Large variability in plasma efavirenz concentration in Papua New Guinea HIV/AIDS patients associated with high frequency of CYP2B6 516T allele.

Papua New Guinea (PNG) has a high HIV/AIDS prevalence and very high frequency of the CYP2B6 c.516G>T (rs3745274) variant. We have conducted the first investigation of the impact of c.516G>T and patient demographics on plasma efavirenz (EFV) and 8-hydroxyefavirenz (8OH-EFV) concentrations, metabolic ratio (8OH-EFV/EFV) (MR), and their association with adverse effects, in PNG patients with HIV/AIDS. For 156 PNG patients with HIV/AIDS taking EFV 600 mg/day (for 3-156 months), plasma EFV and 8OH-EFV concentrations were quantified, CYP2B6 c.516G>T genotyped, and demographic and self-reported adverse effects data recorded. Genotype differences in EFV and 8OH-EFV concentrations, MR, and percent within therapeutic range (1000-4000 ng/ml) were examined, in addition to EFV and 8OH-EFV concentration differences between patients experiencing adverse effects. CYP2B6 c.516T allele frequency was 53%. Plasma EFV (p < 0.0001), 8OH-EFV (p < 0.01), and MR (p < 0.0001) differed significantly between genotypes, with genotype explaining 38%, 10%, and 50% of variability, respectively. Plasma EFV concentrations were significantly higher in T/T (median = 5168 ng/ml) than G/G (1036 ng/ml, post hoc p < 0.0001) and G/T (1502 ng/ml, p < 0.0001) genotypes, with all patients above therapeutic range (n = 23) being T/T genotype (p < 0.0001). EFV and 8OH-EFV concentrations were not significantly higher in patients experiencing adverse effects. In PNG HIV/AIDS population where the 516T frequency is very high, it explains a substantial portion of variability (38%) in EFV disposition; however, at least for the patients receiving EFV long term, this does not translate into significant side effects.

Impact of Efavirenz Mid-dose Plasma Concentration on Long-Term Weight Change Among Virologically Suppressed People Living With HIV.

BACKGROUND: Pharmacogenetic studies have shown that slow and intermediate metabolizers of efavirenz (EFV) gained less weight compared with extensive metabolizers. It is hypothesized that increased EFV exposure suppresses weight gain. We investigated the effect of EFV mid-dose plasma concentration (C12) on long-term weight change among virologically suppressed people living with HIV (PLWH). METHODS: Participants in a prospective EFV pharmacokinetic study were included if they had been taking EFV-containing combination antiretroviral therapy for more than 240 weeks and had 3 or more weight measurements. The weight changes and time to ≥5% of weight gain over 192 weeks were compared between PLWH with higher and those with lower EFV C12 (using mean population C12 as the cutoff). EFV C12 and CYP2B6 516G>T polymorphism were examined in generalized estimating equations and in a Cox proportional hazards model for associations with weight gain, after adjustments for age, sex, companion antiretroviral agent, CD4 lymphocyte count, and plasma HIV RNA. RESULTS: One hundred eighteen PLWH were included. PLWH with higher EFV C12 had less mean weight gain compared with those with lower C12 after 192 weeks (-0.09 vs +1.58 kg, P = 0.033). PLWH with higher C12 were less likely to gain ≥5% weight in Kaplan-Meier analysis (P = 0.0003). In both generalized estimating equations and Cox proportional hazards models, a higher EFV C12 was associated with less weight gain, while CYP2B6 516G>T was not, after adjustments made for confounding factors. CONCLUSIONS: Our findings support that increased EFV exposure was associated with less weight gain.

Higher plasma drug levels in elderly people living with HIV treated with darunavir.

BACKGROUND: The proportion of elderly people living with HIV-1 (PLHIV) is rising. In older patients, comorbidities and concomitant medications are more frequent, increasing the risk of potential drug-drug interactions (PDDIs). Data on the pharmacokinetics of ART in individuals aged ≥ 65 years of age are scarce. We compared plasma drug levels of ART, PDDIs, and side-effects in PLHIV aged ≥ 65 years of age, with controls ≤ 49 years of age. METHODS: Patients ≥ 65 years of age and controls ≤ 49 years of age, all of whom were on stable treatment with atazanavir (ATV), darunavir (DRV), or efavirenz (EFV) were included cross-sectionally. Plasma drug levels of ART were analyzed, comorbidities, concomitant medication, adherence, and side-effects recorded, and PDDIs analyzed using drug interactions databases. RESULTS: Between 2013 and 2015, we included 100 individuals ≥ 65 years of age (study group) and 99 controls (≤ 49 years of age). Steady-state DRV concentrations were significantly higher in the study group than in the control group (p = 0.047). In the ATV group there was a trend towards a significant difference (p = 0.056). No significant differences were found in the EFV arm. The DRV arm had a higher frequency of reported side-effects than the ATV and EFV arms in the study group (36.7% vs. 0% and 23.8% respectively (p = 0.014), with significant differences between DRV vs. ATV, and EFV vs. ATV). CONCLUSIONS: Higher steady-state plasma levels of DRV and ATV (but not EFV) were found in PLHIV aged ≥ 65 years of age, compared to controls ≤ 49 years of age.

High efavirenz levels but not neurofilament light plasma levels are associated with poor neurocognitive functioning in asymptomatic HIV patients.

The aim of this study is to assess the effect of efavirenz exposure on neurocognitive functioning and investigate plasma neurofilament light (Nfl) as a biomarker for neurocognitive damage. Sub-analysis of the ESCAPE-study, a randomised controlled trial where virologically suppressed, cognitively asymptomatic HIV patients were randomised (2:1) to switch to rilpivirine or continue on efavirenz. At baseline and week 12, patients underwent an extensive neuropsychological assessment (NPA), and serum efavirenz concentration and plasma Nfl levels were measured. Subgroups of elevated (≥ 4.0 mg/L) and therapeutic (0.74 to< 4.0 mg/L) baseline efavirenz concentration were made. Differences between these groups in baseline NPA Z-scores and in delta scores after efavirenz discontinuation were assessed. Nfl level was measured using an ELISA analysis using single molecule array (Simoa) technology. Correlation of plasma NFL with NPA Z-scores was evaluated using a linear mixed model. The elevated group consisted of 6 patients and the therapeutic group of 48. At baseline, the elevated group showed lower composite Z-scores (median - 1.03; IQR 0.87 versus 0.27; 0.79. p 0.02). This effect was also seen on the subdomains verbal (p 0.01), executive functioning (p 0.02), attention (p < 0.01) and speed (p 0.01). In the switch group, the elevated group improved more on composite scores after discontinuing efavirenz (mean 0.58; SD 0.32 versus 0.22; 0.54, p 0.15). No association between plasma Nfl and composite Z-score was found. High efavirenz exposure is associated with worse cognitive functioning compared with patients with therapeutic concentrations. Plasma Nfl is not a suitable biomarker to measure cognitive damage in this group.

Dosage Optimization of Efavirenz Based on a Population Pharmacokinetic-Pharmacogenetic Model of HIV-infected Patients in Thailand.

PURPOSE: Efavirenz exhibits high interindividual variability in plasma concentrations, leading to unpredictable efficacy and toxicity. Polymorphism of CYP2B6 516G > T has been found to predominantly contribute to efavirenz variability. However, dosage recommendations incorporating CYP2B6 516G > T polymorphism have not been investigated in the Thai population. This study aimed to develop a population model of the pharmacokinetic properties of efavirenz, and to investigate the impact of patients' characteristics and CYP2B6 516G > T polymorphism on the pharmacokinetic properties of efavirenz. Model-based simulations were performed to provide genotype-based dosage optimization in a Thai population. METHODS: Plasma efavirenz concentrations measured at 12 h post-dose in 360 Thai HIV-infected patients with and without tuberculosis were analyzed by the nonlinear mixed-effects modeling approach. A 1-compartment model with first-order absorption and elimination was used for describing the pharmacokinetic properties of efavirenz. FINDINGS: The allele frequency of CYP2B6 516G > T was 34.17%. The efavirenz oral clearance were 11.9, 8.0, and 2.8 L/h in patients weighing 57 kg and having the CYP2B6 516 GG, 516 GT, and 516 TT genotypes, respectively. The use of rifampicin increased efavirenz oral clearance by 28%. The results from the simulations suggest that efavirenz dosages of 400, 300, and 100 mg once daily in Thai HIV mono-infected patients, and 800, 600, and 200 mg once daily in HIV/tuberculosis co-infected patients carrying CYP2B6 516 GG, 516 GT, and 516 TT, respectively. IMPLICATION: The results from this study provide a rationale for efavirenz dose adjustment based on CYP2B6 516G > T polymorphism in Thai HIV-infected patients, which could help to improve treatment outcomes in this population. ClinicalTrials.gov identifier: NCT01138267.

Influence of a herbal preparation on the pharmacokinetics of highly active antiretroviral therapy drugs in rats.

Shenlin Fuzheng Capsule (SLFZC) is a herbal preparation used for HIV/AIDS in Guangxi, China. This study was designed to evaluate the influence of SLFZC on the pharmacokinetics of highly active antiretroviral therapy (HAART) drugs, zidovudine (3'-azido-3'-deoythymidine, AZT), 2',3'-dideoxy-3'-thiacytidine (3TC) and efavirenz (EFV). Thirty-six male SD rats were divided into three groups. Group A was given a combination of AZT, 3TC and EFV (AZT/3TC/EFV). Group B rats were given AZT/3TC/EFV simultaneously with SLFZC. Group C rats were given AZT/3TC/EFV 2h prior to SLFZC. Blood samples were collected at fixed time intervals. Plasma concentration of each antiretroviral drug was tested for calculation of pharmacokinetic parameters. There was significant difference among groups with respect to t1/2 for AZT (F=3.371, P<0.05), but the Student-Newman-Keuls (SNK) pairwise multiple comparison procedure showed no statistical differences in all pairwise comparisons (P>0.05). There were no significant differences among groups in terms of Cmax, T max, AUC0-12h and CL for AZT, and t1/2, Cmax, Tmax, AUC0-12h and CL for 3TC and EFV, respectively. The results indicate that SLFZC has little impact on pharmacokinetic properties of AZT, 3TC and EFV.

A multicenter, randomized, double-blind study comparing different FK778 doses (manitimus) with tacrolimus and steroids vs. MMF with tacrolimus and steroids in renal transplantation.

BACKGROUND: This multicenter phase II study in renal transplantation compared 3 concentration-controlled ranges of FK778 (manitimus) with mycophenolate mofetil (MMF) both given in combination with tacrolimus and corticosteroids. METHODS: 364 patients were randomized to 12-month treatment: high-level FK778 group (H, N=87) received 4 x 600 mg/day (4 days) followed by 120 mg/day; mid-level FK778 group (M, N=92) received 3 x 600 mg/day (3 days) followed by 110 mg/day, low-level FK778 group (L, N=92) received 2 x 600 mg/day (2 days) followed by 100 mg/day, and control group received MMF 1 g/day (MMF, N=93). After week 6, FK778 doses were adjusted to trough ranges of 75-125 µg/mL (H), 50-100 µg/mL (M) and 25-75 µg/mL (L). Tacrolimus and steroids were administered at the same dose in each of the 4 groups. RESULTS: Biopsy proven acute rejection (BPAR) at 24 weeks, the primary study endpoint, was comparable in the L (22.8%) and MMF (17.2%) groups but higher in the H (34.5%) and M (29.3%) groups. BPAR at 12 months was comparable in the L (23.9%) and MMF (19.4%) groups but higher in the H (34.5%) and M (31.5%) groups. Graft and patient survival were lowest in the H group and renal function was poorest in the H and M groups. Premature study withdrawal was highest in the H group. CONCLUSIONS: Efficacy was similar between the low-level FK778 and MMF groups. Increased FK778 exposure was poorly tolerated and did not improve efficacy.

Esterase inhibitors as ester-containing drug stabilizers and their hydrolytic products: potential contributors to the matrix effects on bioanalysis by liquid chromatography/tandem mass spectrometry.

RATIONALE: Esterase inhibitors are widely used to stabilize ester-containing drugs in biological matrices for quantitative liquid chromatography/tandem mass spectrometry (LC/MS/MS) assays. These co-existing inhibitors could cause matrix effects on bioanalysis and jeopardize the assay performance. We therefore developed an LC/MS/MS methodology to monitor the fate of inhibitors and evaluate their matrix effects, which is described in this study. METHODS: Human plasma containing 20 mM of diisopropylfluorophosphate (DFP), paraoxon, eserine, phenylmethylsulfonyl fluoride (PMSF) or 2-thenoyltrifluoroacetone (TTFA) was extracted by liquid-liquid extraction (LLE) and analyzed by an LC/MS/MS assay for BMS-068645 (a model drug) with additional pre-optimized selected reaction monitoring (SRM) transitions using positive/negative electrospray ionization (ESI) mode for each inhibitor. Hydrolytic products were characterized by product ion or neutral loss scan LC/MS/MS analysis. The matrix effect contribution from each inhibitor was evaluated by post-column infusion of BMS-068645. RESULTS: In the extracted samples by LLE, SRM chromatograms revealed the presence of paraoxon, eserine and TTFA with peak intensity of >2.50E08. Three DFP hydrolytic products, diisopropyl phosphate (DP), triisopropyl phosphate (TP) and DP dimer, and one PMSF hydrolytic product, phenymethanesulfonic acid (PMSA), were identified in the extracted samples. In post-column infusion profiles, ion suppression or enhancement was observed in the retention time regions of eserine (~10% suppression), paraoxon (~70% enhancement) and DP dimer (~20% suppression). CONCLUSIONS: The SRM transitions described here make it possible to directly monitor the inhibitors and their hydrolytic products. In combination with post-column infusion, this methodology provides a powerful tool to routinely monitor the matrix effects-causing inhibitors, so that their matrix effects on the bioanalysis can be evaluated and minimized.

Simultaneous determination of a selective adenosine 2A agonist, BMS-068645, and its acid metabolite in human plasma by liquid chromatography-tandem mass spectrometry--evaluation of the esterase inhibitor, diisopropyl fluorophosphate, in the stabilization of a labile ester-containing drug.

BMS-068645 is a selective adenosine 2A agonist that contains a methyl ester group which undergoes esterase hydrolysis to its acid metabolite. To permit accurate determinations of circulating BMS-068645 and its acid metabolite, blood samples must be rapidly stabilized at the time of collection. A sensitive, rapid and specific liquid chromatography-tandem mass spectrometry (LC/MS/MS) method for the simultaneous quantitation of BMS-068645 and its acid metabolite in human plasma has been developed and validated using diisopropyl fluorophosphate (DFP) as the esterase inhibitor to prevent BMS-068645 from converting to its acid metabolite. The D(5)-stable isotope labeled analogs of BMS-068645 and its metabolite were used as the internal standards (IS). Analytes and IS in plasma containing 20 mM DFP were acidified and extracted into methyl tert-butyl ether. The liquid-liquid extraction effectively eliminated the strong matrix effect caused by the esterase inhibitor. The chromatographic separation was achieved on a Waters Atlantis C18 column with a run time of 4 min. Detection was performed on a Sciex API 4000 with positive ion electrospray mode (ESI/MS/MS), monitoring the ion transitions m/z 487>314 and 473>300 for BMS-068645 and its acid metabolite, respectively. The method was validated over the range from 0.020 to 10.0 ng/mL for BMS-068645 and 0.050 to 10.0 ng/mL for its acid metabolite. Inter- and intra-run precision for the quality control samples during validation were less than 8.7% and 4.0%, respectively, for the two analytes. The assay accuracy was within +/-5.4% of the nominal values. The esterase inhibitor effectively stabilized BMS-068645 during blood collection and storage. Blood collection tubes containing DFP were easily prepared and used at the clinical sites and could be stored at -30 degrees C for 3 months. This method demonstrated adequate sensitivity, specificity, accuracy, precision, stability and ruggedness to support the analysis of human plasma samples in pharmacokinetic studies.

The Vitamin D analogue TX 527 blocks NF-kappaB activation in peripheral blood mononuclear cells of patients with Crohn's disease.

Crohn's disease (CD) is an inflammatory disease characterized by the activation of the immune system in the gut. Since tumor necrosis factor (TNF-alpha) plays an important role in the initiation and perpetuation of intestinal inflammation in CD, we investigated whether TX 527 [19-nor-14,20-bisepi-23-yne-1,25(OH)(2)D(3)], a Vitamin D analogue, could affect peripheral blood mononuclear cells (PBMC) proliferation and exert an immunosuppressive effect on TNF-alpha production in CD patients, and whether this immunosuppressive action could be mediated by NF-kappaB down-regulation. TX 527 significantly decreased cell proliferation and TNF-alpha levels. On activation, NF-kappaB, rapidly released from its cytoplasmatic inhibitor (IKB-alpha), transmigrates into the nucleus and binds to DNA response elements in gene promoter regions. The activation of NF-kappaB, stimulated by TNF-alpha, and its nuclear translocation together with the degradation of IKB-alpha were blocked by TX 527. At the same time, NF-kappaB protein levels present in cytoplasmic extracts decreased in the presence of TNF-alpha and increased when PBMC were incubated with TX 527. The results of our studies indicate that TX 527 inhibits TNF-alpha mediated effects on PBMC and the activation of NF-kappaB and that its action is mediated by Vitamin D receptor (VDR), which is activated when the cells are stimulated with TX 527.

Formation of gamma-glutamylpropargylglycylglycine from propargylglycine in human blood and erythrocytes.

Gamma-Glutamylpropargylglycylglycine (gamma-Glu-PPG-Gly) was isolated as a metabolite of propargylglycine (2-amino-4-pentynoic acid, a natural and synthetic inhibitor of cystathionine gamma-lyase) from human blood incubated with D,L-propargylglycine in the presence of L-glutamate and glycine, and identified by fast-atom-bombardment mass spectrometry, indicating that human blood can metabolize propargylglycine to gamma-Glu-PPG-Gly. When whole blood was incubated with 2 mM D,L-propargylglycine in the presence of 10 mM L-glutamate and 10 mM glycine at 37 degrees C for 16h, 0.094+/-0.013 micromol of gamma-Glu-PPG-Gly was formed per ml of whole blood. When erythrocytes were incubated under the same conditions for 16h, 0.323+/-0.060 micromol of gamma-Glu-PPG-Gly was formed per ml of erythrocytes, suggesting a large contribution of erythrocytes to gamma-Glu-PPG-Gly formation in whole blood. The apparent Km value of gamma-Glu-PPG-Gly formation in human erythrocytes for D,L-propargylglycine was 0.32 mM. The observed rate of gamma-Glu-PPG-Gly formation and the Km value for D,L-propargylglycine suggest that metabolism of propargylglycine to gamma-Glu-PPG-Gly can play a definite biological role in human subjects who are loaded with propargylglycine.

Automated gas chromatography/tandem mass spectrometry with on-line chemical derivatization for the determination of tebufelone and two metabolites in human plasma.

An automated capillary gas chromatography/tandem mass spectrometry (GC/MS/MS) assay for the simultaneous quantitation of tebufelone (TE) and its two major metabolites (PGE-1802413 and PGE-6285825) in plasma was developed. Using a 1:1 BSTFA:pyridine derivatization cocktail to solubilize plasma extracts, trimethylsilylation (TMS) of labile alcohol and carboxylic acid functional groups occurred instantly upon introducing each sample into a 300 degrees C GC injection port. This on-line chemical derivatization process rendered these three diverse analytes equally amenable to GC analysis and circumvented laborious off-line derivatization procedures. The selectivity of MS/MS conducted on a triple-quadrupole instrument allowed minimal sample preparation and rapid analysis. Electron ionization produced molecular ions (M.+) for TMS-TE, TMS2-PGE-1802413, TMS2-PGE-6285825 and their respective stable-isotope-labeled internal standards, which were selected in Q1 to undergo collisionally activated dissociation in Q2. Quantitation was achieved through monitoring product ions in Q3 at m/z 320, 445 and 305 for respective analytes, relative to corresponding internal standard ions at m/z 323, 449 and 305. A 2.5-1000 ng per sample (approximately 25 pg to 10 ng injected) quantitation range provided access to an effective 2.5-10,000 ppb plasma concentration range (0.1-1 ml samples) for each analyte. Based on quality control data accumulated throughout 8 months of method application, the assay showed no bias and composite (N = 212) relative standard deviations of 5.6%, 7.0% and 9.5% for the respective analytes (with quality control levels typically covering a range of 10-250 ng per analyte). During this period, more than 2000 plasma study samples were analyzed, attesting to the reliability and ruggedness of this approach for routine application.

Evaluation of a benchtop ion trap gas chromatographic-tandem mass spectrometric instrument for the analysis of a model drug, tebufelone, in plasma using a stable-isotope internal standard.

The performance of a benchtop GC-ion trap MS-MS instrument, the Varian Saturn 4D, was evaluated for the analysis of a model drug, tebufelone, in plasma. The sample preparation scheme was designed to provide a highly complex extract with matrix-derived interferences eluting near and at the retention time of tebufelone and its stable-isotope-labeled analog. The performance of the ion trap in the selected-reaction-monitoring mode was evaluated and also compared with results obtained on a benchtop GC-MS linear quadrupole instrument operated in the selected-ion-monitoring mode. The ion trap, operated in the selected-reaction-monitoring mode, was found to provide a higher degree of selectivity for the analysis of tebufelone. The increased selectivity obtained on the ion trap operated in the selected-reaction-monitoring mode resulted in superior accuracy and precision, as well as a lower limit of quantitation relative to that obtained by the GC-MS analysis. A linear standard curve was obtained over three orders of magnitude and the limit of quantitation for tebufelone in plasma was 100 pg/ml using the GC-ion trap MS-MS instrument.

Inhibition of dihydropyrimidine dehydrogenase by 5-propynyluracil, a metabolite of the anti-varicella zoster virus agent netivudine.

OBJECTIVE: To study the effects of the anti-herpetic drug netivudine on dihydropyrimidine dehydrogenase activity in elderly volunteers and to relate them to concentrations of netivudine and its metabolite 5-propynyluracil. METHODS: Three groups of eight elderly volunteers received 400 or 800 mg netivudine or placebo once daily for 8 days. Plasma netivudine, 5-propynyluracil, and uracil, an indirect measure of dihydropyrimidine dehydrogenase activity, were assayed before the first dose and on days 2, 3, 5, 7 and 8. Full plasma profiles of netivudine and 5-propynyluracil were determined after the last dose. RESULTS: Plasma uracil was unquantifiable in all subjects before the first dose and at all time points in the placebo group. In recipients of netivudine it reached a plateau between days 3 and 5, with mean values of 23.2 and 23.5 mumol/L on day 8 in the subjects who received 400 and 800 mg. Plasma netivudine concentrations were approximately dose proportional, but 5-propynyluracil concentrations were similar in both groups. The half-maximal rise in plasma uracil occurred after a cumulative 5-propynyluracil exposure of 120 mumol/L.hr; such exposures will be achieved even after doses as low as 50 to 100 mg daily. CONCLUSIONS: Netivudine dosing produces complete inhibition of plasma dihydropyrimidine dehydrogenase. Coadministration with the antimetabolite 5-fluorouracil will require a substantial reduction in 5-fluorouracil dose to avoid toxicity but may also improve the therapeutic index of 5-fluorouracil.

Interspecies scaling of tebufelone pharmacokinetic data and application to preclinical toxicology.

PURPOSE: Retrospective application of allometric scaling techniques to tebufelone, a nonsteroidal antiinflammatory agent, in order to better understand the systemic exposure relationships between the doses administered to the species used in toxicology studies and the doses given to human subjects and patients in clinical studies. METHODS: Non-compartmental estimates of tebufelone's total body volume of distribution during the terminal phase (Vz) and clearance (CL) obtained from intravenous dosing to rat, monkey, dog, and human were allometrically scaled to body weight, and body weight and brain weight, respectively. AUCs determined from single or multiple dose pharmacokinetic studies and from preclinical toxicology studies were plotted versus dose adjusted for bioavailability and divided by allometrically scaled clearance to produce an allometric relationship suggesting a non-linear increase in AUC with dose across the four species. RESULTS: Segmental linear regression analysis of this relationship indicates a change point associated with an AUC of approximately 2,300 ng-hr/mL. Elevations in serum levels of various liver enzymes or associated signs of hepatic toxicity occur in some, but not all of the animals exposed for more than three weeks in repeat dosing studies at the actual dose that this represents. CONCLUSIONS: The analysis suggests that doses producing tebufelone plasma levels above a certain threshold AUC and duration of exposure to parent tebufelone are associated with increased risks of hepatic effects. Whether this is because metabolic shifts occur at these doses cannot be determined from these data.

Studies on hypoxoside and rooperol analogues from Hypoxis rooperi and Hypoxis latifolia and their biotransformation in man by using high-performance liquid chromatography with in-line sorption enrichment and diode-array detection.

Methanol extracts of the corms of Hypoxis rooperi and H. latifolia were studied for their hypoxoside content by an in-line sorption enrichment HPLC technique [Kruger et al., J. Chromatogr., 612 (1993) 191]. Hypoxoside is the trivial name for (E)-1,5-bis(3'-hydroxy-4'-O-beta-D-glucopyranosyl-phenyl) pent-1-en-4-yne and rooperol the aglucone obtained from beta-glucosidase treatment. Hypoxoside and rooperol analogues containing 4, 3 and 2 hydroxyl groups resolved as separate peaks with the proportion of the latter two markedly higher in H. latifolia than in H. rooperi. After oral ingestion of hypoxoside by humans, no hypoxoside or rooperol appeared in the serum. Only rooperol was present in the faeces. The serum and urine contained at least three phase II metabolite peaks. Selective enzyme hydrolysis showed that they represent the diglucuronide, disulfate and glucuronide-sulfate conjugates of all three rooperol analogues.

Automated gas chromatography/tandem mass spectrometry assay for tebufelone and a 13C,(18)O-labeled analog in plasma: applicabilityto absolute bioavailability determination.

An automated capillary gas chromatography/tandem mass spectrometry (GC/MS/MS) assay for the simultaneous quantitation of tebufelone (TE) and 13C, (18)O-labeled TE (TE-CO) in plasma was developed. This method permits the use of stable isotope coadministration (TE and TE-CO dosed concurrently via peroral and intravenous routes, respectively) for the determination of TE absolute bioavailability. The selectivity of MS/MS conducted on a triple-quadrupole instrument allowed minimal sample preparation and rapid analysis. Electron ionization produced molecular ions (M+) for TE, TE-CO, and the 3-methyl-TE internal standard which were selected in Q1 to undergo collisionally activated dissociation in Q2. Quantitation was achieved through monitoring product ions at m/z 248, 251 and 248, respectively, in Q3. A 2-1000 ng per sample (40 pg to 20 ng injected) quantitation range provided access to an effective 1-5000 p.p.b. plasma concentration range (0.2-2 g samples) for both TE and TE-CO. The assay showed no bias and less than 8% relative standard deviation over the entire range. The method was used to determine plasma levels of TE and TE-CO in four dogs receiving 2.5:2.5 mg/kg TE:TE-CO, intravenously. The pharmacokinetics of both isotopomers proved to be identical, indicating no isotope effect and verifying the chemical stability of the (18)O-carbonyl label under these dosing conditions. In addition, the applicability of this analytical approach to the determination of TE peroral bioavailability was initially tested in dogs.