Interpatient variability in the pharmacokinetics of remdesivir and its main metabolite GS-441524 in treated COVID-19 subjects.

BACKGROUND: Remdesivir is the first antiviral drug against SARS-CoV-2 approved for use in COVID-19 patients. OBJECTIVES: To study the pharmacokinetic inter-individual variability of remdesivir and its main metabolite GS-441524 in a real-world setting of COVID-19 inpatients and to identify possible associations with different demographic/biochemical variables. METHODS: Inpatients affected by SARS-CoV-2 infections, undergoing standard-dose remdesivir treatment, were prospectively enrolled. Blood samples were collected on day 4, immediately after (C0) and at 1 h (C1) and 24 h (C24) after infusion. Remdesivir and GS-441524 concentrations were measured using a validated UHPLC-MS/MS method and the AUC0-24 was calculated. At baseline, COVID-19 severity (ICU or no ICU), sex, age, BMI and renal and liver functions were assessed. Transaminases and estimated glomerular filtration rate (e-GFR) were also evaluated during treatment. Linear regression, logistic regression and multiple linear regression tests were used for statistical comparisons of pharmacokinetic parameters and variables. RESULTS: Eighty-five patients were included. The mean (CV%) values of remdesivir were: C0 2091 (99.1%) ng/mL, C1 139.7 (272.4%) ng/mL and AUC0-24 2791 (175.7%) ng·h/mL. The mean (CV%) values of GS-441524 were: C0 90.2 (49.5%) ng/mL, C1 104.9 (46.6%) ng/mL, C24 58.4 (66.9) ng/mL and AUC0-24 1976 (52.6%) ng·h/mL. The multiple regression analysis showed that age (P < 0.05) and e-GFR (P < 0.01) were independent predictors of GS-441524 plasma exposure. CONCLUSIONS: Our results showed a high interpatient variability of remdesivir and GS-441524 likely due to both age and renal function in COVID-19 inpatients. Further research is required to understand whether the pharmacokinetics of remdesivir and its metabolites may influence drug-related efficacy or toxic effect.

Pharmacokinetics of remdesivir and GS-441524 in two critically ill patients who recovered from COVID-19.

BACKGROUND: Remdesivir is a prodrug of the nucleoside analogue GS-441524 and is under evaluation for treatment of SARS-CoV-2-infected patients. OBJECTIVES: To evaluate the pharmacokinetics of remdesivir and GS-441524 in plasma, bronchoalveolar aspirate (BAS) and CSF in two critically ill COVID-19 patients. METHODS: Remdesivir was administered at 200 mg loading dose on the first day followed by 12 days of 100 mg in two critically ill patients. Blood samples were collected immediately after (C0) and at 1 (C1) and 24 h (C24) after intravenous administration on day 3 until day 9. BAS samples were collected on Days 4, 7 and 9 from both patients while one CSF on Day 7 was obtained in one patient. Remdesivir and GS-441524 concentrations were measured in these samples using a validated UHPLC-MS/MS method. RESULTS: We observed higher concentrations of remdesivir at C0 (6- to 7-fold higher than EC50 from in vitro studies) and a notable decay at C1. GS-441524 plasma concentrations reached a peak at C1 and persisted until the next administration. Higher concentrations of GS-441524 were observed in the patient with mild renal dysfunction. Mean BAS/plasma concentration ratios of GS-441524 were 2.3% and 6.4% in Patient 1 and Patient 2, respectively. The CSF concentration found in Patient 2 was 25.7% with respect to plasma. GS-441524 levels in lung and CNS suggest compartmental differences in drug exposure. CONCLUSIONS: We report the first pharmacokinetic evaluation of remdesivir and GS-441524 in recovered COVID-19 patients. Further study of the pharmacokinetic profile of remdesivir, GS-441524 and the intracellular triphosphate form are required.

Plasma trough concentrations of antiretrovirals in HIV-infected persons treated with direct-acting antiviral agents for hepatitis C in the real world.

BACKGROUND: Possible drug-drug interactions (DDIs) between antiretrovirals (ARVs) and direct-acting antiviral agents (DAAs) are of some concern. OBJECTIVES: To investigate ARV plasma trough concentrations (Ctrough) before and during DAAs in patients treated in the real world. METHODS: Single-centre, prospective, observational study including HIV/HCV coinfected persons undergoing DAA treatment. Self-reported adherence was assessed and ARVs Ctrough measured by HPLC-UV. Blood samples were collected before and after 2 months of DAA treatment. RESULTS: One-hundred and thirty-seven patients were included: 21.2% treated with ombitasvir/paritaprevir/ritonavir ± dasabuvir (2D/3D) and 78.8% with sofosbuvir-based regimens. Suboptimal Ctrough before and during DAA was found, respectively, in 3 (10.3%) and 3 (10.3%) cases treated with 2D/3D, and 16 (14.8%) and 11 (10.2%) with sofosbuvir-based regimens, even if self-reported ARV adherence was always ≥93%. In 2D/3D-treated patients, median darunavir Ctrough during DAAs was significantly lower than observed before DAAs [1125 ng/mL (IQR, 810-1616) versus 1903 ng/mL (IQR 1387-3983), respectively] (n = 5; P = 0.009), with a 40.9% decrease. In the same group, no differences in atazanavir or raltegravir concentrations were found. In patients treated with sofosbuvir-based regimens, Ctrough of all ARVs were similar before and during DAAs. CONCLUSIONS: In the real world of HIV/HCV coinfected patients, ARV plasma concentrations during DAAs were generally not different from those found before anti-HCV treatment. Although assessed in a small number of patients, darunavir concentrations during 2D/3D showed a significant reduction when compared with those found before DAAs. ARV plasma concentrations measurement during anti-HCV treatment may give useful information for managing HIV/HCV coinfected persons receiving treatment for both infections.

Development and validation of an HPLC-UV method for quantification of elvitegravir and two other new antiretrovirals, dolutegravir and rilpivirine, in the plasma of HIV-positive patients.

Therapeutic drug monitoring may be crucial in selected clinical conditions for the management of HIV infection. In recent years, new antiretrovirals have been introduced and in particular elvitegravir (EVG) is now recommended for first-line and simplification treatment as well as dolutegravir (DTG) and rilpivirine (RPV). The aim of this study was to develop and validate a high-performance liquid chromatography-ultraviolet (HPLC-UV) method for determining EVG and new antiretrovirals DTG and RPV in human plasma. Solid-phase extraction was applied to a 600 µL plasma sample. Chromatographic separation of the three drugs and internal standard was achieved with a gradient of acetonitrile and phosphate buffer on a C18 reverse-phase analytical column with a 20 min analytical run time. EVG and DTG were detected at 265 nm and RPV at 290 nm. Mean intra- and inter-day precisions were < 10%; the mean accuracy was <15%. Extraction recovery ranged between 105 and 82% for the drugs analyzed. Calibration curves were optimized according to the expected ranges of drug concentrations in patients; the coefficient of determination was >0.997 for all drugs. This method allows for monitoring EVG, DTG and RPV in the plasma of HIV-positive patients using HPLC-UV.

Diagnostic performances of clinical laboratory tests using Triton X-100 to reduce the biohazard associated with routine testing of Ebola virus-infected patients.

BACKGROUND: Ebola virus, an enveloped virus, is the cause of the largest and most complex Ebola virus disease (EVD) outbreak in West Africa. Blood or body fluids of an infected person may represent a biohazard to laboratory workers. Laboratory tests of virus containing specimens should be conducted in referral centres at biosafety level 4, but based on the severity of clinical symptoms, basic laboratories might be required to execute urgent tests for patients suspected of EVD. The aim of this work was to compare the analytical performances of laboratory tests when Triton X-100, a chemical agent able to inactivate other enveloped viruses, was added to specimens. METHODS: Results of clinical chemistry, coagulation and haematology parameters on samples before and after the addition of 0.1% (final concentration) of Triton X-100 and 1 h of incubation at room temperature were compared. RESULTS: Overall, results showed very good agreement by all statistical analyses. Triton X-100 at 0.1% did not significantly affect the results for the majority of the analytes tested. CONCLUSIONS: Triton X-100 at 0.1% can be used to reduce the biohazard in performing laboratory tests on samples from patients with EVD without affecting clinical decisions.

Low-density lipoprotein and ritonavir: an interaction between antiretrovirals and lipids mediated by P-glycoprotein.

BACKGROUND: Antiretroviral therapy has considerably reduced HIV disease progression, but complete eradication of HIV cannot actually be achieved. Moreover, prolonged use of protease inhibitors (PIs) causes profound changes in lipid metabolism with an increased risk of cardiovascular diseases. P-glycoprotein (P-gp) is expressed on many cell types, playing an important role in the efflux of drugs including PIs, limiting their intracellular concentration. Furthermore, several studies showed that P-gp is involved in lipid homeostasis and its activity is regulated by cholesterol. METHODS: THP-1 monocytes were used to study: (i) the influence of low-density lipoprotein (LDL) on P-gp expression and function, assessed by flow cytometry and quantitative real-time PCR analysis and measuring ritonavir and rhodamine-123 dye efflux, respectively; and (ii) the influence of ritonavir on cholesterol mobilization. The intracellular levels of ritonavir or cholesterol were measured by HPLC-UV and filipin staining, respectively. RESULTS: In THP-1 cells, LDL was able to yield an increase in both P-gp expression and activity. THP-1 cells treated with LDL showed a decrease in the intracellular ritonavir concentration in a dose-dependent manner. Notably, ritonavir induced reduced cholesterol mobilization in THP-1 cells, probably due to its inhibitory action on P-gp activity. CONCLUSIONS: Our data indicate a potential interplay between LDL and ritonavir mediated by P-gp. This interaction could influence both therapy effectiveness and cellular lipid metabolism, with important implications in the management of HIV patients treated with boosted PIs.

Analysis of single-nucleotide polymorphisms (SNPs) in human CYP3A4 and CYP3A5 genes: potential implications for the metabolism of HIV drugs.

BACKGROUND: Drug metabolism via the cytochrome P450 (CYP450) system has emerged as an important determinant in the occurrence of several drug interactions (adverse drug reactions, reduced pharmacological effect, drug toxicities). In particular, CYP3A4 and CYP3A5 (interacting with more than 60% of licensed drugs) exhibit the most individual variations of gene expression, mostly caused by single nucleotide polymorphisms (SNPs) within the regulatory region of the CYP3A4 and CYP3A5 genes which might affect the level of enzyme production.In this study, we sought to improve the performance of sensitive screening for CYP3A polymorphism detection in twenty HIV-1 infected patients undergoing lopinavir/ritonavir (LPV/r) monotherapy. METHODS: The study was performed by an effective, easy and inexpensive home-made Polymerase Chain Reaction Direct Sequencing approach for analyzing CYP3A4 and CYP3A5 genes which can detect both reported and unreported genetic variants potentially associated with altered or decreased functions of CYP3A4 and CYP3A5 proteins. Proportions and tests of association were used. RESULTS: Among the genetic variants considered, CYP3A4*1B (expression of altered function) was only found in 3 patients (15%) and CYP3A5*3 (expression of splicing defect) in 3 other patients (15%). CYP3A5*3 did not appear to be associated with decreased efficacy of LPV/r in any patient, since none of the patients carrying this variant showed virological rebound during LPV/r treatment or low levels of TDM. In contrast, low-level virological rebound was observed in one patient and a low TDM level was found in another; both were carrying CYP3A4*1B. CONCLUSIONS: Our method exhibited an overall efficiency of 100% (DNA amplification and sequencing in our group of patients). This may contribute to producing innovative results for better understanding the inter-genotypic variability in gene coding for CYP3A, and investigating SNPs as biological markers of individual response to drugs requiring metabolism via the cytochrome P450 system.

Pharmacokinetics of tecovirimat in subjects with Mpox.

OBJECTIVE: To investigate the pharmacokinetics (PK) of tecovirimat in subjects with Mpox. METHODS: This monocentric, prospective, observational study enrolled subjects with Mpox who received standard treatment with oral tecovirimat. Plasma samples for PK assessment were collected at steady state (5-8 days after initiation of antiviral therapy), before and 3, 5, 7 and 12 h after tecovirimat administration. Drug concentrations were determined by validated liquid chromatography coupled with tandem mass spectrometry. PK parameters were calculated using Phoenix 8.1. RESULTS: Overall, 14 male patients hospitalized for severe Mpox with ongoing tecovirimat treatment were enrolled in this study. Six of the 14 patients were living with human immunodeficiency virus (HIV), all of whom were on antiretroviral therapy (ART) and virologically suppressed at the time of hospitalization. Significant differences in tecovirimat PK were observed in subjects without HIV compared with subjects with HIV. In subjects with HIV, the maximum tecovirimat plasma concentration (39%, P≤0.0001), minimum tecovirimat plasma concentration (42%, P=0.0079) and area under the curve from zero to the last measured time-point (40%, P≤0.0001) were significantly lower compared with subjects without HIV, but all concentrations remained above the in-vitro calculated 90% inhibitory concentration. No significant associations were found between demographic/clinical data and tecovirimat PK. All patients recovered completely within 14 (range 6-36) days of treatment initiation. CONCLUSIONS: This study found a significant decrease in plasma exposure of tecovirimat in Mpox patients with HIV on effective ART compared with those without HIV, with no evident impact on clinical outcomes. Although these results need to be confirmed in larger studies, they may provide useful information on the PK of tecovirimat.

Mpox as AIDS-defining event with a severe and protracted course: clinical, immunological, and virological implications.

A 59-year-old treatment-naive patient with advanced HIV infection presented with a severe and protracted course of mpox (formerly known as monkeypox) that did not respond to the current mpox treatment options. The patient worsened clinically, and developed new mucocutaneous lesions and necrotic evolution of pre-existing ones, along with multiple bilateral lung nodules and the appearance of a tracheal necrotic lesion. Although severe forms of mpox have been observed in people with severe immune system deficiency, including those with advanced HIV presentation, the immunological mechanisms underlying this observation have not yet been fully explained. To our knowledge, this is the first account of a necrotising mpox in a person living with HIV, with viral shedding for more than 11 months and a comprehensive immunological description. Moreover, we documented the virus' persistence by detecting mpox virus DNA from multiple sites and quantified anti-monkeypox virus IgA, IgM, IgG, and neutralising antibodies in serum samples. The severe HIV-driven immune depression and the presence of other co-infections might skew and impair immune responses, thus contributing to the persistence of monkeypox virus infection. Further investigations of immune responses to monkeypox virus infection in people with severe immunosuppression are required to improve management and prevention.

Rapid ART initiation with bictegravir/emtricitabine/tenofovir alafenamide in individuals presenting with advanced HIV disease (Rainbow study).

BACKGROUND: A rapid ART initiation approach can be beneficial in people with advanced HIV disease, in consideration of their high morbidity and mortality. The aim of our study was to evaluate the feasibility, efficacy and safety of rapid ART start with BIC/FTC/TAF in this setting. METHODS: Pilot, single-centre, single-arm, prospective, phase IV clinical trial conducted in a tertiary Italian hospital. Thirty ART-naïve people presenting with advanced HIV-1 diagnosis (defined as the presence of an AIDS-defining event and/or CD4 cell count <200 µL), were enrolled. Main exclusion criteria were active tuberculosis, cryptococcosis and pregnant/breastfeeding women. BIC/FTC/TAF was started within 7 days from HIV diagnosis. The primary endpoint was clinical or virologic failure (VF). Immunological parameters, safety, feasibility, neurocognitive performances and patient-reported outcomes were assessed as well. RESULTS: Over the study period, 40 (34%) of 116 patients diagnosed with HIV infection at INMI Spallanzani had advanced disease, of whom 30 (26%) were enrolled. The proportion of participants with HIV-RNA <50 cp/mL was 9/30 (30%) at week (w) 4, 19/30 (63%) at w12, 24/30 (80%) at w24, 23/30 (77%) at w36 and 27/30 (90%) at w48. Two unconfirmed VF occurred. No ART discontinuation due to toxicity or VF was observed. No ART modification was performed based on the review of genotype and no mutations for the study drugs were detected. Mean CD4 cells count changed by 133 cells/µL at BL to 309 cells/µL at w 48 and 83% of participants had a CD4 > 200 cells/µL at w 48. Two participants developed IRIS and one was diagnosed with disseminated TB and needed an ART switch. INTERPRETATIONS: Our results support the feasibility, efficacy and safety of BIC/FTC/TAF as a rapid ART strategy in patients with advanced HIV disease.

Determination of telaprevir in plasma of HCV-infected patients by HPLC-UV.

Telaprevir is a direct acting antiviral agent, used with pegylated interferon and ribavirin for the management of chronic hepatitis C virus (HCV) genotype 1 infection, in patients not responding to therapy with pegylated interferon and ribavirin only. Although 75% of patients achieve a sustained virological response after treatment with telaprevir, adverse drug-drug interactions and undesirable events often occur. Therefore, telaprevir monitoring is pivotal to improve the management of HCV infection. Here, the first High-Performance Liquid Chromatography-Ultraviolet (HPLC-UV) method to quantify telaprevir in human plasma of HCV-genotype 1-infected patients is reported. The volume of the plasma sample was 700 µL. This method involved automated solid-phase extraction with Oasis HLB Cartridge 1 cc (divinylbenzene and N-vinylpyrrolidone). The extracted samples were reconstituted with 150 µL of 60/40 water/acetonitrile. Thirty microliters of these samples was injected into a HPLC-UV system, and the analytes were eluted on a X Terra(®) RP18 column (250 mm × 4.6 mm i.d.) with a particle size of 5 µm. The mobile phase (ammonium acetate buffer, 150 mM, pH 8.0, and methanol:acetonitrile 50:50) was delivered at 1.0 mL/min with linear gradient elution. The total run time for a single analysis was 16 min; telaprevir was detected by UV at 276 and 286 nm. The calibration curve was linear from 312.5 to 20,000 ng/mL (r(2) > 0.996). The absolute recovery of telaprevir ranged between 89 and 93% at concentrations of quality control samples of 800, 4,000, 8,000, and 16,000 ng/mL. Both precision and accuracy were always <15%. The HPLC-UV method reported here: (i) has been validated, (ii) is currently applied to monitor telaprevir in plasma of HCV-infected patients, and (iii) appears useful in a routine laboratory. ,

Low plasma concentrations of albumin influence the affinity column-mediated immunoassay method for the measurement of tacrolimus in blood during the early period after liver transplantation.

BACKGROUND: Monitoring of tacrolimus (TAC) concentrations in transplanted patients is necessary to ensure effective immunosuppression and to avoid adverse side effects. The fully automated analysis of TAC by the affinity column-mediated immunoassay (ACMIA), which does not require a precipitation step, may represent an efficient alternative to liquid chromatography-tandem mass spectrometry (LC-MS/MS), including in the clinically urgent situation. The aim of this work was to compare the analytical performances of ACMIA with those of LC-MS/MS and to evaluate the influence of hematological parameters, time posttransplant, and type of transplant on the results obtained from routine blood samples. METHODS: Performance characteristics of ACMIA were evaluated using quality control materials and samples spiked with TAC from the International Proficiency Testing Scheme. One hundred and fifty-eight whole-blood samples from patients who received a liver (n = 55) or kidney (n = 14) transplant were assayed by ACMIA and LC-MS/MS, and hematologic, biochemical, and demographic data were collected. Univariate and multivariate statistical analyses were also performed to assess associations between the interassay differences with clinical and laboratory parameters. RESULTS: For artificially spiked samples, the average difference between results obtained by ACMIA and LC-MS/MS was 0.24 ± 0.51 ng/mL (2.91 ± 7.03%). Crosschecking of calibrators and controls by both methods was in accordance with the nominal concentrations of TAC. The lower limit of quantification of ACMIA was found to be 3.0 ng/mL. The results with the 2 methods using routine samples from the transplant recipients correlated well (Spearman's r = 0.90). However, the ACMIA method demonstrated a positive mean bias of 1.78 ng/mL in comparison with LC-MS/MS. Multivariate analysis showed that liver transplant and albumin plasma concentrations significantly and independently affected ACMIA results (P = 0.033 and P = 0.001, respectively). Samples from liver transplant recipients early postsurgery were associated with a larger method bias than those from renal transplant recipients. CONCLUSIONS: Results obtained by ACMIA must be interpreted cautiously, particularly at lower TAC concentrations. Patients with low plasma concentrations of albumin are likely to display higher concentrations of TAC compared with LC-MS/MS in the early postsurgery period.

Association of Sofosbuvir and Daclatasvir Plasma Trough Concentrations with Patient-, Treatment-, and Disease-Related Factors Among HIV/HCV-Coinfected Persons.

BACKGROUND: Sofosbuvir plus daclatasvir achieves high rates of sustained virologic response (SVR), with no differences according to HIV serostatus. However, only limited information is available on the pharmacokinetic variability of sofosbuvir and daclatasvir in HIV/HCV-coinfected patients. OBJECTIVES: The aim of this study was to identify patient-, treatment-, and disease-related factors that are significantly associated with sofosbuvir and daclatasvir plasma trough concentrations (Ctrough), including liver and renal function, among HIV/HCV-coinfected persons. METHODS: In this observational cohort pilot study, HIV/HCV-coinfected patients undergoing sofosbuvir plus daclatasvir treatment were prospectively enrolled. Biochemical and viro-immunological parameters were assessed at baseline, week 4 (W4), end of treatment (EOT), and after EOT. The FIB-4 score and CKD-EPI equation were used to estimate liver disease and glomerular filtration rate (eGFR), respectively. For sofosbuvir, sofosbuvir metabolite (GS-331007), and daclatasvir, Ctrough was measured at W4 and week 8 (W8), and the mean of the values at those two time points (mean-Ctrough) was calculated. The Mann-Whitney test and Spearman's rank correlation were used to evaluate the correlations between the mean-Ctrough of each direct-acting antiviral (DAA) and the considered variables. RESULTS: Thirty-five patients were included (SVR 94%). An increased GS-331007 mean-Ctrough was significantly correlated with a decreased eGFR at W4 (rho = -0.36; p = 0.037) and EOT (rho = -0.34; p = 0.048). There was a significant correlation between daclatasvir mean-Ctrough and FIB-4 at all time points: baseline (rho = -0.35; p = 0.037), W4 (rho = -0.44; p = 0.008), EOT (rho = -0.40; p = 0.023), and after EOT (rho = -0.39; p = 0.028). CONCLUSIONS: In HIV/HCV-coinfected patients in a real-world setting, exposure to a high GS-331007 Ctrough was associated with a slight decrease in renal function, while advanced hepatic impairment was significantly associated with a lower daclatasvir Ctrough. Though the clinical and therapeutic relevance of these findings may be limited, increasing clinicians' knowledge regarding DAA exposure in difficult-to-treat patients could be relevant in single cases, and further investigations are warranted.

Marked increase in etravirine and saquinavir plasma concentrations during atovaquone/proguanil prophylaxis.

The case of a 32-year-old Caucasian female with multi-drug resistant HIV-1 subtype B infection treated with a salvage regimen including maraviroc, raltegravir, etravirine and unboosted saquinavir who started atovaquone/proguanil prophylaxis, is reported. The potential interactions between atovaquone/proguanil and these anti-retroviral drugs are investigated. Pharmacokinetic analyses documented a marked increase in etravirine and saquinavir plasma concentrations (+55% and +274%, respectively), but not in raltegravir and maraviroc plasma concentrations. The evidence that atovaquone/proguanil significantly interacts with etravirine and saquinavir, but not with raltegravir and maraviroc, suggests that the mechanism of interaction is related to cytochrome P450.

Multicompartment vectors as novel drug delivery systems: selective activation of Tγδ lymphocytes after zoledronic acid delivery.

Multicompartment nanoscopic carriers can be easily assembled by inducing the aggregation of anionic "hybrid" niosomes by means of cationic biocompatible polyelectrolytes. The resulting vesicle clusters, whose size and overall net charge can be easily controlled by varying the polyelectrolyte-to-particle charge ratio, show an interesting potential for multidrug delivery. In this article we provide strong evidence for their effective use in vitro as multicompartment vectors selectively directed toward monocyte/macrophage cells, showing that the monocyte/macrophage-mediated activation of Tγδ lymphocytes induced by zoledronic acid is enhanced by a factor 10(3) when the zoledronic acid is intracellularly delivered through these carriers. Furthermore, the multicompartment ɛ-polylysine niosome clusters, with their intrinsic selectivity toward macrophages, appear particularly suitable for implementing therapeutic strategies against chronically infected macrophages. FROM THE CLINICAL EDITOR: ɛ-polylysine niosome clusters, with their intrinsic selectivity toward macrophages, offer the potential for multidrug delivery. The effectiveness of aminobisphosphonate zoledronate is demonstrated to enhance the recruitment of Tγδ lymphocytes by macrophages by 2 orders of magnitude, suggesting a new therapeutic strategy for addressing pathologies featuring chronically infected macrophages.

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.

Determination of antituberculosis drug concentration in human plasma by MALDI-TOF/TOF.

Therapeutic drug monitoring allows to determine the best dosage regimen adapted to each patient optimizing the therapeutic benefits, while minimizing the risk for side effects. Here, the first methodological approach based on matrix-assisted laser desorption/ionization source equipped with tandem time-of-flight (MALDI-TOF/TOF) mass spectrometry for the determination of the antituberculosis (anti-TB) drugs ethambutol, pyrazinamide, rifampicin, and streptomycin concentration in the plasma of tuberculosis-infected patients is reported. The volume of the plasma sample was 200 microL. Plasma samples were cleaned-up by protein precipitation and evaporated in a water bath under a nitrogen stream. The extracted samples were reconstituted with 200 microL of 50% methanol-0.03% formic acid solution (v/v), spiked with known amounts of anti-TB drugs, mixed (1:1) with a saturated matrix solution (4-hydroxybenzoic acid in 50% acetonitrile-0.1% trifluoracetic acid solution; v/v), and spotted onto the MALDI-TOF/TOF sample target plate. The anti-TB drug concentration was determined by standard additions analysis. Regression of standard additions was linear over the whole anti-TB drug concentration range explored (the final anti-TB drug concentration ranged from 0.20 to 200 pmol/microL). The absolute recovery of the anti-TB drugs ranged between 87 and 110%. The minimal ethambutol, pyrazinamide, rifampicin, and streptomycin concentration detectable by MALDI-TOF/TOF is 0.08, 0.20, 0.12, and 0.15 pmol/microL, respectively.