The development and validation of a novel LC-MS/MS method for the quantification of cenicriviroc in human plasma and cerebrospinal fluid

A high performance liquid chromatography tandem mass spectrometric method was developed and validated cenicriviroc quantification in human plasma and cerebrospinal fluid. The method involved precipitation with acetonitrile and injecting supernatants onto the column. Separation was achieved on an XBridge C18 column with a gradient elution of 0.1% formic acid in water and acetonitrile. Analyte detection was conducted in positive ion mode using SRM. The m/z transitions were: CVC (697.3→574.3) and CVC-d7 (704.4→574.3). Calibration curve ranged from 5-1000 ng/ml for plasma and 0.241-15.0 ng/ml for CSF. The intra and inter day precision and accuracy were 90%. The method was utilised for the measurement of patients' plasma and CSF samples taking a dose of 50, 150 and 300mg qd

Pharmacodynamic modeling of bacillary elimination rates and detection of bacterial lipid bodies in sputum to predict and understand outcomes in treatment of pulmonary tuberculosis

This work was supported by a Wellcome Trust Clinical PhD Fellowship (086757/Z/08/A to D. J. S.), the Malawi Liverpool Wellcome Trust Core grant, and Medical Research Council (grant number G0300403 to M. R. B.).Background. Antibiotic-tolerant bacterial persistence prevents treatment shortening in drug-susceptible tuberculosis, and accumulation of intracellular lipid bodies has been proposed to identify a persister phenotype of Mycobacterium tuberculosis cells. In Malawi, we modeled bacillary elimination rates (BERs) from sputum cultures and calculated the percentage of lipid body-positive acid-fast bacilli (%LB + AFB) on sputum smears. We assessed whether these putative measurements of persistence predict unfavorable outcomes (treatment failure/relapse). Methods. Adults with pulmonary tuberculosis received standard 6-month therapy. Sputum samples were collected during the first 8 weeks for serial sputum colony counting (SSCC) on agar and time-to positivity (TTP) measurement in mycobacterial growth indicator tubes. BERs were extracted from nonlinear and linear mixed-effects models, respectively, fitted to these datasets. The %LB + AFB counts were assessed by fluorescence microscopy. Patients were followed until 1 year posttreatment. Individual BERs and %LB + AFB counts were related to final outcomes. Results. One hundred and thirty-three patients (56% HIV coinfected) participated, and 15 unfavorable outcomes were reported. These were inversely associated with faster sterilization phase bacillary elimination from the SSCC model (odds ratio [OR], 0.39; 95% confidence interval [CI], .22-.70) and a faster BER from the TTP model (OR, 0.71; 95% CI, .55-.94). Higher %LB + AFB counts on day 21-28 were recorded in patients who suffered unfavorable final outcomes compared with those who achieved stable cure (P = .008). Conclusions. Modeling BERs predicts final outcome, and high %LB + AFB counts 3-4 weeks into therapy may identify a persister bacterial phenotype. These methods deserve further evaluation as surrogate endpoints for clinical trials.Publisher PDFPeer reviewe

Pharmacokinetics of dolutegravir with and without darunavir/cobicistat in healthy volunteers (vol 74, pg 149, 2019)

Background:Dolutegravir combined with darunavir/cobicistat is a promising NRTI-sparing and/or salvage strategy for the treatment of HIV-1 infection. Methods:This Phase I, open-label, 57 day, crossover, pharmacokinetic (PK) study, enrolled healthy volunteers aged 18-65 years, who were randomized to one of two groups. Group 1 received dolutegravir (50 mg) once daily for 14 days followed by a 7 day washout, then a 14 day dolutegravir/darunavir/cobicistat (DTG/DRV/COBI) once-daily co-administration period followed by a 7 day washout and finally a 14 day period of darunavir/cobicistat (800/150 mg) once daily. Group 2 followed the same sequence starting with darunavir/cobicistat and concluding with dolutegravir. Each group underwent intensive PK sampling over 24 h on day 14 of each drug period and DTG/DRV/COBI concentrations were measured using validated LC-MS/MS methods. Results:Twenty participants completed all PK phases. Thirteen were female and median age and BMI were 33.5 years and 27 kg/m2. Dolutegravir geometric mean ratios (GMR, DTG/DRV/COBI versus dolutegravir alone) and 90% CI for Cmax, AUC0-24 and C24 were 1.01 (0.92-1.11), 0.95 (0.87-1.04) and 0.9 (0.8-1.0), respectively. Darunavir GMR (DRV/COBI/DTG versus darunavir/cobicistat alone) and 90% CI for Cmax, AUC0-24 and C24 were 0.90 (0.83-0.98), 0.93 (0.86-1.00) and 0.93 (0.78-1.11), respectively. No grade 3 or 4 adverse events or laboratory abnormalities were observed. Conclusions:Concentrations of dolutegravir and darunavir, when boosted with cobicistat, decreased by <10% during co-administration, suggesting this combination can be prescribed safely in the treatment of HIV-1, with no adjustment to current guideline-recommended dosages

A novel LC-MS/MS method for the determination of favipiravir ribofuranosyl-5'-triphosphate (T-705-RTP) in human peripheral mononuclear cells.

Favipiravir is a broad-spectrum antiviral that is metabolised intracellularly into the active form, favipiravir ribofuranosyl-5'-triphosphate (F-RTP). Measurement of the intracellular concentration of F-RTP in mononuclear cells is a crucial step to characterising the pharmacokinetics of F-RTP and to enable more appropriate dose selection for the treatment of COVID-19 and emerging infectious diseases. The described method was validated over the range 24 - 2280 pmol/sample. Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood and lysed using methanol-water (70:30, v/v) before cellular components were precipitated with acetonitrile and the supernatant further cleaned by weak anion exchange solid phase extraction. The method was found to be both precise and accurate and was successfully utilised to analyse F-RTP concentrations in patient samples collected as part of the AGILE CST-6 clinical trial

Development and validation of an LC-MS/MS method for quantification of favipiravir in human plasma

Favipiravir (FVP) is a broad-spectrum antiviral that selectively inhibits viral RNA-dependent RNA polymerase, first trialled for the treatment of influenza infection. It has been shown to be effective against a number of RNA virus families including arenaviruses, flaviviruses and enteroviruses. Most recently, FVP has been investigated as a potential therapeutic for severe acute respiratory syndrome coronavirus 2 infection. A liquid chromatography tandem mass spectrometry method for the quantification of FVP in human plasma has been developed and validated for use in clinical trials investigating favipiravir as treatment for coronavirus disease-2019. Samples were extracted by protein precipitation using acetonitrile, using C, N- Favipiravir as internal standard. Elution was performed on a Synergi Polar-RP 150 × 2.1 mm 4 µm column using a gradient mobile phase programme consisting of 0.2% formic acid in water and 0.2% formic acid in methanol. The assay was validated over the range 500-50,000 ng/mL; this method was found to be precise and accurate and recovery of FVP from the matrix was high. Stability experiments confirmed and expanded on the known stability of FVP, including under heat treatment and for a period of 10 months at - 80 °C

High intrapulmonary rifampicin and isoniazid concentrations are associated with rapid sputum bacillary clearance in patients with pulmonary tuberculosis

This work was supported by a Wellcome Trust Clinical PhD Fellowship [grant number 105392/B/14/Z to A.D.M. and L69AGB to JM]. ELC was supported by Wellcome [200901/Z/16/Z]. The Malawi-Liverpool-Wellcome Clinical Research Programme is supported by a strategic award from the Wellcome Trust [206545/Z/17/Z]. We also acknowledge infrastructural support for bioanalysis from the Liverpool Biomedical Research Centre funded by Liverpool Health Partners.Background Intrapulmonary pharmacokinetics may better explain response to tuberculosis (TB) treatment than plasma pharmacokinetics. We explored these relationships by modelling bacillary clearance in sputum in adult patients on first-line treatment in Malawi. Methods Bacillary elimination rates (BER) were estimated using linear mixed-effects modelling of serial time-to-positivity in mycobacterial growth indicator tubes for sputum collected during the intensive phase of treatment (weeks 0 to 8) for microbiologically confirmed TB. Population pharmacokinetic models used plasma and intrapulmonary drug levels at 8 and 16 weeks. Pharmacokinetic-pharmacodynamic relationships were investigated using individual-level measures of drug exposure (AUC and Cmax) for rifampicin, isoniazid, pyrazinamide, and ethambutol, in plasma, epithelial lining fluid, and alveolar cells as covariates in the bacillary elimination models. Results Among 157 participants (58% HIV co-infected), drug exposure in plasma or alveolar cells was not associated with sputum bacillary clearance. Higher peak concentrations (Cmax) or exposure (AUC) to rifampicin or isoniazid in epithelial lining fluid was associated with more rapid bacillary elimination and shorter time to sputum negativity. More extensive disease on baseline chest radiograph was associated with slower bacillary elimination. Clinical outcome was captured in 133 participants, with 15 (11%) unfavourable outcomes recorded (recurrent TB, failed treatment, or death). No relationship between BER and late clinical outcome was identified. Conclusions Greater intrapulmonary drug exposure to rifampicin or isoniazid in the epithelial lining fluid was associated with more rapid bacillary clearance. Higher doses of rifampicin and isoniazid may result in sustained high intrapulmonary drug exposure, rapid bacillary clearance, shorter treatment duration and better treatment outcomes.Publisher PDFPeer reviewe

Host Immune Transcriptional Profiles Reflect the Variability in Clinical Disease Manifestations in Patients with Staphylococcus aureus Infections

Staphylococcus aureus infections are associated with diverse clinical manifestations leading to significant morbidity and mortality. To define the role of the host response in the clinical manifestations of the disease, we characterized whole blood transcriptional profiles of children hospitalized with community-acquired S. aureus infection and phenotyped the bacterial strains isolated. The overall transcriptional response to S. aureus infection was characterized by over-expression of innate immunity and hematopoiesis related genes and under-expression of genes related to adaptive immunity. We assessed individual profiles using modular fingerprints combined with the molecular distance to health (MDTH), a numerical score of transcriptional perturbation as compared to healthy controls. We observed significant heterogeneity in the host signatures and MDTH, as they were influenced by the type of clinical presentation, the extent of bacterial dissemination, and time of blood sampling in the course of the infection, but not by the bacterial isolate. System analysis approaches provide a new understanding of disease pathogenesis and the relation/interaction between host response and clinical disease manifestations

Platform adaptive trial of novel antivirals for early treatment of COVID-19 In the community (PANORAMIC): protocol for a randomised, controlled, open-label, adaptive platform trial of community novel antiviral treatment of COVID-19 in people at increased risk of more severe disease

Introduction: There is an urgent need to determine the safety, effectiveness and cost-effectiveness of novel antiviral treatments for COVID-19 in vaccinated patients in the community at increased risk of morbidity and mortality from COVID-19. // Methods and analysis: PANORAMIC is a UK-wide, open-label, prospective, adaptive, multiarm platform, randomised clinical trial that evaluates antiviral treatments for COVID-19 in the community. A master protocol governs the addition of new antiviral treatments as they become available, and the introduction and cessation of existing interventions via interim analyses. The first two interventions to be evaluated are molnupiravir (Lagevrio) and nirmatrelvir/ritonavir (Paxlovid). Eligibility criteria: community-dwelling within 5 days of onset of symptomatic COVID-19 (confirmed by PCR or lateral flow test), and either (1) aged 50 years and over, or (2) aged 18–49 years with qualifying comorbidities. Registration occurs via the trial website and by telephone. Recruitment occurs remotely through the central trial team, or in person through clinical sites. Participants are randomised to receive either usual care or a trial drug plus usual care. Outcomes are collected via a participant-completed daily electronic symptom diary for 28 days post randomisation. Participants and/or their Trial Partner are contacted by the research team after days 7, 14 and 28 if the diary is not completed, or if the participant is unable to access the diary. The primary efficacy endpoint is all-cause, non-elective hospitalisation and/or death within 28 days of randomisation. Multiple prespecified interim analyses allow interventions to be stopped for futility or superiority based on prespecified decision criteria. A prospective economic evaluation is embedded within the trial. // Ethics and dissemination: Ethical approval granted by South Central–Berkshire REC number: 21/SC/0393; IRAS project ID: 1004274. Results will be presented to policymakers and at conferences, and published in peer-reviewed journals. // Trial registration number: ISRCTN30448031; EudraCT number: 2021-005748-31

Molnupiravir Plus Usual Care Versus Usual Care Alone as Early Treatment for Adults with COVID-19 at Increased Risk of Adverse Outcomes (PANORAMIC): Preliminary Analysis from the United Kingdom Randomised, Controlled Open-Label, Platform Adaptive Trial

Background: The safety, effectiveness and cost-effectiveness of molnupiravir, an oral antiviral medication for SARS-CoV-2, in patients in the community who are multiply-vaccinated and at increased risk of morbidity and mortality from COVID-19, has not been established. We aimed to determine whether molnupiravir added to usual care reduced hospital admissions/deaths among people at higher risk from COVID-19, and here report our preliminary analyses. Methods: Participants in this UK multicentre, open-label, adaptive, multi-arm, platform, randomised controlled trial were aged ≥50, or ≥18 years with comorbidities, and unwell ≤5 days with confirmed COVID-19 in the community, and were randomised to usual care or usual care plus molnupiravir (800mg twice daily for 5 days). The primary outcome measure was all-cause hospitalisation/death within 28 days, analysed using Bayesian models. The main secondary outcome measure was time to first self-reported recovery. A sub-set of participants in each group were assessed for the virology primary outcome measure of day seven SARS-CoV-2 viral load. Trial registration: ISRCTN30448031 Findings: Between December 8, 2021 and April 27, 2022, 25783 participants were randomised to molnupiravir plus usual care (n=12821) or usual care alone (n=12962). Mean (range) age of participants was 56·6 years (18 to 99), 58·6% were female, and 99% had at least one dose of a SARS-CoV-2 vaccine. The median duration of symptoms prior to randomisation was two days (IQR 1 – 3), the median number of days from symptom onset to starting to take the medication was three days (IQR 3 – 4), 87% (11109/11997) received their medication within five days of symptom onset, and 95·4% (n=11857) of participants randomised to molnupiravir reported taking molnupiravir for five days. Primary outcome measure data were available in 25000 (97%) participants and included in this analysis. 103/12516 (0·8%) hospitalisations/deaths occurred in the molnupiravir group versus 96/12484 (0·8%) in usual care alone with a posterior probability of superiority of 0·34 (adjusted odds ratio 1·061 (95% Bayesian credible interval [BCI]) 0·80 to 1·40). Estimates were similar for all subgroups. The observed median (IQR) time-to-first-recovery from randomisation was 9 (5–23) days in molnupiravir and 15 (7–not reached) days in usual care. There was an estimated benefit of 4·2 (95% BCI: 3·8 – 4·6) days in time-to-first-recovery (TTR) giving a posterior probability of superiority of >0·999 (estimated median TTR 10·3 [10·2 – 10·6] days vs 14·5 [14·2 – 14·9] days respectively; hazard ratio [95% BCI], 1·36 [1·3–1·4] days), which met the pre-specified superiority threshold. On day 7, SARS-CoV-2 virus was below detection levels in 7/34 (21%) of the molnupiravir group, versus 1/39 (3%) in the usual care group (p=0.039), and mean viral load was lower in the molnupiravir group compared with those receiving usual care [(SD) of log10(viral load) 3·82 (1·40) in the molnupiravir group and 4.93 (1·38) in the usual care group, (P<0·001)]. 59 (0·4%) participants experienced serious adverse events in the molnupiravir group and 52 (0·4%) in usual care. Interpretation: In this preliminary analysis, we found that molnupiravir did not reduce already low hospitalisations/deaths among higher risk, vaccinated adults with COVID-19 in the community, but resulted in faster time to recovery, and reduced viral detection and load. Funding: This project is funded by the NIHR (NIHR135366). The views expressed are those of the authors and not necessarily those of the NIHR or the Department of Health and Social Care