A Rapid and Sensitive Liquid Chromatography-Tandem Mass Spectrometry Bioanalytical Method for the Quantification of Encorafenib and Binimetinib as a First-Line Treatment for Advanced (Unresectable or Metastatic) Melanoma-Application to a Pharmacokinetic Study.

The combination regimen targeting BRAF and MEK inhibition, for instance, encorafenib (Braftovi™, ENF) plus binimetinib (Mektovi®, BNB), are now recommended as first-line treatment in patients with unresectable or metastatic melanoma with a BRAF V600-activating mutation. Patients treated with combination therapy of ENF and BNB demonstrated a delay in resistance development, increases in antitumor activity, and attenuation of toxicities compared with the activity of either agent alone. However, the pharmacokinetic profile of the FDA-approved ENF and BNB is still unclear. In this study, a rapid and sensitive LC-MS/MS bioanalytical method for simultaneous quantification of ENF and BNB in rat plasma was developed and validated. Chromatography was performed on an Agilent Eclipse plus C18 column (50 mm × 2.1 mm, 1.8 µm), with an isocratic mobile phase composed of 0.1% formic acid in water/acetonitrile (67:33, v/v, pH 3.2) at a flow rate of 0.35 mL/min. A positive multiple reaction monitoring (MRM) mode was chosen for detection and the process of analysis was run for 2 min. Plasma samples were pre-treated using protein precipitation with acetonitrile containing spebrutinib as the internal standard (IS). Method validation was assessed as per the FDA guidelines for the determination of ENF and BNB over concentration ranges of 0.5-3000 ng/mL (r2 ≥ 0.997) for each drug (plasma). The lower limits of detection (LLOD) for both drugs were 0.2 ng/mL. The mean relative standard deviation (RSD) of the results for accuracy and precision was ≤ 7.52%, and the overall recoveries of ENF and BNB from rat plasma were in the range of 92.88-102.28%. The newly developed approach is the first LC-MS/MS bioanalytical method that can perform simultaneous quantification of ENF and BNB in rat plasma and its application to a pharmacokinetic study. The mean result for Cmax for BNB and ENF was found to be 3.43 ± 0.46 and 16.42 ± 1.47 µg/mL achieved at 1.0 h for both drugs, respectively. The AUC0-∞ for BNB and ENF was found to be 18.16 ± 1.31 and 36.52 ± 3.92 µg/mL.h, respectively. On the other hand, the elimination half-life (t1/2kel) parameters for BNB and ENF in the rat plasma were found to be 3.39 ± 0.43 h and 2.48 ± 0.24 h, and these results are consistent with previously reported values.

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.

A rapid ultra high performance liquid chromatography-tandem mass spectrometry method for the quantification of daidzein, its valine carbamate prodrug, and glucuronide in rat plasma samples: Comparison of the pharmacokinetic behavior of daidzine valine carbamate prodrugs.

Two valine carbamate prodrugs of daidzein were designed to improve its bioavailability. To compare the pharmacokinetic behavior of these prodrugs with different protected phenolic hydroxyl groups of daidzein, a rapid and sensitive method for simultaneous quantification of daidzein, its valine carbamate prodrug, and daidzein-7-O-glucuronide in rat plasma was developed and validated in this study. The samples were processed using a fast one-step protein precipitation method with methanol added to 50 µL of plasma and were analyzed by ultra-high performance liquid chromatography with tandem mass spectrometry. To improve the selectivity, peak shape, and peak elution, several key factors, especially stationary phase and the composition of the mobile phase, were tested, and the analysis was performed using the Kinetex® C18 column (100 × 2.1 mm, 2.6 µm) within only 2.6 min under optimal conditions. The established method exhibited good linearity over the concentration range of 2.0-1000 ng/mL for daidzein, and 8.0-4000 ng/mL for the prodrug and daidzein-7-O-glucuronide. The accuracy of the quality control samples was between 95.5 and 110.2% with satisfactory intra- and interday precision (relative standard deviation values < 10.85%), respectively. This sensitive, rapid, low-cost, and high-throughput method was successfully applied to compare the pharmacokinetic behavior of different daidzein carbamate prodrugs.

Development and optimization of a high-throughput HPLC-MS/MS method for the simultaneous determination of naringenin and its valine carbamate prodrug in rat plasma.

A valine carbamate prodrug of naringenin (NAR) called 4'V was synthesized to enhance its oral bioavailability because of low water solubility and poor membrane permeability of NAR. This study developed and fully validated a sensitive, rapid, and robust HPLC-MS/MS method for the simultaneous determination of NAR and 4'V in plasma. The analytes were treated using liquid-liquid extraction, separated on a Phenomenex Kinetex XB-C18 column, and detected using a triple-quadrupole tandem mass spectrometer equipped with an electrospray ionization interface. The analytes were eluted within only 4 min by gradient procedure. The excellent linear correlations were validated over the range of 4-400 ng/mL (r = 0.9990) for NAR and 2-2000 ng/mL (r = 0.9951) for 4'V, with lower limits of quantification of 4 and 2 ng/mL, respectively. For all quality control samples, the intra-day and inter-day precision and accuracy were within ±15%. The validated method was economical, high throughput, and reliable and was first successfully applied to a pharmacokinetic study of NAR and 4'V after oral administration to Sprague-Dawley rats. The results of the pharmacokinetic study demonstrated that the idea of amino acid carbamate prodrug is a promising strategy to improve the bioavailability of NAR.

Relatively mild symptoms after chronic overdose with a double-dose encorafenib: a case report.

Encorafenib (Braftovi) is indicated for the treatment of adult patients with unresectable or metastatic melanoma with a BRAF V600 mutation, in combination with binimetinib (Mektovi). According to the product label of encorafenib, there are no specific treatment recommendations in case of an overdose. We report on a 63-year-old man who ingested a double dose (900 mg) of encorafenib for 16 days. He developed overall minor chronic overdose symptoms such as nausea and vomiting grade 1 and muscle pain. Based on the most occurring adverse events of encorafenib, liver values, kidney function parameters and QTc interval were measured. Kidney function parameters were normal, whereas liver values were slightly increased (grade 1) and QTc slightly prolonged. The plasma concentration 3 h after the last dose was 2110 ng/mL. We describe the course of a case with a chronic overdose during 16 days of the double dose of encorafenib as well as the followed approach, which could be taken into account when observing an encorafenib overdose. Providing information in times of Covid-19 is challenging, but remains necessary for good clinical care.

Cenobamate for the treatment of focal epilepsies.

INTRODUCTION: Antiseizure drugs (ASDs) play a central and crucial role in the treatment of epilepsy patients because the majority require anticonvulsant treatment for an extended period of time. Due to the fact that 30% of patients are refractory to medical treatment, new therapeutic options are necessary. Cenobamate is the latest approved antiepileptic drug in focal epilepsy, and its mode of action is thought to be mediated by blocking voltage-gated sodium channels and interaction with the GABAergic system. AREAS COVERED: This article reviews animal studies, pharmacokinetics, pharmacodynamics, and the phase 1 to 3 trials and open-label extension data on cenobamate. EXPERT OPINION: Cenobamate has the potential to perform as an important ASD because trial data are indicative of remarkable responder and seizure freedom rates so far not seen with other ASDs. Cenobamate demonstrated significant efficacy at a dosage between 100 and 400 mg per day. The side-effect profile of this drug is comparable to other ASDs and is mainly CNS related; in particular, somnolence, dizziness, headache, diplopia, and nystagmus. However, slow titration is mandatory to decrease the risk of drug rash with eosinophilia and systemic symptoms (DRESS) that was observed in several patients with fast uptitration schemes.

Predicted values for human total clearance of a variety of typical compounds with differently humanized-liver mouse plasma data.

Prediction of human pharmacokinetics is important in the preclinical stage. Values for total clearance of compounds from plasma should be one of the most important pharmacokinetic parameters for predictions. Although several physiological and empirical methods including single-species allometry for prediction of values for human clearance of compounds using humanized-liver mice have been reported, further improvement of prediction accuracies would be still expected. To optimize these approaches, we proposed methods for unbound intrinsic clearance in virtually 100% humanized-liver mouse by incorporating unbound plasma fractions of compounds in differently humanized-liver mice. Comparisons of prediction accuracies of values for human clearance of 15 model compounds were performed among our current physiological and previously reported models and single-species allometry using humanized-liver mice. Incorporation of the actual unbound plasma fractions of compounds and correction of residual mice hepatocyte in humanized-liver mice showed comparable prediction accuracy to that by single-species allometry. After exclusion of 3 compounds with large species differences in values of clearance and unbound plasma fractions between mice and humans out of 15 compounds, prediction accuracies were improved in the methods investigated. The previously and present reported physiological methods could show the good prediction accuracy of values for clearance of drugs from plasma.

Pharmacokinetics of daclatasvir in Egyptian adolescents with genotype-4 HCV infection.

BACKGROUND: Daclatasvir has potent antiviral activity against HCV infection when used in combination with sofosbuvir, however, its pharmacokinetics have not been described in adolescents. The aim is to determine the pharmacokinetic parameters of daclatasvir in adolescents, and to develop a population pharmacokinetic (PopPK) model. METHODS: Seventeen adolescent patients with genotype-4 chronic HCV infection received once daily oral daclatasvir 60 mg in combination with 400 mg sofosbuvir for 12 weeks. Steady state concentrations were determined. Non-compartmental and population PK were determined. RESULTS: The average PK parameters calculated by non-compartmental analysis (NCA): maximum plasma concentration (Cmax), area under the curve (AUC), apparent oral volume of distribution (V/F), apparent oral clearance (CL/F) and half-life (T1/2) were 1,092 ng/ml, 11,178 ng/ml•h, 55 l, 4.5 l/h and 8.5 h, respectively. Daclatasvir was best described by one compartment structural PK model with zero order absorption and first-order elimination. The absorption rate constant (K0), V/F, and CL/F of the final PopPK model of daclatasvir were 1.5/h, 52 l and 4.7 l/h, respectively. Body weight and serum albumin had significant effect on the V/F parameter. CONCLUSIONS: Body weight and serum albumin were the major determinants of daclatasvir V/F in this population. PK parameters were comparable to those reported in adult HCV patients, demonstrating that 60 mg daclatasvir is an appropriate dose for adolescents. ClinicalTrials.gov NCT03540212.

Cenobamate (YKP3089) as adjunctive treatment for uncontrolled focal seizures in a large, phase 3, multicenter, open-label safety study.

OBJECTIVE: During the development of cenobamate, an antiseizure medication (ASM) for focal seizures, three cases of drug reaction with eosinophilia and systemic symptoms (DRESS) occurred. To mitigate the rate of DRESS, a start-low, go-slow approach was studied in an ongoing, open-label, multicenter study. Also examined were long-term safety of cenobamate and a method for managing the pharmacokinetic interaction between cenobamate, a 2C19 inhibitor, and concomitant phenytoin or phenobarbital. METHODS: Patients 18-70 years old with uncontrolled focal seizures taking stable doses of one to three ASMs were enrolled. Cenobamate 12.5 mg/d was initiated and increased at 2-week intervals to 25, 50, 100, 150, and 200 mg/d. Additional biweekly 50 mg/d increases to 400 mg/d were allowed. During titration, patients taking phenytoin or phenobarbital could not have their cenobamate titration rate or other concomitant ASMs adjusted; phenytoin/phenobarbital doses could be decreased by 25%-33%. RESULTS: At data cutoff (median treatment duration = 9 months), 1347 patients were enrolled, of whom 269 (20.0%) discontinued, most commonly due to adverse events (n = 137) and consent withdrawn for reason other than adverse event (n = 74); 1339 patients received ≥1 treatment dose (median modal dose = 200 mg). The most common treatment-emergent adverse events (TEAEs) were somnolence (28.1%), dizziness (23.6%), and fatigue (16.6%). Serious TEAEs occurred in 108 patients (8.1%), most commonly seizure (n = 14), epilepsy (n = 5), and pneumonia, fall, and dizziness (n = 4 each). No cases of DRESS were identified. In the phenytoin/phenobarbital groups, 43.4% (36/114) and 29.7% (11/51) of patients, respectively, had their doses decreased. At the end of titration, mean plasma phenytoin/phenobarbital levels were generally comparable to baseline. SIGNIFICANCE: No cases of DRESS were identified in 1339 patients exposed to cenobamate using a start-low (12.5 mg/d), go-slow titration approach. Cenobamate was generally well tolerated in the long term, with no new safety issues found. Phenytoin/phenobarbital dose reductions (25%-33%), when needed during cenobamate titration, maintained stable plasma levels.

Mass Balance, Metabolism, and Excretion of Cenobamate, a New Antiepileptic Drug, After a Single Oral Administration in Healthy Male Subjects.

BACKGROUND AND OBJECTIVE: Cenobamate is an antiepileptic drug for the treatment of partial-onset seizures. The current study was designed to assess the mass balance and the metabolic profiling of cenobamate in humans. METHODS: Absorption, metabolism, and excretion of cenobamate were investigated in healthy male subjects after a single oral dose of 400 mg of cenobamate containing 50 µCi of [14C]-cenobamate as capsule formulation. RESULTS: Cenobamate was rapidly (median time to maximum plasma concentration of 1.25 h) and extensively (≥ 88% of dose) absorbed. The mean cenobamate plasma concentration-time profile revealed a multiphasic elimination profile whereas the mean plasma/blood concentration-time curve for total radioactivity did not appear to be multiphasic, suggesting that elimination mechanisms for cenobamate and its metabolites may be different. Blood/plasma ratios observed for the area under the concentration-time curve (AUC) and peak concentration (both ~ 0.60) suggest a limited penetration of cenobamate and metabolites into red blood cells (RBCs). Eight cenobamate metabolites were identified across plasma, urine, and feces. Cenobamate was the main plasma radioactive component and M1 was the only metabolite detected in plasma (> 98% and < 2% total radioactivity AUC, respectively). All detected metabolites were found in urine, with M1 as the major radioactive component (mean cumulative recovery 37.7% of dose); unchanged cenobamate accounted for 6%. Metabolites comprised ~ 88% of the dose recovered in urine, indicating extensive metabolism by the kidneys and/or metabolites formed in the liver were rapidly eliminated from the bloodstream. However, cenobamate metabolites appear to be formed slowly. Minor amounts of cenobamate (0.48%) and five metabolites (≤ 1.75% each; M1, M3, M6, M7, M11) were recovered in feces. CONCLUSION: This study indicates that cenobamate is primarily eliminated in urine as metabolites. Cenobamate is the major circulating component in plasma after oral administration and has a limited penetration into RBCs.

Pharmacokinetics, Safety, and Tolerability of Ledipasvir/Sofosbuvir and Sofosbuvir/Velpatasvir in Healthy Chinese Subjects.

PURPOSE: Ledipasvir/sofosbuvir and sofosbuvir/velpatasvir have been approved worldwide for the treatment of chronic hepatitis C virus (HCV) infection. Although both have been approved in China, there are currently no data on their pharmacokinetic profiles in Chinese individuals. Two studies investigated the pharmacokinetic properties, safety, and tolerability of ledipasvir/sofosbuvir and sofosbuvir/velpatasvir, respectively, in healthy Chinese subjects. METHODS: Two Phase I, open-label, single- and multiple-dose studies were conducted in healthy Chinese subjects. Ledipasvir/sofosbuvir (90/400 mg) or sofosbuvir/velpatasvir (400/100 mg), respectively, was administered orally once daily under fasted conditions. Subjects received a single dose (day 1) and multiple doses (days 8-17 [ledipasvir/sofosbuvir]; days 8-14 [sofosbuvir/velpatasvir]). Plasma pharmacokinetic parameters were estimated by using noncompartmental models, and safety was assessed through clinical evaluation and monitoring of adverse events. FINDINGS: Fourteen subjects were enrolled in each study (7 men, 7 women each; mean age, 30 years [ledipasvir/sofosbuvir] and 29 years [sofosbuvir/velpatasvir]). The pharmacokinetic parameters for sofosbuvir, GS-566500, GS-331007, and ledipasvir or velpatasvir were similar to historical values in non-Chinese subjects. Consistent with the t1/2 of ledipasvir relative to 24-h dosing, accumulation of 177% (AUC) and 107% (Cmax) was observed. There was no significant accumulation of velpatasvir, sofosbuvir, GS-566500, or GS-331007. Both drugs were generally well tolerated; no serious adverse events or discontinuations due to adverse events were reported. IMPLICATIONS: Overall, ledipasvir/sofosbuvir and sofosbuvir/velpatasvir exhibited pharmacokinetic and safety profiles in healthy Chinese subjects similar to those in non-Chinese subjects in historical studies, supporting their use in the Chinese population with HCV infection. ChinaDrugTrials.org.cn identifiers: CTR20160149 (ledipasvir/sofosbuvir); CTR20160602 (sofosbuvir/velpatasvir).

Revefenacin, a Long-Acting Muscarinic Antagonist, Does Not Prolong QT Interval in Healthy Subjects: Results of a Placebo- and Positive-Controlled Thorough QT Study.

Revefenacin is a novel once-daily, lung-selective, long-acting muscarinic antagonist developed as a nebulized inhalation solution for the maintenance treatment of chronic obstructive pulmonary disease. In a randomized, 4-way crossover study, healthy subjects received a single inhaled dose of revefenacin 175 µg (therapeutic dose), revefenacin 700 µg (supratherapeutic dose), and placebo via standard jet nebulizer, and a single oral dose of moxifloxacin 400 mg (open-label) in separate treatment periods. Electrocardiograms were recorded, and pharmacokinetic samples were collected serially after dosing. The primary end point was the placebo-corrected change from baseline QT interval corrected for heart rate using Fridericia's formula, analyzed at each postdose time. Concentration-QTc modeling was also performed. Following administration of revefenacin 175  and 700 µg, placebo-corrected change from baseline QTcF (ΔΔQTcF) values were close to 0 at all times, with the largest mean ΔΔQTcF of 1.0 millisecond (95% confidence interval [CI], -1.2 to 3.1 milliseconds) 8 hours postdose and 1.0 millisecond (95%CI, -1.1 to 3.1 milliseconds) 1 hour postdose after inhalation of revefenacin 175 and 700 µg, respectively. Revefenacin did not have a clinically meaningful effect on heart rate (within ±5 beats per minute of placebo), or PR and QRS intervals (within ±3 and ±1 milliseconds of placebo, respectively). Using concentration-QTc modeling, an effect of revefenacin > 10 milliseconds can be excluded within the observed plasma concentration range of up to ≈3 ng/mL. Both doses of revefenacin were well tolerated. These results demonstrate that revefenacin does not prolong the QT interval.

Development and Validation of a New LC-MS/MS Analytical Method for Direct-Acting Antivirals and Its Application in End-Stage Renal Disease Patients.

BACKGROUND AND OBJECTIVE: The effectiveness of direct-acting antivirals (DAAs) is not well established in end-stage renal disease (ESRD) patients. Assessment of the plasma concentrations may support understanding of their therapeutic outcomes in this population. The aim of this study is to develop a direct, yet matrix-effect tolerant, analytical method for determining DAAs in the plasma of ESRD patients while maintaining a moderate cost per sample and with an improved analyte extraction recovery. METHODS: In this study, a liquid chromatography-tandem mass spectrometric (LC-MS/MS) method was developed for the analysis of ombitasvir (OMB), paritaprevir (PRT) and ritonavir (RIT) in plasma. Sample preparation was performed using the liquid-liquid extraction (LLE) method. Isocratic separation was performed using a mixture of methanol and 10 mM ammonium acetate (79:21, v/v) followed by MS/MS detection. The method was validated and applied to determine DAAs in the plasma of ESRD patients (n = 7). RESULTS: The developed method was linear (r2 > 0.995), accurate (89.4 ± 7.8 to 108.3 ± 3.0) and precise (% CV 0.9-15.0) and showed improved recovery (> 80) over previously published ones in the range 5-250, 30-1,500, 20-1,000 ng/mL for OMB, PRT and RIT, respectively. Relative matrix effect was absent, and the method accurately determined the three DAAs in real-life samples (n = 7). CONCLUSIONS: An efficient analytical method for the determination of DAAs is presented. The method overcomes the potential analytical response fluctuation in ESRD. The developed method show improved extraction recoveries and is suitable for routine application in developing economies where hepatitis C virus is most prevalent.

Single-Dose Pharmacokinetics and Safety of Solriamfetol in Participants With Normal or Impaired Renal Function and With End-Stage Renal Disease Requiring Hemodialysis.

Solriamfetol (JZP-110), a selective dopamine and norepinephrine reuptake inhibitor with wake-promoting effects, is renally excreted ∼90% unchanged within 48 hours. Effects of renal impairment and hemodialysis on the pharmacokinetics and safety of 75-mg single-dose solriamfetol were evaluated in adults with normal renal function (n = 6); mild (n = 6), moderate (n = 6), or severe (n = 6) renal impairment; and end-stage renal disease (ESRD) with and without hemodialysis (n = 7). Relative to normal renal function, geometric mean area under the plasma concentration-time curve from time zero to infinity increased 53%, 129%, and 339%, and mean half-life was 1.2-, 1.9-, and 3.9-fold higher with mild, moderate, and severe renal impairment, respectively. Renal excretion of unchanged solriamfetol over 48 hours was 85.8%, 80.0%, 66.4%, and 57.1% in normal, mild, moderate, and severe renal impairment groups, respectively; mean maximum concentration and time to maximum concentration did not vary substantially. Decreases in solriamfetol clearance were proportional to decreases in estimated glomerular filtration rate. Geometric mean area under the plasma concentration-time curve from time zero to time of last quantifiable concentration increased 357% and 518% vs normal in ESRD with and without hemodialysis, respectively, with half-life >100 hours in both groups. Over the 4-hour hemodialysis period, ∼21% of solriamfetol dose was removed. Adverse events included headache (n = 1) and nausea (n = 1). Six days after dosing, 1 participant had increased alanine and aspartate aminotransferase, leading to study discontinuation. While these adverse events were deemed study-drug related, they were mild and resolved. Results from this study combined with population pharmacokinetic modeling/simulation suggest that solriamfetol dosage adjustments are necessary in patients with moderate or severe but not with mild renal impairment. Due to significant exposure increase/prolonged half-life, dosing is not recommended in patients with ESRD.

Colloidal Drug Aggregate Stability in High Serum Conditions and Pharmacokinetic Consequence.

Colloidal drug aggregates have been a nuisance in drug screening, yet, because they inherently comprise drug-rich particles, they may be useful in vivo if issues of stability can be addressed. As the first step toward answering this question, we optimized colloidal drug aggregate formulations using a fluorescence-based assay to study fulvestrant colloidal formation and stability in high (90%) serum conditions in vitro. We show, for the first time, that the critical aggregation concentration of fulvestrant depends on media composition and increases with serum concentration. Excipients, such as polysorbate 80, stabilize fulvestrant colloids in 90% serum in vitro for over 48 h. Using fulvestrant and an investigational pro-drug, pentyloxycarbonyl-( p-aminobenzyl) doxazolidinylcarbamate (PPD), as proof-of-concept colloidal formulations, we demonstrate that the in vivo plasma half-life for stabilized colloids is greater than their respective monomeric forms. These studies demonstrate the potential of turning the nuisance of colloidal drug aggregation into an opportunity for drug-rich formulations.

Simultaneous determination of amitraz, chlordimeform, formetanate and metabolites in human blood by liquid chromatography tandem mass spectrometry with phospholipid-removal pretreatment.

In this study, a reliable ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method coupled with an easy, fast and effective sample pretreatment procedure was developed for simultaneous determination of amitraz, chlordimeform, formetanate and their metabolites in human blood. With the procedures of protein precipitation and a phospholipid-removal step, the endogenous compound interference was significantly reduced, and matrix effects were significantly reduced. The linear ranges of matrix-matched standard curves were from 0.5 to 1000 ng/mL with coefficients of determination >0.996. Very low limits of detection (0.05-0.12 ng/mL) and limits of quantitation (0.15-0.4 ng/mL) were achieved. Reasonable recoveries ranging from 88.1 to 103.5% were obtained. The intra-day RSDs ranging from 3.2 to 8.6% and inter-day RSDs ranging from 4.8 to 9.2% indicated good precision. With the introduction of a phospholipid-removal step, the ME ranged from 90.1 to 98.5%. The established method was successfully applied to the analysis of a blood sample from a formetanate poisoning case. This method possesses the advantages of high sensitivity, reduced matrix effects and rapidity.

Micelle and inclusion complex enhanced spectrofluorimetric methods for determination of Retigabine: Application in pharmaceutical and biological analysis.

Two new, simple, selective, and highly sensitive spectrofluorimetric methods were developed and validated for the determination of the antiepileptic drug; retigabine (RTG). The first method (Method-I) depends on enhancement of the weak native fluorescence of RTG via the use of an organized medium; sodium dodecyl sulphate (SDS) in acetate buffer (pH 3.74). The second method (Method-II) depends on the enhancement of RTG weak native fluorescence through complexation with a macromolecule; beta cyclodextrin (ß-CD) in phosphate buffer (pH 3.20). A full study of different experimental parameters influencing the fluorescence intensity was carried out. In addition, a thorough investigation of the fluorescence quantum yield, fluorophore brightness and mechanism of fluorescence enhancement was performed. A seven-fold improvement in the fluorescence intensity was brought by the first method, whereas a six and half-fold enhancement of the fluorescence intensity was obtained by the second one. Linearity was achieved over wide ranges (0.05-12.5 µg mL-1) and (0.05-15 µg mL-1) with low limits of detection (LOD) of 10.6 and 14.3 ng mL-1, and limits of quantification (LOQ) of 32.0 and 43.2 ng mL-1 for (Method-I) and (Method-II), respectively. The proposed methods were validated according to ICH and US-FDA guidelines. The applicability of the proposed methods was tested for determination of RTG in its pharmaceutical dosage forms, and to study the stability of RTG under different stress conditions according to ICH guidelines including alkaline, acidic, oxidative, thermal, and photolytic stress conditions. Moreover, the high sensitivity achieved by the proposed methods permitted the determination and detection of RTG in both spiked and real rabbit plasma samples utilizing a simple protein precipitation step followed by liquid-liquid extraction method. Percentage recoveries from rabbit plasma samples were within the acceptable limits; (93.47-104.74%) and (91.33-105.70%) for (Method-I) and (Method-II), respectively.

Análise do impacto do uso de organofosforados e carbamatos em trabalhadores rurais de um município da região noroeste do estado do Rio Grande do Sul / Impact analysis of the use of organophosphates and carbamates rural workers of the municipality in a northwest region of Rio Grande do Sul

Anualmente milhões de agricultores são intoxicados no mundo, e destes, mais de 20 mil morrem em consequência da exposição a agrotóxicos. Intoxicações por organofosforados (OF) e carbamatos (CAR) representam as maiores ameaças à saúde dos trabalhadores rurais. Os OF e CAR atuam na inibição da enzima colinesterase, sendo assim a inibição desta mostra-se um excelente indicador da severidade da intoxicação. O objetivo deste estudo foi analisar o impacto do uso de OF e CAR em trabalhadores rurais na cidade de Mato Queimado/RS. Foi realizado um estudo transversal, prospectivo e experimental. Investigaramse 27 trabalhadores rurais expostos. Foram realizadas coletas sanguíneas e dados epidemiográficos nos meses de fevereiro e março de 2014. A atividade da colinesterase foi determinada através do método bioquímico cinético colorimétrico. A faixa etária média dos participantes foi 34,6 anos (± 8,5). A forma de contato mais prevalente foi a aplicação do produto (88,9%). O tempo médio de exposição foi de 10,7 anos. 70,4% relataram usar equipamentos de proteção individual (EPI), sendo mais frequente o uso de máscara (55,5%). A média dos valores de colinesterase para foi de 3244,45 U/I (± 345,8), níveis estes abaixo dos de referência. Através dos resultados obtidos nesta pesquisa torna-se imprescindível a utilização de meios de monitoramento biológico dos trabalhadores rurais na finalidade de prevenção e promoção da saúde.

Annually millions of rural workers are intoxicated in the world, and of these, more than 20,000 die as a result of exposure to pesticides. Intoxication by insecticides organophosphate (OF) and carbamates (CAR) represent the greatest threats to the health of rural workers. OF CAR and act on the inhibition of cholinesterase enzyme, thus inhibition of this proves to be an excellent indicator of the severity of the intoxication. The objective of this study was to analyze the impact of using OF CAR and in rural workers in the city of Mato Queimado/RS. A cross-sectional, prospective and experimental study was conducted. Twenty-three rural workers exposed were investigated. Sample collection and data demographic were conducted in February and March 2014. The cholinesterase activity was determined by biochemical kinetic colorimetric method. The average age of participants was 34.6 years (± 8.5). The most prevalent form of contact is via the application of the product (88.9%). The mean duration of exposure was 10.7 years. Still, 70.4% reported using personal protective equipment (PPE), more frequent use of mask (55.5%). The average values for cholinesterase was 3244.45 U/l (± 345.8) levels below those of the reference. The results obtained in this study are essential to use biological monitoring means of rural workers in purpose of prevention and health promotion.

Rapid bioanalytical LC-MS/MS method for the simultaneous determination of sofosbuvir and velpatasvir in human plasma-application to a pharmacokinetic study in Egyptian volunteers.

A novel, rapid and validated LC-MS/MS bioanalytical method has been developed for the extraction and determination of sofosbuvir and velpatasvir simultaneously in human plasma using ledipasvir as an internal standard (IS). The simple and reproducible protein precipitation technique with acetonitrile was successfully used for the deproteinization and extraction of the analytes from human plasma matrix. The developed method achieved consistent recoveries over different concentrations with average extraction recoveries of 81.72% and 80.46% for sofosbuvir and velpatasvir, respectively. The chromatographic separation was performed within only 2.80 min as a run time by an isocratic elution through C18 Zorbox eclipse plus (100 × 4.6 mm, 5 µm). Optimum mobile phase consisted of 0.1% formic acid in water: acetonitrile: methanol (30:60:10, v/v/v) pumped at a flow rate of 0.55 mL min-1 and injection volume was 10 µL. LC-MS/MS detection was done by multiple reaction monitoring transitions operating at positive ionization mode for both analytes and IS. Bioanalytical method validation as per EMA guidelines was carried out where the proposed method revealed linearity over the concentration range of 5-5000 and 10-1500 ng mL-1 for sofosbuvir and velpatasvir, respectively. After validation, the method was applied to the analysis of the two drugs after a single oral administration of Epclusa 400/100 mg tablets to Egyptian healthy volunteers.