Microanalysis of the antiretroviral nevirapine in human hair from HIV-infected patients by liquid chromatography-tandem mass spectrometry.

Sufficient drug exposure is crucial for maintaining durable responses to HIV treatments. However, monitoring drug exposure using single blood samples only provides short-term information and is highly subject to intra-individual pharmacokinetic variation. Drugs can accumulate in hair over a long period of time, so hair drug levels can provide drug exposure information over prolonged periods. We now report on a specific, sensitive, and reproducible liquid chromatography-tandem mass spectrometry method for measuring nevirapine (NVP), a widely used antiretroviral drug, levels in human hair using even a single short strand of hair. Hair samples are cut into small segments, and the drug is extracted in methanol/trifluoroacetic acid (v/v, 9:1) shaken at 37 °C in a water bath overnight, followed by liquid-liquid extraction under alkaline conditions. The extracted samples are then separated on a BDS-C(18) column with a mobile phase composed of 50% acetonitrile containing 0.15% acetic acid and 4 mM ammonium acetate with an isocratic elution for a total run time of 3 min and detected by triple quadrupole electrospray multiple reaction mode at precursor/product ion at 267.0 > 225.9 m/z. Deuterated nevirapine-d5 was used as an internal standard. This method was validated from 0.25 to 100 ng/mg using 2 mg hair samples. The accuracies for spiked NVP hair control samples were 98-106% with coefficients of variation (CV) less than 10%. The CV for incurred hair control samples was less than 7%. The extraction efficiency for incurred control hair samples was estimated at more than 95% by repeated extractions. This method has been successfully applied to analyze more than 1,000 hair samples from participants in a large ongoing cohort study of HIV-infected participants. We also showed that NVP in human hair can easily be detected in a single short strand of hair. This method will allow us to identify drug non-adherence using even a single strand of hair.

3-Iodothyronamine is an endogenous and rapid-acting derivative of thyroid hormone.

Thyroxine (T(4)) is the predominant form of thyroid hormone (TH). Hyperthyroidism, a condition associated with excess TH, is characterized by increases in metabolic rate, core body temperature and cardiac performance. In target tissues, T(4) is enzymatically deiodinated to 3,5,3'-triiodothyronine (T(3)), a high-affinity ligand for the nuclear TH receptors TR alpha and TR beta, whose activation controls normal vertebrate development and physiology. T(3)-modulated transcription of target genes via activation of TR alpha and TR beta is a slow process, the effects of which manifest over hours and days. Although rapidly occurring effects of TH have been documented, the molecules that mediate these non-genomic effects remain obscure. Here we report the discovery of 3-iodothyronamine (T(1)AM), a naturally occurring derivative of TH that in vitro is a potent agonist of the G protein-coupled trace amine receptor TAR1. Administering T(1)AM in vivo induces profound hypothermia and bradycardia within minutes. T(1)AM treatment also rapidly reduces cardiac output in an ex vivo working heart preparation. These results suggest the existence of a new signaling pathway, stimulation of which leads to rapid physiological and behavioral consequences that are opposite those associated with excess TH.

Pharmacokinetics and tolerability of oseltamivir combined with probenecid.

Oseltamivir is an inhibitor of influenza virus neuraminidase, which is approved for use for the treatment and prophylaxis of influenza A and B virus infections. In the event of an influenza pandemic, oseltamivir supplies may be limited; thus, alternative dosing strategies for oseltamivir prophylaxis should be explored. Healthy volunteers were randomized to a three-arm, open-label study and given 75 mg oral oseltamivir every 24 h (group 1), 75 mg oseltamivir every 48 h (q48h) combined with 500 mg probenecid four times a day (group 2), or 75 mg oseltamivir q48h combined with 500 mg probenecid twice a day (group 3) for 15 days. Pharmacokinetic data, obtained by noncompartmental methods, and safety data are reported. Forty-eight subjects completed the pharmacokinetic analysis. The study drugs were generally well tolerated, except for one case of reversible grade 4 thrombocytopenia in a subject in group 2. The calculated 90% confidence intervals (CIs) for the geometric mean ratios between groups 2 and 3 and group 1 were outside the bioequivalence criteria boundary (0.80 to 1.25) at 0.63 to 0.89 for group 2 versus group 1 and 0.57 to 0.90 for group 3 versus group 1. The steady-state apparent oral clearance of oseltamivir carboxylate was significantly less in groups 2 (7.4 liters/h; 90% CI, 6.08 to 8.71) and 3 (7.19 liters/h; 90% CI, 6.41 to 7.98) than in group 1 (9.75 liters/h; 90% CI, 6.91 to 12.60) (P < 0.05 for both comparisons by analysis of variance). The (arithmetic) mean concentration at 48 h for group 2 was not significantly different from the mean concentration at 24 h for group 1 (42 +/- 76 and 81 +/- 54 ng/ml, respectively; P = 0.194), but the mean concentration at 48 h for group 3 was significantly less than the mean concentration at 24 h for group 1 (23 +/- 26 and 81 +/- 54 ng/ml, respectively; P = 0.012). Alternate-day dosing of oseltamivir plus dosing with probenecid four times daily achieved trough oseltamivir carboxylate concentrations adequate for neuraminidase inhibition in vitro, and this combination should be studied further.

Effect of sex and AIDS status on the plasma and intrapulmonary pharmacokinetics of rifampicin.

OBJECTIVE: To compare the steady-state plasma and intrapulmonary concentrations of oral rifampicin (rifampin) in men and women with and without AIDS. DESIGN: Prospective nonblinded pharmacokinetic study. PARTICIPANTS: Ten men with AIDS, ten men without AIDS, ten women with AIDS, and ten women without AIDS. METHODS: Rifampicin 600 mg was administered orally once daily for 5 days to 40 adult volunteers. Blood was obtained 2 hours after the last dose and at the time of bronchoalveolar lavage (BAL) performed 4 hours after the last dose. Rifampicin was measured in plasma, epithelial lining fluid (ELF) and alveolar cells. Standardised BAL was performed without systemic sedation. The volume of ELF was calculated by the urea dilution method, and alveolar cells were recovered by a standardised centrifugation technique. The volume of alveolar cells was calculated from the cell count and differential performed on the BAL fluid. Rifampicin was measured by high-performance liquid chromatography. RESULTS: Sex or AIDS status had no effect on plasma concentrations of rifampicin at 2 hours, 4 hours, or in ELF. Plasma concentrations (mean +/- SD) of rifampicin at 2 hours (9.15 +/- 5.4 mg/L) were not significantly different (p > 0.05) from those at 4 hours (9.10 +/- 5.6 mg/L) following the last dose. The ELF concentration was 2.0 +/- 1.6 mg/L with a range of 0-7.3 mg/L and the ELF/plasma ratio at 4 hours was 0.2 +/- 0.2. Rifampicin was not detectable in ELF in eight subjects (three with AIDS and five without AIDS) or in alveolar cells in three subjects without AIDS. There was no significant effect of AIDS on alveolar cell concentrations of rifampicin. Alveolar cell concentrations of rifampicin were significantly greater in women (13.9 +/- 6.7 mg/L) than in men (6.6 +/- 4.1 mg/L) [p = 0.0003]. Alveolar cell rifampicin concentrations were 78% greater in smoking women (17.8 +/- 7.0 mg/L) than in nonsmoking women (10.0 +/- 2.4 mg/L), but the difference was not significant (p > 0.05). CD4+ cell counts in the AIDS subjects were not correlated with the concentrations of rifampicin in plasma, ELF or alveolar cells. CONCLUSIONS: The absorption of oral rifampicin was not affected by sex or AIDS. Plasma and alveolar cell concentrations were not significantly different, were both greater than ELF concentrations, and were adequate to inhibit Mycobacterium tuberculosis. Considerable interpatient variability was detected despite witnessed drug administration. The clinical significance of these findings is unknown but merits further investigation.

Pharmacokinetics and subjective effects of sublingual buprenorphine, alone or in combination with naloxone: lack of dose proportionality.

OBJECTIVE: Buprenorphine and buprenorphine/naloxone combinations are effective pharmacotherapies for opioid dependence, but doses are considerably greater than analgesic doses. Because dose-related buprenorphine opioid agonist effects may plateau at higher doses, we evaluated the pharmacokinetics and pharmacodynamics of expected therapeutic doses. DESIGN: The first experiment examined a range of sublingual buprenorphine solution doses with an ascending dose design (n = 12). The second experiment examined a range of doses of sublingual buprenorphine/naloxone tablets along with one dose of buprenorphine alone tablets with a balanced crossover design (n = 8). PARTICIPANTS: Twenty nondependent, opioid-experienced volunteers. METHODS: Subjects in the solution experiment received sublingual buprenorphine solution in single ascending doses of 4, 8, 16 and 32 mg. Subjects in the tablet experiment received sublingual tablets combining buprenorphine 4, 8 and 16 mg with naloxone at a 4 : 1 ratio or buprenorphine 16 mg alone, given as single doses. Plasma buprenorphine, norbuprenorphine and naloxone concentrations and pharmacodynamic effects were measured for 48-72 hours after administration. RESULTS: Buprenorphine concentrations increased with dose, but not proportionally. Dose-adjusted areas under the concentration-time curve for buprenorphine 32 mg solution, buprenorphine 1 6 mg tablet and buprenorphine/naloxone 16/4 mg tablet were only 54 +/- 16%, 70 +/- 25% and 72 +/- 17%, respectively, of that of the 4 mg dose of sublingual solution or tablet. No differences were found between dose strengths for most subjective and physiological effects. Pupil constriction at 48 hours after administration of solution did, however, increase with dose. Subjects reported greater intoxication with the 32 mg solution dose, even though acceptability of the 4 mg dose was greatest. Naloxone did not change the bioavailability or effects of the buprenorphine 16 mg tablet. CONCLUSION: Less than dose-proportional increases in plasma buprenorphine concentrations may contribute to the observed plateau for most pharmacodynamic effects as the dose is increased.

Pharmacokinetics of quinacrine in the treatment of prion disease.

BACKGROUND: Prion diseases are caused by the accumulation of an aberrantly folded isoform of the prion protein, designated PrPSc. In a cell-based assay, quinacrine inhibits the conversion of normal host prion protein (PrPC) to PrPSc at a half-maximal concentration of 300 nM. While these data suggest that quinacrine may be beneficial in the treatment of prion disease, its penetration into brain tissue has not been extensively studied. If quinacrine penetrates brain tissue in concentrations exceeding that demonstrated for in vitro inhibition of PrPSc, it may be useful in the treatment of prion disease. METHODS: Oral quinacrine at doses of 37.5 mg/kg/D and 75 mg/kg/D was administered to mice for 4 consecutive weeks. Plasma and tissue (brain, liver, spleen) samples were taken over 8 weeks: 4 weeks with treatment, and 4 weeks after treatment ended. RESULTS: Quinacrine was demonstrated to penetrate rapidly into brain tissue, achieving concentrations up to 1500 ng/g, which is several-fold greater than that demonstrated to inhibit formation of PrPSc in cell culture. Particularly extensive distribution was observed in spleen (maximum of 100 microg/g) and liver (maximum of 400 microg/g) tissue. CONCLUSIONS: The documented extensive brain tissue penetration is encouraging suggesting quinacrine might be useful in the treatment of prion disease. However, further clarification of the distribution of both intracellular and extracellular unbound quinacrine is needed. The relative importance of free quinacrine in these compartments upon the conversion of normal host prion protein (PrPC) to PrPSc will be critical toward its potential benefit.

Relative bioavailability of ketoprofen 20% in a poloxamer-lecithin organogel.

PURPOSE: The bioavailability of a single, topically applied, 200-mg dose of ketoprofen (delivered in a ketoprofen 20% gel) relative to a single 50-mg oral dose in healthy volunteers was studied. METHODS: This was an open-label crossover study. The subjects were randomized to receive an oral 50-mg ketoprofen capsule or a single topical dose of ketoprofen 20% in a poloxamer-lecithin organogel (PLO). Treatment was followed by a one-week washout period. Blood samples were collected at intervals up to 10 hours after administration, and plasma ketoprofen concentrations were determined by high-performance liquid chromatography with ultraviolet or mass spectrometry detection. Noncompartmental pharmacokinetic values were obtained after each dose, and relative bioavailability was calculated. RESULTS: Eight healthy volunteers enrolled in and completed the study. Topical absorption of ketoprofen was highly variable among the subjects over the 10-hour sampling period. The median oral maximum plasma concentration (Cmax) exceeded the topical Cmax by nearly 200-fold (4.15 versus 0.021 microg/mL) (p = 0.001). The median relative bioavailability of topical ketoprofen was 0.48%, with individual subjects' values ranging from 0.18% to 2.1%. CONCLUSION: The relative bioavailability of ketoprofen was low and highly variable when the drug was administered as a single dose in a PLO-based ketoprofen 20% gel.

A high-performance liquid chromatographic-tandem mass spectrometric method for the determination of cethromycin (ABT-773) in human plasma, bronchoalveolar lavage fluid, and alveolar cells.

A method is developed for the specific and sensitive determination of cethromycin concentrations in plasma, bronchoalveolar lavage (BAL), and alveolar cells (AC), using a high-performance liquid chromatographic-tandem mass spectrometry (MS) method. The mobile phase consists of 50% acetonitrile-0.05% acetic acid-5mM ammonium acetate; the column used is a C(8) reversed-phase stationary phase. The preparation of samples requires a solvent extraction step. The retention times for cethromycin and the internal standard are approximately 2.0 and 2.7 min, respectively, with a total run time of 3.5 min. Detection is carried out using electrospray MS in a multiple reaction monitor mode. The detection limits for cethromycin are 1 ng/mL for plasma and 0.2 ng/mL for BAL supernatants and AC suspensions. The assay has excellent performance characteristics and has been used to support a study of the intrapulmonary pharmacokinetics of cethromycin in human subjects.

Sensitive analysis of anti-HIV drugs, efavirenz, lopinavir and ritonavir, in human hair by liquid chromatography coupled with tandem mass spectrometry.

A highly sensitive and selective method using liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS) was developed and validated for the measurement of three antiretroviral agents, efavirenz, lopinavir and ritonavir, in human hair. Hair samples from adherent HIV-infected patients on antiretroviral therapies were cut into about 1 mm length segments and drugs were extracted by first shaking the samples with methanol in a 37 degrees C water bath overnight (>14 h), followed by methyl tert-butyl ether/ethyl acetate (1:1) extraction under weak alkaline conditions. The extracted lopinavir and ritonavir were separated by reversed-phase chromatography and detected by tandem mass spectrometry in electrospray positive ionization mode with multiple reaction monitoring (MRM), while efavirenz was monitored in negative ionization MRM mode. This method was validated from 0.01 to 4.0 ng/mg hair for ritonavir and 0.05-20 ng/mg hair for lopinavir and efavirenz by using 2 mg of a human hair sample. The interday and intraday assay precision (coefficients of variation, CV) for spiked quality control (QC) samples at low, medium and high concentrations were within 15% and accuracy ranged from 89% to 110%. Assay reproducibility was also demonstrated by analysis of incurred hair QC samples (CV <14%). No significant matrix ionization suppression was observed. This developed method allowed for the monitoring of these target medications in the hair samples of HIV-infected women on antiretroviral therapy in an observational study using small amounts of hair.

Cytochrome P450 eicosanoids are activators of peroxisome proliferator-activated receptor alpha.

Cytochrome P450 (P450) eicosanoids regulate vascular tone, renal tubular transport, cellular proliferation, and inflammation. Both the CYP4A omega-hydroxylases, which catalyze 20-hydroxyeicosatetraenoic acid (20-HETE) formation, and soluble epoxide hydrolase (sEH), which catalyzes epoxyeicosatrienoic acid (EET) degradation to the dihydroxyeicosatrienoic acids (DHETs), are induced upon activation of peroxisome proliferator-activated receptor alpha (PPARalpha) by fatty acids and fibrates. In contrast, the CYP2C epoxygenases, which are responsible for EET formation, are repressed after fibrate treatment. We show here that P450 eicosanoids can bind to and activate PPARalpha and result in the modulation of PPARalpha target gene expression. In transactivation assays, 14,15-DHET, 11,2-EET, and 20-HETE were potent activators of PPARalpha. Gel shift assays showed that EETs, DHETs, and 20-HETE induced PPARalpha-specific binding to its cognate response element. Expression of apolipoprotein A-I was decreased 70% by 20-HETE, whereas apolipoprotein A-II expression was increased up to 3-fold by 11,12-EET, 14,15-DHET, and 20-HETE. In addition, P450 eicosanoids induced CYP4A1, sEH, and CYP2C11 expression, suggesting that they can regulate their own levels. Given that P450 eicosanoids have multiple cardiovascular effects, pharmacological modulation of their formation and/or degradation may yield therapeutic benefits.

Quinacrine is mainly metabolized to mono-desethyl quinacrine by CYP3A4/5 and its brain accumulation is limited by P-glycoprotein.

Quinacrine (QA), an antimalarial drug used for over seven decades, has been found to have potent antiprion activity in vitro. To determine whether QA can be used to treat prion diseases, we investigated its metabolism and ability to traverse the blood-brain barrier in mice. In vitro and in vivo, we identified by liquid chromatography-tandem mass spectrometry the major metabolic pathway of QA as N-desethylation and compared our results with an authentic reference compound. The major human cytochrome (P450) isoforms involved in QA mono-desethylation were identified as CYP3A4/5 by using specific chemical and antibody inhibition as well as cDNA-expressed P450 studies. QA transport from the basolateral to apical side in multidrug resistance protein 1 gene (MDR1)-transfected Madin-Darby canine kidney (MDCK) cells was markedly greater than in control MDCK cells and was inhibited by the potent P-glycoprotein (P-gp) inhibitor GG918 (N-(4-[2-(1,2,3,4-tetrahydro-6,7-dimethoxy-2-iso-1-quinolynyl)-ethyl]-phenyl)-9,10-dihydro-5-methoxy-9-oxo-4-acridine carboxamine). In MDR1-knockout (KO) mice, QA brain levels were 6 to 9 times higher after a single i.v. dose of 2 mg/kg QA and 49 times higher after multiple oral doses of 10 mg/kg/day QA for 7 days, compared with those in wild-type (WT) FVB mice. In contrast, the QA levels in plasma, liver, spleen, and kidney were similar after a single 2 mg/kg i.v. dose and <2 times greater after 10 mg/kg oral doses in MDR1-KO mice compared with WT mice. These results indicate that P-gp plays a critical role in transporting QA from the brain.

Functional analysis of polymorphisms in the organic anion transporter, SLC22A6 (OAT1).

OBJECTIVES: The organic anion transporter, OAT1 (SLC22A6), plays a role in the renal elimination of many drugs and environmental toxins. The goal of this study was to identify and functionally characterize OAT1 variants as a first step towards understanding whether genetic variation in OAT1 may contribute to interindividual differences in renal elimination of xenobiotics. METHODS: As part of a larger study, 276 DNA samples from an ethnically diverse population were screened and 12 coding region variants of OAT1 were identified. The non-synonymous variants were then constructed and characterized in Xenopus laevis oocytes. A small family-based clinical study was conducted to determine the renal elimination of a model OAT1 substrate, adefovir (an antiviral agent) in human subjects who possessed a non-functional variant, OAT1-R454Q. RESULTS: Six non-synonymous variants were identified; two (OAT1-R50 H and OAT1-R293W) were present at > or = 1% in at least one ethnic population. These two variants exhibited normal uptake of p-aminohippurate, ochratoxin A and methotrexate assayed in X. laevis oocytes. One variant, OAT1-R454Q, was non-functional with respect to the above substrates. In the clinical study, there was no significant decrease in the renal secretory clearance of adefovir in family members heterozygous for OAT1-454Q in comparison to those with the reference transporter, OAT1-454R. CONCLUSIONS: These data indicate that the coding region of OAT1 has low genetic and functional diversity and suggest that coding region variants of OAT1 may not contribute substantially to interindividual differences in renal elimination of xenobiotics.

Intra- and interindividual variabilities of valacyclovir oral bioavailability and effect of coadministration of an hPEPT1 inhibitor.

Variability in valacyclovir bioavailability and the potential for cephalexin-valacyclovir interaction were evaluated. The intraindividual acyclovir area under the concentration-time curve (AUC) varied minimally, whereas interindividual differences were substantial. Coadministration of the human peptide transporter 1 (hPEPT1) substrates valacyclovir and cephalexin minimally reduced the acyclovir AUC. These results suggest a stable valacyclovir absorption phenotype, significant interindividual variability, and minimal interaction between these hPEPT1 substrates.

Basal signaling activity of mu opioid receptor in mouse brain: role in narcotic dependence.

Narcotic analgesics cause addiction by poorly understood mechanisms, involving mu opoid receptor (MOR). Previous cell culture studies have demonstrated significant basal, spontaneous MOR signaling activity, but its relevance to narcotic addiction remained unclear. In this study, we tested basal MOR-signaling activity in brain tissue from untreated and morphine-pretreated mice, in comparison to antagonist-induced withdrawal in morphine-dependent mice. Using guanosine 5'-O-(3-[(35)S]thio)triphosphate ([(35)S]GTP gamma S) binding and adenylyl cyclase activity assay in brain homogenates, we demonstrated that morphine pretreatment of mice enhanced basal MOR signaling in mouse brain homogenates and, moreover, caused persistent changes in the effects of naloxone and naltrexone, antagonists that elicit severe withdrawal in dependent subjects. Naloxone and naltrexone suppressed basal [(35)S]GTP gamma S binding (acting as "inverse agonists") only after morphine pretreatment, but not in drug-naive animals. Moreover, naloxone and naltrexone stimulated adenylyl cyclase activity in striatum homogenates only after morphine pretreatment, by reversing the inhibitory effects of basal MOR activity. After cessation of morphine treatment, the time course of inverse naloxone effects on basal MOR signaling was similar to the time course of naltrexone-stimulated narcotic withdrawal over several days. The neutral antagonist 6 beta-naltrexol blocked MOR activation without affecting basal signaling (G protein coupling and adenylyl cyclase regulation) and also elicited substantially less severe withdrawal. These results demonstrate long-lasting regulation of basal MOR signaling as a potential factor in narcotic dependence.