Off-label use of vemurafenib (VMF) to treat
mutation-positive, refractory, childhood Langerhans cell histiocytosis (LCH) was evaluated.
Fifty-four patients from 12 countries took VMF 20 mg/kg/d. They ...were classified according to risk organ involvement: liver, spleen, and/or blood cytopenia. The main evaluation criteria were adverse events (Common Terminology Criteria for Adverse Events version 4.3) and therapeutic responses according to Disease Activity Score.
LCH extent was distributed as follows: 44 with positive and 10 with negative risk organ involvement. Median age at diagnosis was 0.9 years (range, 0.1 to 6.5 years). Median age at VMF initiation was 1.8 years (range, 0.18 to 14 years), with a median follow-up of 22 months (range, 4.3 to 57 months), whereas median treatment duration was 13.9 months (for 855 patient-months). At 8 weeks, 38 complete responses and 16 partial responses had been achieved, with the median Disease Activity Score decreasing from 7 at diagnosis to 0 (
< .001). Skin rash, the most frequent adverse event, affected 74% of patients. No secondary skin cancer was observed. Therapeutic plasma VMF concentrations (range, 10 to 20 mg/L) seemed to be safe and effective. VMF discontinuation for 30 patients led to 24 LCH reactivations. The blood
allele load, assessed as circulating cell-free DNA, decreased after starting VMF but remained positive (median, 3.6% at diagnosis, and 1.6% during VMF treatment;
< .001) and was associated with a higher risk of reactivation at VMF discontinuation. None of the various empirical therapies (hematopoietic stem-cell transplantation, cladribine and cytarabine, anti-MEK agent, vinblastine, etc) used for maintenance could eradicate the
clone.
VMF seemed safe and effective in children with refractory
-positive LCH. Additional studies are needed to find effective maintenance therapy approaches.
A method based on liquid chromatography coupled to triple quadrupole mass spectrometry detection using 50 µL of plasma was developed and fully validated for quantification of remdesivir and its ...active metabolites GS-441524.
A simple protein precipitation was carried out using 75 µL of methanol containing the internal standard (IS) remdesivir-13C6 and 5 µL ZnSO4 1 M. After separation on Kinetex® 2.6 µm Polar C18 100A LC column (100 × 2.1 mm i.d.), both compounds were detected by a mass spectrometer with electrospray ionization in positive mode. The ion transitions used were m/z 603.3 → m/z 200.0 and m/z 229.0 for remdesivir, m/z 292.2 → m/z 173.1 and m/z 147.1 for GS-441524 and m/z 609.3 → m/z 206.0 for remdesivir-13C6.
Calibration curves were linear in the 1-5000 μg/L range for remdesivir and 5-2500 for GS-441524, with limit of detection set at 0.5 and 2 μg/L and limit of quantification at 1 and 5 μg/L, respectively. Precisions evaluated at 2.5, 400 and 4000 μg/L for remdesivir and 12.5, 125, 2000 μg/L for GS-441524 were lower than 14.7% and accuracy was in the 89.6-110.2% range. A slight matrix effect was observed, compensated by IS. Higher stability of remdesivir and metabolite was observed on NaF-plasma. After 200 mg IV single administration, remdesivir concentration decrease rapidly with a half-life less than 1 h while GS-441524 appeared rapidly and decreased slowly until H24 with a half-life around 12 h.
This method would be useful for therapeutic drug monitoring of these compounds in Covid-19 pandemic.
The rapid global emergence of SARS-CoV-2 has been the cause of significant health concern, highlighting the immediate need for antivirals. Viral RNA-dependent RNA polymerases (RdRp) play essential ...roles in viral RNA synthesis, and thus remains the target of choice for the prophylactic or curative treatment of several viral diseases, due to high sequence and structural conservation. To date, the most promising broad-spectrum class of viral RdRp inhibitors are nucleoside analogues (NAs), with over 25 approved for the treatment of several medically important viral diseases. However, Coronaviruses stand out as a particularly challenging case for NA drug design due to the presence of an exonuclease (ExoN) domain capable of excising incorporated NAs and thus providing resistance to many of these available antivirals. Here we use the available structures of the SARS-CoV RdRp and ExoN proteins, as well as Lassa virus N exonuclease to derive models of catalytically competent SARS-CoV-2 enzymes. We then map a promising NA candidate, GS-441524 (the active metabolite of Remdesivir) to the nucleoside active site of both proteins, identifying the residues important for nucleotide recognition, discrimination, and excision. Interestingly, GS-441524 addresses both enzyme active sites in a manner consistent with significant incorporation, delayed chain termination, and altered excision due to the ribose 1′-CN group, which may account for the increased antiviral effect compared to other available analogues. Additionally, we propose structural and function implications of two previously identified RdRp resistance mutations in relation to resistance against Remdesivir. This study highlights the importance of considering the balance between incorporation and excision properties of NAs between the RdRp and ExoN.
•Sequence comparison of SARS-CoV and SARS-CoV-2 for nsp12 (RdRp) and nsp14 (ExoN) show near structural & functional identity.•Base & ribose modifications of GS-441524 5′-TP vs ATP point to specific discrimination mechanisms & active site residues.•Lassa virus N & SARS-CoV nsp14 ExoN domains allow modeling catalytically competent SARS-CoV-2 nsp14 Exonuclease.•RNA terminated with GS-441524 5′-MP at the 3′-end indicate a problematic accommodation at the nsp14 ExoN active site.•Anti-SARS-CoV-2 nucleoside analog design must consider balance between incorporation & excision by nsp12 & nsp14.
The guanosine analog AT-527 represents a promising candidate against Severe Acute Respiratory Syndrome coronavirus type 2 (SARS-CoV-2). AT-527 recently entered phase III clinical trials for the ...treatment of COVID-19. Once in cells, AT-527 is converted into its triphosphate form, AT-9010, that presumably targets the viral RNA-dependent RNA polymerase (RdRp, nsp12), for incorporation into viral RNA. Here we report a 2.98 Å cryo-EM structure of the SARS-CoV-2 nsp12-nsp7-nsp8
-RNA complex, showing AT-9010 bound at three sites of nsp12. In the RdRp active-site, one AT-9010 is incorporated at the 3' end of the RNA product strand. Its modified ribose group (2'-fluoro, 2'-methyl) prevents correct alignment of the incoming NTP, in this case a second AT-9010, causing immediate termination of RNA synthesis. The third AT-9010 is bound to the N-terminal domain of nsp12 - known as the NiRAN. In contrast to native NTPs, AT-9010 is in a flipped orientation in the active-site, with its guanine base unexpectedly occupying a previously unnoticed cavity. AT-9010 outcompetes all native nucleotides for NiRAN binding, inhibiting its nucleotidyltransferase activity. The dual mechanism of action of AT-527 at both RdRp and NiRAN active sites represents a promising research avenue against COVID-19.
An LC‐MS/MS method for hair testing of glyphosate and aminomethylphosphonic acid (AMPA), its main biodegradation product, has been developed. After decontamination, 50 mg of hair was ground and ...sonicated in water for 2 h. The method was fully validated in the 5–500 pg/mg range for glyphosate and 10–500 pg/mg for AMPA, and the limits of detection were 2 and 5 pg/mg, respectively. Matrix effect for glyphosate and AMPA was compensated by an isotope‐labeled internal standard. Hair samples from four farmers who regularly used glyphosate and one farmer who used glyphosate but not his wife and 14 hair samples from nonoccupationally exposed subjects were tested. Glyphosate was found in head hair of three farmers, with concentration in the range 14–188 pg/mg. The fourth was found negative but with hair colored in red. Glyphosate was detected in 10 of 14 hair samples from nonoccupationally exposed subjects at concentrations of 11.5 pg/mg or lower and only in one segment (0–3 cm) of the farmer's spouse (6 pg/mg). AMPA was detected in five subjects, above the limit of quantification only in two of three occupationally exposed subjects with positive glyphosate.
Further studies should be conducted to validate this potential new biomarker that could be useful for assessing long‐term exposure to glyphosate.
Background
Baclofen is a GABA‐B receptor agonist currently used in the treatment of spasticity. In recent years, baclofen has been used to reduce craving, voluntary alcohol intake and withdrawal ...syndrome of alcoholic patients. To date, there are no data available to estimate the relationship between baclofen exposure and the variation of craving. The first objective of this study was to investigate the variation of craving as a function of exposure, and the second was to explore the possible existence of baclofen responders and nonresponders.
Methods
Sixty‐seven outpatients, 43 men/24 women (weight: 73 kg 42 to 128; age: 49 years old 29 to 68) followed in the addictology unit, were studied during 3 months after treatment initiation. Baclofen was administered by oral route. Therapeutic drug monitoring enabled the measurement of plasma concentrations. Craving level was assessed by the Obsessive–Compulsive Drinking Scale (OCDS). A population pharmacokinetic (PK)–pharmacodynamic analysis of the OCDS variation following baclofen administration was performed. Demographic data, biological data, and tobacco consumption were tested for their influence on the parameters.
Results
Data were modeled with a 1‐compartment model with first‐order absorption and elimination. PK analysis confirms the results of our previous study. An Emax model best‐described the exposure–OCDS relationship. None of the covariates tested was able to improve the fit or decrease intersubject variability. However, 2 subpopulations were defined for the exposure corresponding to half the maximal effect (BE50). The proportion of patients being classified as responders was 38% (95% confidence interval CI 7 to 76), the maximal decrease in OCDS (Emax) was 72% (95% CI 25 to 85), and the BE50 was 12.6 (95% CI 0.02 to 74.3) or 4,390 (95% CI 20.4 to 31,800) h mg/l for responders and nonresponders, respectively.
Conclusions
We defined the relationship between baclofen exposure and craving in patients with alcohol use disorder. Baclofen treatment decreased craving in all patients. However, we drew up the hypothesis of 2 subpopulations of patients differentiated by their speed of response. A wide interindividual variability in response was depicted, making it currently impossible to predict which group a patient will belong to.
•Contamination by body fluid is possible with ostarine.•Description of the first AAF with body fluid contamination.•Urine quantification of ostarine after controlled body fluid contamination.
...Ostarine, also known as MK-2866 or enobosarm, is a selective androgen receptor modulator (SARM). It has anabolic properties and as such is widely used in doping, accounting in 2021 for 25 % of the adverse analytical findings (AAF) among the class S1.2 “Other anabolic agents” of products banned by the World Anti-Doping Agency, to which it belongs. But in some cases, it can be responsible for an AAF following contamination. We report the case of an athlete who contaminated herself by exchanging body fluids while kissing her boyfriend, who took 25 mg per day of MK-2866 for 9 days prior to the athlete's AAF (urinary concentration evaluated at 13 ng/mL) without her knowledge. Both subjects came to our lab for hair testing. The athlete's hair was black and slightly frizzy. Six segments of 2 cm then 7 × 3 cm (33 cm) were analysed and showed increasing concentrations, from 2 pg/mg on the first segment to 17.8 pg/mg on the last segment. The boyfriend's hair, light-brown, analyzed on 4 × 2 cm, also showed increasing values, from 65 to 143 pg/mg. These gradients of concentration in the hair’s athlete and in her boyfriend were compatible with external contamination of the hair, confirmed by analysis of washing baths, pillowcases (150 pg on each), and the athlete's hairbrush (250 pg).
Fingernails were also contaminated, with 21 pg/mg in the athlete and 1041 pg/mg in the boyfriend, with highly contaminated washing baths, and toenails were less contaminated, with 2 pg/mg in the athlete and 17.3 pg/mg in the boyfriend. Urine samples taken 35 days after the start of MK-2866 treatment showed a value of 3690 ng/mL in the boyfriend and 5.7 ng/mL in the athlete. After 6 days off, these concentrations were 3.3 ng/mL and 0.1 ng/mL, respectively. A controlled transfer study was carried out 12 days after discontinuation (urine concentrations returned to negative level). After administration of 17 mg (the 25 mg/mL vial having been controlled at 17 mg/mL), urine samples were taken from the boyfriend and the athlete (n = 10 for each) for more than 25 h after they had been living normally with each other (regular kissing in particular). The boyfriend's urine concentrations ranged from 681 ng/mL to 12822 ng/mL (Tmax = 8:30 hrs), and the athlete's from 0.3 ng/mL to 13 ng/mL with Tmax = 8:30 hrs, i.e. at 22:30 hrs, which corresponded exactly to the time of collection of the urine that showed AAF, with a similar concentration. The dose ingested by the athlete was estimated at 15 µg. These results demonstrate the transfer of ostarine via body fluids between two subjects, with a high risk of AAF in one athlete, as observed in our case.
Untargeted liquid chromatography–high resolution mass spectrometry (LC–HRMS) techniques have become indispensable tools for systematic toxicological analysis. They allow the research of an almost ...unlimited number of drugs within a single analytical cycle, but shared mass spectra libraries are still missing to identify newly marketed compounds, along with defined analytical procedures. This article describes the optimization, validation, and application of an untargeted screening method devoted to hair analysis using data‐dependent analysis (DDA) and a shared HRMS database. This method used an ultra‐high performance liquid chromatography coupled to a benchtop Orbitrap. Raw MS data were processed with Compound Discoverer software coupled to the mzCloud™ library. Optimizations were performed on blank hair spiked with 19 analytes having different physical and chemical properties. To validate the effectiveness of a shared spectra database, 20 compounds spectra were added and then retrospectively screened. Sensitivity and reliability were evaluated on 317 compounds of interest in toxicology. The method was then applied to 11 hair samples. The matrix effect range by ion suppression/enhancement was 40%–110%. The method allows the detection of 284 among the 317 screened compounds, including 72 new psychoactive substances (NPS). Lower limit of identification (LLOI) and lower limit of detection (LLOD) were 1 to 1000 pg/mg and 1 to 500 pg/mg, respectively. The method was successfully applied to 11 clinical cases and 144 compounds were identified including 24 NPS including AKB48‐5F for the first time in hair. We developed and validated an LC–HRMS untargeted screening of 284 compounds and successfully applied it to 11 real hair samples.
This article describes the optimization, validation, and application of an untargeted screening method devoted to hair analysis using data‐dependent analysis and a shared HRMS database. This method used an ultra‐high performance liquid chromatography coupled to a benchtop Orbitrap. Raw MS data were processed with Compound Discoverer software coupled to the mzCloud™ library. Sensitivity and reliability were evaluated on 317 compounds of interest in toxicology. The method was then applied to two hair samples from patients who consumed NPS.
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