Abstract
Background
Remdesivir has received significant attention for its potential application in the treatment of COVID-19, caused by SARS-CoV-2. Remdesivir has already been tested for Ebola virus ...disease treatment and found to have activity against SARS and MERS coronaviruses. The remdesivir core contains GS-441524, which interferes with RNA-dependent RNA polymerases alone. In non-human primates, following IV administration, remdesivir is rapidly distributed into PBMCs and converted within 2 h to the active nucleoside triphosphate form, while GS-441524 is detectable in plasma for up to 24 h. Nevertheless, remdesivir pharmacokinetics and pharmacodynamics in humans are still unexplored, highlighting the need for a precise analytical method for remdesivir and GS-441524 quantification.
Objectives
The validation of a reliable UHPLC-MS/MS method for remdesivir and GS-441524 quantification in human plasma.
Methods
Remdesivir and GS-441524 standards and quality controls were prepared in plasma from healthy donors. Sample preparation consisted of protein precipitation, followed by dilution and injection into the QSight 220 UHPLC-MS/MS system. Chromatographic separation was obtained through an Acquity HSS T3 1.8 μm, 2.1 × 50 mm column, with a gradient of water and acetonitrile with 0.05% formic acid. The method was validated using EMA and FDA guidelines.
Results
Analyte stability has been evaluated and described in detail. The method successfully fulfilled the validation process and it was demonstrated that, when possible, sample thermal inactivation could be a good choice in order to improve biosafety.
Conclusions
This method represents a useful tool for studying remdesivir and GS-441524 clinical pharmacokinetics, particularly during the current COVID-19 outbreak.
Vitamin D (Vit D) is a fat-soluble molecule acting like a hormone, and it is involved in several biological mechanisms such as gene expression, calcium homeostasis, bone metabolism, immune ...modulation, viral protection, and neuromuscular functions. Vit D deficiency can lead to chronic hypocalcemia, hyperparathyroidism, and many other pathological conditions; in this context, low and very low levels of 25-hydroxy-vitamin D (25-OH-D) were found to be associated with an increased risk of COVID-19 infection and the likelihood of many severe diseases. For all these reasons, it is important to quantify and monitor 25-OH-D levels to ensure that the serum/blood concentrations are not clinically suboptimal. Serum concentration of 25-OH-D is currently the main indicator of Vit D status, and it is currently performed by different assays, but the most common quantitation techniques involve immunometric methods or chromatography. Nevertheless, other quantitation techniques and instruments are now emerging, such as AFIAS-1
and AFIAS-10
(Boditech and Menarini) based on the immunofluorescence analyzer, that guarantee an automated system with cartridges able to give quick and reliable results as a point-of-care test (POCT). This work aims to compare AFIAS-1
and AFIAS-10
(Boditech and Menarini) Vit D quantitation with Ultra High-Performance Liquid Chromatography coupled with tandem mass spectrometry that currently represents the gold standard technique for Vit D quantitation. The analyses were performed in parallel on 56 samples and in different conditions (from fresh and frozen plasma) to assess the reliability of the results. Any statistically significant differences in methods, the fixed error, and the error proportional to concentration were reported. Results obtained in all conditions showed a good correlation between both AFIAS
instruments and LC-MS/MS, and we can affirm that AFIAS-1
and AFIAS-10
are reliable instruments for measuring 25-OH-D with accuracy and in a fast manner.
The phytocomplex of Cannabis is made up of approximately 500 substances: terpeno-phenols metabolites, including Δ-9-tetrahydrocannabinol and cannabidiol, exhibit pharmacological activity.
Medical ...Cannabis has several pharmacological potential applications, in particular in the management of chronic and neuropathic pain. In the literature, a few data are available concerning cannabis pharmacokinetics, efficacy and safety.
Thus, aim of the present study was the evaluation of cannabinoid pharmacokinetics in a cohort of patients, with chronic and neuropathic pain, treated with inhaled medical cannabis and decoction, as a galenic preparation.
In this study, 67 patients were enrolled. Dried flower tops with different THC and CBD concentrations were used: Bedrocan® medical cannabis with THC level standardized at 19% and with a CBD level below 1%, Bediol® medical cannabis with THC and CBD level standardized at similar concentration of 6.5% and 8%, respectively.
Cannabis was administered as a decoction in 47 patients and inhaled in 11 patients. The blood withdrawn was obtained before the new dose administration at the steady state and metabolites plasma concentrations were measured with an UHPLC-MS/MS method.
Statistically significant differences were found in cannabinoids plasma exposure between inhaled and oral administration of medical cannabis, between male and female and cigarette smokers.
For the first time, differences in cannabinoid metabolites exposures between different galenic formulations were suggested in patients. Therapeutic drug monitoring could be useful to allow for dose adjustment, but further studies in larger cohorts of patients are required in order to confirm these data.
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•Cannabis shows different pharmacological applications, in particular in the management of pain.•Cannabinoids pharmacokinetics description in patients with chronic and neuropathic pain.•Differences in cannabinoids plasma exposure between route of administration.
Schizophrenia affects approximately 24 million people worldwide and clozapine is the most effective antipsychotic drug. Nevertheless, its use in therapy is limited due to adverse effects.Therapeutic ...drug monitoring is a clinical tool useful to reduce the clozapine toxicity. In the literature, papers showed how psychiatric disorders could be associated with low vitamin D levels, but a few studies focusing on its role in affecting clozapine exposure are available. A TDM repository was analyzed: clozapine and vitamin D levels measured with liquid chromatography were considered. 1261 samples obtained from 228 individuals were evaluated: 624 patients (49.5%) showed clozapine plasma levels in therapeutic range (350–600 ng/mL). Clozapine toxic plasma levels (>1000 ng/mL) were more present in winter (p = 0.025), compared to other seasons. Concerning vitamin D, a sub-analysis of 859 samples was performed: 326 (37.81%) were deficient ( ng/mL), 490 (57.12%) had insufficient concentrations (10–30 ng/mL), while 43 (5.02%) had sufficient (>30 ng/mL) levels. A correlation between vitamin D and clozapine plasma levels (p = 0.007, Pearson coefficient=0.093) was observed. The role of seasonal variation in clozapine plasma exposure in psychiatric patients treated with clozapine was suggested. Further studies in larger cohorts are needed in order to clarify these aspects.
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•Vitamin D in psychiatric patients treated with clozapine.•Clozapine therapeutic Drug Monitoring in treated patients.•Impact of seasonality in Vitamin D and clozapine plasma exposure.•Clozapine toxicity in treated patients and the role of seasonality.
Tacrolimus (TAC) pharmacokinetics is influenced by the donor CYP3A5 genotype and the age of pediatric liver recipients. However, an optimization of a genotype-based algorithm for determining TAC ...starting is needed to earlier achieve stable target levels. As the graft itself is responsible for its metabolism, the Graft-to-Recipient Weight Ratio (GRWR) might play a role in TAC dose requirements. A single-center study was carried out in a cohort of 49 pediatric recipients to analyse the impact of patient and graft characteristics on TAC pharmacokinetics during the first 15 post-transplant days. Children < 2 years received grafts with a significantly higher GRWR (4.2%) than children between 2-8 (2.6%) and over 8 (2.7%). TAC concentration/weight-adjusted dose ratio was significantly lower in recipients from CYP3A5*1/*3 donors or with extra-large (GRWR > 5%) or large (GRWR 3-5%) grafts. The donor CYP3A5 genotype and GRWR were the only significant predictors of the TAC weight adjusted doses. Patients with a GRWR > 4% had a higher risk of acute rejection, observed in 20/49 (41%) patients. In conclusion, TAC starting dose could be guided according to the donor CYP3A5 genotype and GRWR, allowing for a quicker achievement of target concentrations and eventually reducing the risk of rejection.
Background: Vitamin D deficiency has been associated with the severity of COVID-19. The role of vitamin D in pregnant women with COVID-19 has been poorly investigated to date. The aim of this study ...was to evaluate the influence of vitamin D in affecting some clinical features in pregnancy between SARS-CoV-2 positive and negative patients. Methods: Vitamin D pathway related polymorphisms and 25-hydroxyvitamin D levels were quantified in pregnant women followed from the first to the third trimester of pregnancy. Vitamin D deficiency was considered with values ≤ 30 ng/mL. Results: In total, 160 women were enrolled: 23 resulted positive for at least one SARS-CoV-2 related test (molecular swab or antibody tests). Vitamin D-associated polymorphisms were able to affect vitamin D levels in SARS-CoV-2 negative and positive subjects: remarkably, all the VDR TaqICC genotype patients were negative for SARS-CoV-2. In a sub-population (118 patients), vitamin D levels correlated with pregnancy-related factors, such as alpha-fetoprotein levels. Third-trimester vitamin D levels were lower in preterm births compared to full-term pregnancy: this trend was highlighted for SARS-CoV-2 positive patients. Conclusions: This is the first study demonstrating a role of vitamin D in affecting the clinical characteristics of pregnant women during the COVID-19 era. Further studies in larger and different cohorts of patients are required to confirm these findings.
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•Analysis of concordance between EMIT and LC–MS/MS for TDM of Mycophenolic Acid.•Accuracy of LC–MS/MS method was tested by proficiency and inter-laboratory tests.•LC–MS/MS resulted ...concordant with a home-made validated HPLC-UV method.•Compared to LC–MS/MS, analytical results by EMIT appeared overestimated.•Overestimation by EMIT would result in a diagnostic mismatch between 12.4 and 31.4 %.
Therapeutic drug monitoring (TDM) of immunosuppressive drugs is crucial in organ-transplanted patients to prevent rejection or toxic effects due to inadequate dosage. Mycophenolic acid (MPA) is a commonly used immunosuppressant in this setting.
Nowadays, MPA concentrations are monitored by Enzyme Multiplied Immunoassay Technology (EMIT), and Liquid Chromatography (LC)-based techniques, particularly coupled to Tandem Mass Spectrometry (LC–MS/MS).
This study evaluates the concordance between TDM results for MPA obtained through CE-IVD EMIT and LC–MS/MS assays in plasma samples. LC–MS/MS quantification was based on a commercial kit and the analytical performance in terms of accuracy was tested through external proficiency tests and inter-laboratory comparison with a home-made HPLC-UV method.
Both these evaluations confirmed the reliability of the LC–MS/MS method (1.6 % and 9.0 % of bias, respectively). Conversely, the comparison between EMIT and LC–MS/MS showed overestimation by EMIT of 33.5 %. This bias resulted concentration-dependent, ranging from 46.4 % in the concentration range of 1–2 mg/L, to 21.4 % over 4 mg/L. Considering the theoretical clinical impact of this overestimation, a fraction comprised between 12.4 % and 31.4 % of samples which resulted over three different minimum effective concentration values by EMIT (no indication for dose adjustment) had discordant indications by LC–MS/MS (dose adjustment needed). Concluding, this study highlights a clinically relevant systematic overestimation of MPA concentration by EMIT, supporting the switch to LC–MS/MS techniques for TDM purpose. However, further prospective studies are needed in order to evaluate the clinical impact of switching the TDM activity from EMIT to LC–MS/MS in a larger cohort in a long period.
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.
Adalimumab (ADA) is a human anti-tumor necrosis factor (TNF-α) monoclonal antibody used in inflammatory bowel diseases, such as Crohn's disease (CD). Vitamin-D (VD) is important for biological ...functions, such as the modulation of expression of genes encoding enzymes and transporters involved in drug metabolism and transport. ADA trough levels were associated with VD concentrations in patients with IBD, but no data are present in the literature concerning VD pathway-related gene single-nucleotide polymorphisms (SNPs) in affecting clinical outcomes. For this reason, the aim of this study was to evaluate the ability of VD-related genetics to predict clinical remission at 3 and 12 months in patients affected by CD treated with ADA. Patients affected by CD were included in this study. SNPs in
,
,
, and
genes were analyzed through real-time PCR. A total of 63 patients were enrolled. Calprotectin, hemoglobin, and C-reactive protein levels were influenced by SNPs in
,
, and
genes. After 3 months of therapy, clinical remission was predicted by smoke, systemic steroids, and
BsmI, whereas at 12 months by
1296AA/AC and VD supplementation. This study reports the association between VD pathway-related genetics and ADA treatment. Further studies are needed to confirm these promising data.
Recently, anti-HIV treatment has achieved high efficacy and tolerability. Nevertheless, few data are available about the intracellular penetration of antiretrovirals, partly due to the technical ...challenges related to intracellular quantification. This work aimed to validate an ultra-high performance liquid chromatography (UHPLC) tandem mass spectrometry (MS/MS) method for the simultaneous quantification of maraviroc, nevirapine, rilpivirine, dolutegravir, raltegravir, cobicistat, darunavir, ritonavir, atazanavir, efavirenz, elvitegravir, and etravirine within peripheral blood mononuclear cells (PBMCs) and apply it to samples from patients. PBMCs were isolated by density gradient on cell preparation tubes (CPT). Samples were prepared by addition of internal standards (IS), sonication, centrifugation, and drying. Reconstituted extracts underwent chromatographic separation by reversed phase UHPLC and detection was performed by electrospray ionization and multiple reaction monitoring. Method validation followed FDA and EMA guidelines, showing acceptable accuracy, precision, recovery and IS-normalized matrix effect. The application to 56 samples from patients undergoing antiretroviral treatment provided description of intracellular penetration, showing method eligibility for future studies.
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