Tenofovir Douche as HIV Preexposure Prophylaxis for Receptive Anal Intercourse: Safety, Acceptability, Pharmacokinetics, and Pharmacodynamics (DREAM 01).

BACKGROUND: Despite highly effective HIV preexposure prophylaxis (PrEP) options, no options provide on-demand, nonsystemic, behaviorally congruent PrEP that many desire. A tenofovir-medicated rectal douche before receptive anal intercourse may provide this option. METHODS: Three tenofovir rectal douches-220 mg iso-osmolar product A, 660 mg iso-osmolar product B, and 660 mg hypo-osmolar product C-were studied in 21 HIV-negative men who have sex with men. We sampled blood and colorectal tissue to assess safety, acceptability, pharmacokinetics, and pharmacodynamics. RESULTS: The douches had high acceptability without toxicity. Median plasma tenofovir peak concentrations for all products were several-fold below trough concentrations associated with oral tenofovir disoproxil fumarate (TDF). Median colon tissue mucosal mononuclear cell (MMC) tenofovir-diphosphate concentrations exceeded target concentrations from 1 hour through 3 to 7 days after dosing. For 6-7 days after a single product C dose, MMC tenofovir-diphosphate exceeded concentrations expected with steady-state oral TDF 300 mg on-demand 2-1-1 dosing. Compared to predrug baseline, HIV replication after ex vivo colon tissue HIV challenge demonstrated a concentration-response relationship with 1.9 log10 maximal effect. CONCLUSIONS: All 3 tenofovir douches achieved tissue tenofovir-diphosphate concentrations and colorectal antiviral effect exceeding oral TDF and with lower systemic tenofovir. Tenofovir douches may provide a single-dose, on-demand, behaviorally congruent PrEP option, and warrant continued development. Clinical Trials Registration . NCT02750540.

Development and validation of a multiplexed assay for the measurement of long-acting hormonal contraceptives in plasma via liquid chromatography-tandem mass spectrometry.

BACKGROUND: Exogenous progestins are an effective tool for hormonal contraception and family planning. Progestins may be delivered as oral pills, intramuscular or subcutaneous injections, vaginal rings, or intrauterine devices. Drug concentrations may vary based on the route and duration of delivery. Measurement of synthetic steroids in blood plasma can aid in determination of product adherence, evaluation of drug-drug interactions, and investigation of unintended pregnancies. METHODS: Drug-free K2EDTA plasma was spiked with the synthetic steroids etonogestrel (ETO), levonorgestrel (LNG), medroxyprogesterone acetate (MPA), and norethisterone (NET). Plasma was combined with isotopically labeled internal standards, and drugs were extracted via liquid-liquid extraction. Samples were then subjected to liquid chromatographic-tandem mass spectrometric (LC-MS/MS) analysis. The method was validated in accordance with regulatory recommendations. The assay was evaluated in a cohort of remnant plasma samples in individuals using one of the aforementioned progestins. RESULTS: The analytical measuring range for ETO, MPA, and NET was 20-10,000 pg/mL; the primary linearity for LNG was 20-20,000 pg/mL. The method showed acceptable precision and accuracy for all progestins. Stability was established for 72 h with room temperature storage and through 3 freeze-thaw cycles. All analytes were stable in whole blood incubated at room temperature for 25 h, and at 40°C and 100% humidity for 2 h. Ion suppression was observed for all analytes spiked in plasma; average ion suppression was 31.6%, 66.6%, 32.1% and 41.2% for ETO, LNG, MPA, and NET, respectively. However, internal standards showed comparable ion suppression, and relative matrix effects were minimal. ETO, LNG, MPA, and NET could also be quantified accurately in K3EDTA plasma and serum. Progestins were successfully measured in remnant samples from individuals using hormonal contraceptives. CONCLUSIONS: A multiplexed LC-MS/MS assay for the quantification of ETO, LNG, MPA, and NET has been developed and validated. The assay met acceptable performance characteristics and may be used in downstream studies to evaluate progestin pharmacology.

Development and validation of a liquid chromatographic-tandem mass spectrometric assay for the quantification of cabotegravir and rilpivirine from dried blood spots.

BACKGROUND: Dried blood spots (DBS) have been utilized as a blood plasma alternative for therapeutic drug monitoring and pharmacologic analysis. There are analytical and physiochemical considerations in bridging drug concentrations from plasma to DBS. Recently, the long-acting antiretroviral cabotegravir (CAB) has been approved for HIV prevention, and a co-packaged regimen of long-acting CAB and rilpivirine (RPV) has been approved for HIV treatment. Measurement of these drugs in blood collected as DBS may offer increased capacity and flexibility in translational applications. METHODS: Whole blood was spiked with CAB and RPV and spotted on DBS cards. Following extraction and addition of isotopically labeled internal standards, samples were subjected to liquid chromatographic-tandem mass spectrometric (LC-MS/MS) analysis. The method was validated according to regulatory recommendations, and the assay was evaluated in remnant samples from an HIV prevention trial in which paired DBS and plasma samples were collected. RESULTS: DBS CAB and RPV concentrations were linear from 25 to 20,000 ng/mL and 2-2500 ng/mL, respectively. Precision, accuracy, and matrix effect results were acceptable. DBS RPV demonstrated stability under all tested conditions; DBS CAB showed mean biases of - 23.5% when stored at room temperature for 36 days, and - 18.0% at 40 °C and 100% humidity for two days. DBS measurements for CAB and RPV were an average 54.0% and 14.1% lower, respectively, as compared to paired plasma samples. Derived conversion factors of 1.79 and 1.16 were applied to DBS CAB and RPV measurements, respectively, to estimate plasma concentrations. Estimated plasma CAB and RPV concentrations showed mean biases of 2.2% and 0.6%, respectively. In a CAB clinical trial, application of the conversion factor resulted in agreement between estimated plasma CAB concentrations from DBS and plasma CAB concentrations (y = 1.08x - 79.2, r = 0.932; mean bias of -3.2%; 95% CI: -48.2% to 41.9%). CONCLUSIONS: We developed and validated a novel LC-MS/MS assay for the quantification of CAB and RPV from DBS, and identified conversion factors to estimate plasma concentrations from spotted blood.

Development and validation of assays for the quantification of ß-D-N4-hydroxycytidine in human plasma and ß-D-N4-hydroxycytidine-triphosphate in peripheral blood mononuclear cell lysates.

The novel antiviral prodrug molnupiravir is under evaluation for the treatment of SARS-CoV-2. Molnupiravir is converted to ß-D-N4-hydroxycytidine (NHC), which is the primary form found in systemic circulation. ß-D-N4-hydroxycytidine-triphosphate (NHCtp) is the bioactive anabolite produced intracellularly. Sensitive and accurate bioanalytical methods are required to characterize NHC and NHCtp pharmacokinetics in clinical trials. Human K2EDTA plasma or peripheral blood mononuclear cell (PBMC) lysates were spiked with NHC (plasma) or NHCtp (PBMC), respectively. Following the addition of isotopically-labeled internal standards and sample extraction via protein precipitation or lysate dilution, respectively, samples were subjected to liquid chromatographic-tandem mass spectrometric (LC-MS/MS) analysis. Methods were validated in accordance with FDA Bioanalytical Method Validation recommendations. NHC can be quantified in plasma with a lower limit of quantification (LLOQ) of 1 ng/mL; the primary linearity of the assay is 1-5000 ng/mL. Assay precision and accuracy were ≤ 6.40% and ≤ ± 6.37%, respectively. NHC is unstable in whole blood and has limited stability in plasma at room temperature. The calibration range for NHCtp in PBMC lysates is 1-1500 pmol/sample, and the assay has an LLOQ of 1 pmol/sample. Assay precision and accuracy were ≤ 11.8% and ≤± 11.2%. Ion suppression was observed for both analytes; isotopically-labeled internal standards showed comparable ion suppression, resulting in negligible (<5%) relative matrix effects. Sensitive, specific, and dynamic LC-MS/MS assays have been developed and validated for the quantification of NHC in plasma and NHCtp in PBMC lysates. The described methods are appropriate for use in clinical trials.

Interspecies Differences in Tenofovir Alafenamide Fumarate Stability in Plasma.

Tenofovir (TFV) alafenamide fumarate (TAF) is an antiretroviral that has been evaluated in alternative drug delivery systems in several species. The ex vivo stability of TAF was evaluated, and TAF was stable in dog-, sheep-, and macaque-spiked plasma. A negative bias was observed in TAF recovery in rabbit-spiked plasma; there was complete loss of TAF and corresponding overestimation of TFV in rodent-spiked plasma. These data highlight considerations when evaluating TAF and TFV concentrations in preclinical studies.

Rifampicin effect on intracellular and plasma pharmacokinetics of tenofovir alafenamide.

OBJECTIVES: Tenofovir alafenamide produces lower plasma tenofovir and higher intracellular tenofovir diphosphate (DP) concentrations than tenofovir disoproxil fumarate but it is likely a victim of interactions with rifampicin. We aimed to investigate the pharmacokinetics of tenofovir alafenamide/emtricitabine with rifampicin. PATIENTS AND METHODS: Healthy volunteers received tenofovir alafenamide/emtricitabine at 25/200 mg once daily, followed by tenofovir alafenamide/emtricitabine + rifampicin daily followed by tenofovir disoproxil fumarate. Plasma tenofovir alafenamide, tenofovir, emtricitabine and intracellular tenofovir-DP and emtricitabine triphosphate pharmacokinetics and genetic polymorphisms were assessed. RESULTS: Tenofovir alafenamide exposure decreased when tenofovir alafenamide/emtricitabine + rifampicin was used compared with tenofovir alafenamide/emtricitabine [geometric mean ratio (GMR) (90% CI): 0.45 (0.33-0.60)]. Plasma tenofovir and intracellular tenofovir-DP concentrations decreased with rifampicin [GMR (90% CI): 0.46 (0.40-0.52) and 0.64 (0.54-0.75), respectively]. GMR (90% CI) of intracellular tenofovir-DP AUC0-24 for tenofovir alafenamide/emtricitabine + rifampicin versus tenofovir disoproxil fumarate was 4.21 (2.98-5.95). Rifampicin did not affect emtricitabine pharmacokinetics. CYP3A4*22 rs35599367 was associated with higher plasma tenofovir alafenamide AUC0-24 at day 56. CONCLUSIONS: Following tenofovir alafenamide/emtricitabine administration with rifampicin, intracellular tenofovir-DP concentrations were still 4.21-fold higher than those achieved by tenofovir disoproxil fumarate, supporting further study during HIV/TB co-infection.

Pharmacokinetics of rifapentine and rifampin in a rabbit model of tuberculosis and correlation with clinical trial data.

In clinical trials of two rifamycin antibiotics (rifampin and rifapentine) for treating tuberculosis (TB), patients with cavitary lung lesions did not appear to derive benefit from rifapentine. Rifapentine was found not to outperform rifampin, despite a lower minimum inhibitory concentration against Mycobacterium tuberculosis in mouse models of TB. To understand these findings, we have developed a rabbit model of TB that reliably develops lung cavities with features similar to those of patients with pulmonary cavitary TB. After single or multiple doses of rifampin or rifapentine that produced human-equivalent plasma exposures, rabbits were sacrificed at different time points after dosing. We measured site-of-disease drug pharmacokinetics and tissue drug distribution. We used pharmacokinetic-pharmacodynamic (PK/PD) modeling to estimate drug penetration into different types of tubercular lesions. Both drugs penetrated rabbit lung cellular lesions, as well as the fibrotic cavity wall of cavitary lesions (penetration coefficients ≥1 compared to plasma). For the necrotic liquefied material inside cavitary lesions known as caseum (which contains high numbers of bacteria), the penetration coefficient was 1.0 for rifampin but only 0.25 for rifapentine. When estimates of site-of-disease drug PK were substituted into clinical PK/PD models, the relationship between site-of-action exposure and sputum culture conversion was significant (P < 10-7). We propose that poor penetration of rifapentine into lung cavitary lesions explains, in part, why rifapentine doses required to improve treatment outcomes in two phase 2 clinical trials were four times higher in TB patients with large cavities compared to TB patients without cavitary lung disease.

Validation and implementation of liquid chromatographic-mass spectrometric (LC-MS) methods for the quantification of tenofovir prodrugs.

BACKGROUND: The nucleotide reverse transcriptase inhibitor tenofovir (TFV) is widely administered in a disoproxil prodrug form (tenofovir disoproxil fumarate, TDF) for HIV management and prevention. Recently, novel prodrugs tenofovir alafenamide fumarate (TAF) and hexadecyloxypropyl tenofovir (CMX157) have been pursued for HIV treatment while minimizing adverse effects associated with systemic TFV exposure. Dynamic and sensitive bioanalytical tools are required to characterize the pharmacokinetics of these prodrugs in systemic circulation. Two parallel methods have been developed, one to combinatorially quantify TAF and TFV, and a second method for CMX157 quantification, in plasma. METHODS: K2EDTA plasma was spiked with TAF and TFV, or CMX157. Following the addition of isotopically labeled internal standards and sample extraction via solid phase extraction (TAF and TFV) or protein precipitation (CMX157), samples were subjected to liquid chromatographic-tandem mass spectrometric (LC-MS/MS) analysis. For TAF and TFV, separation occurred using a Zorbax Eclipse Plus C18 Narrow Bore RR, 2.1 × 50 mm, 3.5 µm column and analytes were detected on an API5000 mass analyzer; CMX157 was separated using a Kinetex C8, 2.1 × 50 mm, 2.6 µm column and quantified using an API4500 mass spectrometer. Methods were validated according to FDA Bioanalytical Method Validation guidelines. RESULTS: Analytical methods: were optimized for the multiplexed monitoring of TAF and TFV, and CMX157 in plasma. The lower limits of quantification (LLOQs) for TAF, TFV, and CMX157 were 0.03, 1.0, and 0.25 ng/mL, respectively. Calibration curves were generated via weighted linear regression of standards. Intra- and inter-assay precision and accuracy studies demonstrated %CVs ≤ 14.4% and %DEVs ≤ ±â€¯7.95%, respectively. Stability and matrix effects studies were also performed. All results were acceptable and in accordance with the recommended guidelines for bioanalytical methods. Assays were also applied to quantify in vivo concentrations of prodrugs and TFV in a preclinical study post-rectal administration. CONCLUSIONS: Sensitive, specific, and dynamic LC-MS/MS assays have been developed and validated for the multiplexed quantification TAF and TFV, as well as an independent assay for CMX157 quantification, in plasma. The described methods meet sufficient throughput criteria to support large research trials.

An Ultraperformance LC-MS/MS Method for the Quantification of the Antimalarial Atovaquone in Plasma.

BACKGROUND: A primary modality in the treatment and prevention of malaria is the administration of antimalarial agents. Atovaquone (ATQ) has been used in single-drug and multidrug antimalarial applications; however, studies have demonstrated high interindividual drug variability. With the scarcity of analytical methodologies available in the literature, we have developed and optimized a rapid, ultraperformance (UP) LC-MS/MS method for the quantification of ATQ in human plasma. METHODS: ATQ was extracted from 25 µL K2-EDTA human plasma via protein precipitation with acetonitrile. Sample solutions were separated on a Synergi 2.5-µm Polar-RP 100A (100 × 2 mm) column. ATQ and its internal standard were detected over 1.3 min on an API 4000 mass analyzer using an electrospray ionization source operated in negative ionization and selected reaction monitoring modes. The method was validated in accordance with the Food and Drug Administration (FDA) Guidance for Industry: Bioanalytical Method Validation recommendations. RESULTS: Owing to pharmacokinetic parameters associated with ATQ, 2 calibration curves were generated to quantify the drug across a dynamic concentration range. Two standard curves were established ranging from 250 to 5000 ng/mL and 5000 to 50000 ng/mL, respectively. QC levels for both lower and higher concentration ranges prepared at low (750 ng/mL, 12000 ng/mL), mid (2000 ng/mL, 22500 ng/mL), and high (4250 ng/mL, 42500 ng/mL) concentrations yielded interassay precision ≤9.1% and accuracy ≤±9.4%. Dilutional, stability, and matrix effects studies were also performed, and results were within acceptability limits. CONCLUSIONS: This work describes the development and analytical evaluation of a UPLC-MS/MS method for ATQ quantification in plasma. The described method is sufficiently sensitive for ATQ quantification in plasma to support preclinical and clinical trials.

Development and validation of a liquid chromatographic-tandem mass spectrometric method for the multiplexed quantification of etravirine, maraviroc, raltegravir, and rilpivirine in human plasma and tissue.

BACKGROUND: Analytical methodologies for antiretroviral (ARV) quantification are important in determining both systemic and localized drug concentrations. The CCR5-antagonist maraviroc (MVC), the non-nucleoside reverse transcriptase inhibitors (NNRTIs) etravirine (ETV) and rilpivirine (RPV), as well as the integrase strand transfer inhibitor (INSTI) raltegravir (RAL), have all been evaluated using both oral and non-oral dosing regimens, demonstrating a need for dynamic and sensitive bioanalytical tools for drug quantification in plasma and tissue. METHODS: K2EDTA plasma or blank luminal tissue lysate were spiked with ETV, MVC, RAL, and RPV. Following the addition of isotopically-labeled internal standards and sample extraction via protein precipitation or solid phase extraction, respectively, samples were subjected to liquid chromatographic-tandem mass spectrometric (LC-MS/MS) analysis. Chromatographic separation was performed using a Waters BEH C8, 50×2.1mm, 1.7µm particle size column, and detected on an API 5000 mass analyzer operated in selective reaction monitoring mode. The method was validated according to FDA Bioanalytical Method Validation guidelines. RESULTS: Analytical methods were optimized for the multiplexed monitoring of ETV, MVC, RAL, and RPV in plasma and homogenized tissue lysate. The lower limits of quantification (LLOQs) for ETV, RAL, and RPV were 1ng/mL and the LLOQ for MVC was 0.1ng/mL in plasma; the LLOQs for all ARVs in homogenized tissue lysate was 0.05ng/sample. Standard curves were generated via weighted quadratic (plasma) or linear (tissue) regression of calibrators. Intra- and inter-assay precision and accuracy studies demonstrated %CVs≤15.93% and %DEVs ≤±13.52%, respectively. Stability and matrix effects studies, as well as external proficiency testing assessment, were also performed. All results were acceptable and in accordance with the guidelines recommended by the FDA, Guidance for Industry: Bioanalytical Method Validation document. CONCLUSIONS: LC-MS/MS assays that are sensitive, specific, and dynamic have been developed and validated for the multiplexed quantification of ETV, MVC, RAL, and RPV in plasma and homogenized tissue lysate. The described methods meet sufficient throughput criteria to support large research trials.

The ICN Leadership For Change™ Programme--20 years of growing influence.

AIM: The aim of this article was to present experiences from the field in the context of the International Council of Nurses' Leadership for Change™ programme, which celebrates 20 years of excellence in 2016 for developing the leadership and management capacity of nurses worldwide. BACKGROUND: The programme was launched in 1996 in order to boost nurse participation in the healthcare policy-making process, globally, and to foster within the nursing profession the requisite skills for nurses to lobby for and assume a greater responsibility in the leadership and management of health care services. INTRODUCTION: Over the course of two decades, the programme has been implemented in cooperation between ICN, national nurses associations, the World Health Organization, Ministries of Health and a variety of donor organizations such as the W.K. Kellogg Foundation and development agencies such as USAID and AUSAID. The programme has been implemented in more than 60 nations throughout Africa, Asia, Europe, the Middle East, Latin America and the Pacific Islands, to name a few regions. METHODS: This article offers an overview of the impact that certified ICN LFC nurse trainers and their colleagues have had in the United Arab Emirates, Vietnam and the United States of America and is affiliated islands and the North Pacific Islands. RESULTS: Twenty years of growth and empowerment are now the ongoing legacy of the ICN LFC Program, which has graduated and deployed nurse trainers around the world and achieved significant advances in the professional development of nurse leaders on an international scale. IMPLICATIONS FOR NURSING AND HEALTH POLICY: Nurse leaders can improve the health and well-being of their nations in collaboration with consumers and other key stakeholders. Nurse leaders are critical in improving health systems, their work places and broader societal challenges through sound nursing practice, education, research and evidence-based health and social policy change.

Improvements in the continuum of HIV care in an inner-city emergency department.

OBJECTIVE: The Johns Hopkins Hospital Emergency Department has served as a window on the HIV epidemic for 25 years, and as a pioneer in emergency department-based screening/linkage-to-care (LTC) programs. We document changes in the burden of HIV and HIV care metrics to the evolving HIV epidemic in inner-city Baltimore. DESIGN/METHODS: We analyzed seven serosurveys conducted on 18 ,144 adult Johns Hopkins Hospital Emergency Department patients between 1987 and 2013 as well as our HIV-screening/LTC program (2007, 2013) for trends in HIV prevalence, cross-sectional annual incidence estimates, undiagnosed HIV, LTC, antiretrovirals treatment, and viral suppression. RESULTS: HIV prevalence in 1987 was 5.2%, peaked at more than 11% from 1992 to 2003 and declined to 5.6% in 2013. Seroprevalence was highest for black men (initial 8.0%, peak 20.0%, last 9.9%) and lowest for white women. Among HIV-positive individuals, proportion of undiagnosed infection was 77% in 1987, 28% in 1992, and 12% by 2013 (P < 0.001). Cross-sectional annual HIV incidence estimates declined from 2.28% in 2001 to 0.16% in 2013. Thirty-day LTC improved from 32% (2007) to 72% (2013). In 2013, 80% of HIV-positive individuals had antiretrovirals ARVs detected in sera, markedly increased from 2007 (27%) (P < 0.001). Proportion of HIV-positive individuals with viral suppression (<400 copies/ml) increased from 23% (2001) to 59% (2013) (P < 0.001). CONCLUSION: Emergency department-based HIV testing has evolved from describing the local epidemic to a strategic interventional role, serving as a model for early HIV detection and LTC. Our contribution to community-based HIV-screening and LTC program parallels declines in undiagnosed HIV infection and incidence, and increases in antiretroviral use with associated viral suppression in the community.

Impact of mild traumatic brain injury on auditory brain stem dysfunction in mouse model.

The auditory brainstem response (ABR) is an electrophysiological test that examines the functionality of the auditory nerve and brainstem. Traumatic brain injury (TBI) can be detected if prolonged peak latency is observed in ABR measurements, since latency measures the neural conduction time in the brainstem, and an increase in latency can be a sign of pathological lesion at the auditory brainstem level. The ABR is elicited by brief sounds that can be used to measure hearing sensitivity as well as temporal processing. Reduction in peak amplitudes and increases in latency are indicative of dysfunction in the auditory nerve and/or central auditory pathways. In this study we used sixteen young adult mice that were divided into two groups: sham and mild traumatic brain injury (mTBI), with ABR measurements obtained prior to, and at 2, 6, and 14 weeks after injury. Abnormal ABRs were observed for the nine TBI cases as early as two weeks after injury and the deficits lasted for fourteen weeks after injury. Results indicated a significant reduction in the Peak 1 (P1) and Peak 4 (P4) amplitudes to the first noise burst, as well as an increase in latency response for P1 and P4 following mTBI. These results are the first to demonstrate auditory sound processing deficits in a rodent model of mild TBI.

Quantification of rifapentine, a potent antituberculosis drug, from dried blood spot samples using liquid chromatographic-tandem mass spectrometric analysis.

The quantification of antituberculosis drug concentrations in multinational trials currently requires the collection of modest blood volumes, centrifugation, aliquoting of plasma, freezing, and keeping samples frozen during shipping. We prospectively enrolled healthy individuals into the Tuberculosis Trials Consortium Study 29B, a phase I dose escalation study of rifapentine, a rifamycin under evaluation in tuberculosis treatment trials. We developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for quantifying rifapentine in whole blood on dried blood spots (DBS) to facilitate pharmacokinetic/pharmacodynamic analyses in clinical trials. Paired plasma and whole-blood samples were collected by venipuncture, and whole blood was spotted on Whatman protein saver 903 cards. The methods were optimized for plasma and then validated for DBS. The analytical measuring range for quantification of rifapentine and its metabolite was 50 to 80,000 ng/ml in whole-blood DBS. The analyte was stable on the cards for 11 weeks with a desiccant at room temperature and protected from light. The method concordance for paired plasma and whole-blood DBS samples was determined after correcting for participant hematocrit or population-based estimates of bias from Bland-Altman plots. The application of either correction factor resulted in acceptable correlation between plasma and whole-blood DBS (Passing-Bablok regression corrected for hematocrit; y = 0.98x + 356). Concentrations of rifapentine may be determined from whole-blood DBS collected via venipuncture after normalization in order to account for the dilutional effects of red blood cells. Additional studies are focused on the application of this methodology to capillary blood collected by finger stick. The simplicity of processing, storage, shipping, and low blood volume makes whole-blood DBS attractive for rifapentine pharmacokinetic evaluations, especially in international and pediatric trials.

Dual quantification of dapivirine and maraviroc in cervicovaginal secretions from ophthalmic tear strips and polyester-based swabs via liquid chromatographic-tandem mass spectrometric (LC-MS/MS) analysis.

BACKGROUND: Topical microbicidal agents are being actively pursued as a modality to prevent HIV viral transmission during sexual intercourse. Quantification of antiretroviral agents in specimen sources where antiviral activity is elicited is critical, and drug measurements in cervicovaginal fluid can provide key information on local drug concentrations. Two antiretroviral drugs, dapivirine and maraviroc, have gained interest as vaginal microbicidal agents, and rugged methods are required for their quantification in cervicovaginal secretions. METHODS: Cervicovaginal fluid spiked with dapivirine and maraviroc were applied to ophthalmic tear strips or polyester-based swabs to mimic collection procedures used in clinical studies. Following sample extraction and the addition of isotopically labeled internal standards, samples were subjected to liquid chromatographic-tandem mass spectrometric (LC-MS/MS) analysis using a Waters BEH C8, 50mm×2.1mm, 1.7µm particle size column, on an API 4000 mass analyzer operated in selective reaction monitoring mode. The method was validated according to FDA Bioanalytical Method Validation guidelines. RESULTS: Due to the disparate saturation capacity of the tested collection devices, the analytical measuring ranges for dapivirine and maravirocin cervicovaginal fluid on the ophthalmic tear strip were 0.05-25ng/tear strip, and 0.025-25ng/tear strip, respectively. As for the polyester-based swab, the analytical measuring ranges were 0.25-125ng/swab for dapivirine and 0.125-125ng/swab for maraviroc. Dilutional studies were performed for both analytes to extended ranges of 25,000ng/tear strip and 11,250ng/swab. Standard curves were generated via weighted (1/x(2)) linear or quadratic regression of calibrators. Precision, accuracy, stability and matrix effects studies were all performed and deemed acceptable according to the recommendations of the FDA Bioanalytical Method Validation guidelines. CONCLUSIONS: A rugged LC-MS/MS method for the dual quantification of dapivirine and maraviroc in cervicovaginal fluid using two unique collection devices has been developed and validated. The described method meets the criteria to support large research trials.

The development and validation of a method using high-resolution mass spectrometry (HRMS) for the qualitative detection of antiretroviral agents in human blood.

BACKGROUND: Antiretroviral drugs are used for the treatment and prevention of HIV infection. Non-adherence to antiretroviral drug regimens can compromise their clinical efficacy and lead to emergence of drug-resistant HIV. Clinical trials evaluating antiretroviral regimens for HIV treatment and prevention can also be compromised by poor adherence and non-disclosed off-study antiretroviral drug use. This report describes the development and validation of a high throughput, qualitative method for the identification of antiretroviral drugs using high-resolution mass spectrometry (HRMS) for the retrospective assessment of off-study antiretroviral drug use and the determination of potential antiretroviral therapy (ART) non-compliance. METHODS: Serum standards were prepared that contained 15 antiretroviral drugs: 9 protease inhibitors (PIs), 4 nucleotide/nucleoside reverse transcriptase inhibitors (NRTIs), and 2 non-nucleoside/nucleotide reverse transcriptase inhibitors (NNRTIs). Analytical separation was achieved on a Hypersil Gold PFP (100×3mm) column and the eluent was analyzed using the Thermo Exactive Orbitrap mass spectrometer (Exactive-MS) operated in full scan mode. Limit of identification, signal intensity precision, retention time analysis, selectivity, and carryover studies were conducted. Concordance with liquid chromatographic-tandem mass spectrometric (LC-MS/MS) methods was evaluated using remnant plasma samples from a clinical trial. RESULTS: The limit of identification ranged from 5 to 10ng/ml for 14 drugs (9 PIs, 1 NNRTI, 4 NRTIs) and was 150ng/ml for 1 NNRTI. Precision studies with high and low control mixtures revealed signal intensity coefficients of variation of 3.0-27.5%. The Exactive-MS method was selective for the compounds of interest. Overall, concordance ranged from 89.1% to 100% for the screening of antiretroviral drugs in clinical plasma specimens as compared to LC-MS/MS methods. CONCLUSION: Using the Exactive-MS, we developed and validated a highly selective, robust method for the multiplexed detection of 15 antiretroviral drugs.

Preliminary study of quinine pharmacokinetics in pregnant women with malaria-HIV co-infection.

Pregnant women bear the greatest burden of malaria-human immunodeficiency virus co-infection. Previous studies suggest that interaction with antiretroviral drugs may compromise antimalarial pharmacokinetics and treatment outcomes. We conducted a preliminary clinical study to assess quinine pharmacokinetics in Malian pregnant women with acute malaria who reported taking nevirapine-based antiretroviral therapy. Of seven women, six had stable concentrations of nevirapine in the plasma and one had none. Quinine concentrations were lower, and its metabolite 3-hydroxyquinine higher, in the six women with nevirapine than in the one without, and quinine concentrations were below the recommended therapeutic range in 50% of the women. This preliminary observation warrants further research to understand the impact of long-term antiretroviral therapy on the treatment of acute malaria.

Pharmacokinetics and safety of tenofovir in HIV-infected women during labor and their infants during the first week of life.

BACKGROUND: Data describing the pharmacokinetics and safety of tenofovir in neonates are lacking. METHODS: The HIV Prevention Trials Network 057 protocol was a phase 1, open-label study of the pharmacokinetics and safety of tenofovir disoproxil fumarate (TDF) in HIV-infected women during labor and their infants during the first week of life with 4 dosing cohorts: maternal 600 mg doses/no infant dosing; no maternal dosing/infant 4 mg/kg doses on days 0, 3, and 5; maternal 900 mg doses/infant 6 mg/kg doses on days 0, 3, and 5; maternal 600 mg doses/infant 6 mg/kg daily for 7 doses. Pharmacokinetic sampling was performed on cohort 1 and 3 mothers and all infants. Plasma, amniotic fluid, and breast milk tenofovir concentrations were determined by liquid chromatographic-tandem mass spectrometric assay. The pharmacokinetic target was for infant tenofovir concentration throughout the first week of life to exceed 50 ng/mL, the median trough tenofovir concentration in adults receiving standard chronic TDF dosing. RESULTS: One hundred twenty-two mother-infant pairs from Malawi and Brazil were studied. Tenofovir exposure in mothers receiving 600 and 900 mg exceeded that in nonpregnant adults receiving standard 300 mg doses. Tenofovir elimination in the infants was equivalent to that in older children and adults, and trough tenofovir plasma concentrations exceeded 50 ng/mL in 74%-97% of infants receiving daily dosing. CONCLUSIONS: A TDF dosing regimen of 600 mg during labor and daily infant doses of 6 mg/kg maintains infant tenofovir plasma concentration above 50 ng/mL throughout the first week of life and should be used in the studies of TDF efficacy for HIV prevention of mother-to-child transmission and early infant treatment.

Understanding collision-induced dissociation of dofetilide: a case study in the application of density functional theory as an aid to mass spectral interpretation.

Fragmentation of molecules under collision-induced dissociation (CID) conditions is not well-understood. This may make interpretation of MSMS spectra difficult and limit the effectiveness of software tools intended to aid mass spectral interpretation. Density Functional Theory (DFT) has been successfully applied to explain the thermodynamics of fragmentation in the gas phase by the modelling the effect that protonation has on the bond lengths (and hence bond strengths). In this study, dofetilide and four methylated analogues were used to investigate further the potential for using DFT to understand and predict the CID fragmentation routes. The products ions present in the CID spectra of all five compounds were consistent with charge-directed fragmentation, with protonation adjacent to the cleavage site being required to initiate fragmentation. Protonation at the dissociative site may have occurred either directly or via proton migration. A correlation was observed between protonation-induced bond lengthening and the bonds which were observed to break in the CID spectra. This correlation was quantitative in that the bonds calculated to elongate to the greatest extent gave rise to the most abundant of the major product ions. Thus such quantum calculations may offer the potential for contributing to a predictive tool for aiding the accuracy and speed mass spectral interpretation by generating numerical data in the form of bond length increases to act as descriptors flagging potential bond cleavages.