The multidrug resistance protein (MRP) 4 is expressed and functionally active in isolated bladder from pig.

Multidrug resistance proteins type 4 (MRP4) and 5 (MRP5) play pivotal roles in the transport of cyclic nucleotides in various tissues. However, their specific functions within the lower urinary tract remain relatively unexplored. This study aimed to investigate the effect of pharmacological inhibition of MRPs on cyclic nucleotide signaling in isolated pig bladder. The relaxation responses of the bladder were assessed in the presence of the MRP inhibitor, MK571. The temporal changes in intra- and extracellular levels of cAMP and cGMP in stimulated tissues were determined by mass spectrometry. The gene (ABCC4) and protein (MRP4) expression were also determined. MK571 administration resulted in a modest relaxation effect of approximately 26% in carbachol-pre-contracted bladders. The relaxation induced by phosphodiesterase inhibitors such as cilostazol, tadalafil, and sildenafil was significantly potentiated in the presence of MK571. In contrast, no significant potentiation was observed in the relaxation induced by substances elevating cAMP levels or stimulators of soluble guanylate cyclase. Following forskolin stimulation, both intracellular and extracellular cAMP concentrations increased by approximately 15.8-fold and 12-fold, respectively. Similarly, stimulation with tadalafil + BAY 41-2272 resulted in roughly 8.2-fold and 3.4-fold increases in intracellular and extracellular cGMP concentrations, respectively. The presence of MK571 reduced only the extracellular levels of cGMP. This study reveals the presence and function of MRP4 transporters within the porcine bladder and paves the way for future research exploring the role of this transporter in both underactive and overactive bladder disorders.

Metabolic Stability and Metabolite Identification of N-Ethyl Pentedrone Using Rat, Mouse and Human Liver Microsomes.

New Psychoactive Substances (NPSs) are defined as a group of substances produced from molecular modifications of traditional drugs. These molecules represent a public health problem since information about their metabolites and toxicity is poorly understood. N-ethyl pentedrone (NEP) is an NPS that was identified in the illicit market for the first time in the mid-2010s, with four intoxication cases later described in the literature. This study aims to evaluate the metabolic stability of NEP as well as to identify its metabolites using three liver microsomes models. To investigate metabolic stability, NEP was incubated with rat (RLM), mouse (MLM) and human (HLM) liver microsomes and its concentration over time evaluated by liquid chromatography-mass spectrometry. For metabolite identification, the same procedure was employed, but the samples were analyzed by liquid chromatography-high resolution mass spectrometry. Different metabolism profiles were observed depending on the model employed and kinetic parameters were determined. The in vitro NEP elimination half-lives (t1/2) were 12.1, 187 and 770 min for the rat, mouse and human models, respectively. Additionally, in vitro intrinsic clearances (Cl int, in vitro) were 229 for rat, 14.8 for mouse, and 3.6 µL/min/mg in the human model, and in vivo intrinsic clearances (Cl int, in vivo) 128, 58.3, and 3.7 mL/min/kg, respectively. The HLM model had the lowest rate of metabolism when compared to RLM and MLM. Also, twelve NEP metabolites were identified from all models, but at different rates of production.

In vitro metabolism of the new antifungal dapaconazole using liver microsomes.

Dapaconazole is a new antifungal imidazole that has been shown a high efficacy against several pathogenic fungi. This study aimed to investigate the interspecies variation in the in vitro metabolic profiles and in vivo hepatic clearance (CLH,in vivo) prediction of dapaconazole using liver microsomes from male Sprague Dawley rat, male Beagle dog and mixed gender human using a liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS) method. In addition, the produced metabolites were identified by ultra-high-performance liquid chromatography with quadrupole time-of-flight mass spectrometer (UHPLC-QTOF-MS/MS). The microsomal protein concentration of 0.1 mg/mL and the incubation time of 10 min were employed for the kinetics determination, resulting in a sigmoidal kinetic profile for all species evaluated. The predicted CLH,in vivo was 6.5, 11.6 and 7.5 mL/min/kg for human, rat and dog, respectively. Furthermore, five metabolized products were identified. These findings provide preliminary information for understanding dapaconazole metabolism and the interspecies differences in catalytic behaviours, supporting the choice of a suitable laboratory animal for future pharmacokinetics and metabolism studies.

Quantification of cyclic AMP and cyclic GMP levels in Krebs-Henseleit solution by LC-MS/MS: Application in washed platelet aggregation samples.

Cyclic Nucleotides are important in regulating platelet function. Increases in 3'5'-cyclic adenosine monophosphate (cAMP) and 3'5'-cyclic guanosine monophosphate (cGMP) inhibit platelet aggregation and are pharmacological targets for antiplatelet therapy. Here we report an improved method for determining cAMP and cGMP concentrations and, for the first time, in washed platelet supernatants by combining high-performance liquid chromatography and tandem mass spectrometry (LC-MS/MS). Characteristic peaks of the substrates, cGMP or cAMP and their internal standards were identified in negative-ion electrospray ionisation using multiple reaction monitoring. Compared with previously reported methods, the method presented here shows high precision with the lowest lower limit of quantification (LLoQ) to date (10 pg/mL). The effect of a novel catecholamine, 6-nitrodopamine, on cyclic nucleotide levels was quantified. Our results showed that this new method was fast, sensitive, and highly reproducible.

Comparative bioavailability study with two sodium valproate tablet formulations administered under fasting conditions in healthy subjects.

AIM: To assess the bioequivalence of two sodium valproate formulations in healthy subjects of both sexes. MATERIALS AND METHODS: The study was conducted using an open, randomized, two-period crossover design with a 2-week washout interval. Plasma samples were obtained over a 96-hour period. Plasma concentrations of valproate were analyzed by liquid chromatography coupled to tandem mass spectrometry (LC/MS) with negative ion electrospray ionization. From the sodium valproate plasma concentration vs. time curves, the following pharmacokinetic parameters were obtained Cmax, AUC, tmax, Ke, and T1/2. RESULTS: The geometric mean with corresponding 90% confidence interval for test/reference percent ratios were 104.43% (90% CI 100.42 - 108.61%) for Cmax, 98.11% (90% CI = 94.66 - 101.70%) for AUClast, and 96.71% (90% CI = 92.97 - 100.60%) for AUC0-inf. CONCLUSION: Since the 90% CI for Cmax and AUClast ratios were all inside the 80 - 125% interval proposed by the US Food and Drug Administration Agency (FDA), it was concluded that the new sodium valproate formulation (epilenil 500-mg coated tablet) without food elaborated by Biolab Sanus Farmaceutica Ltda is bioequivalent to depakene formulation for both the rate and the extent of absorption.

TECHNIQUE OF EXPOSURE OF THE ESOPHAGOGASTRIC JUNCTION OBTAINED BY THE FLEXIBLE LIVER RETRACTOR IN BARIATRIC SURGERY: A RANDOMIZED CONTROLLED TRIAL. / TÉCNICA DE EXPOSIÇÃO DA JUNÇÃO ESOFAGOGÁSTRICA OBTIDA POR MEIO DE AFASTADOR FLEXÍVEL DE FÍGADO EM CIRURGIA BARIÁTRICA: ENSAIO CLÍNICO RANDOMIZADO.

AIM: In the Roux-en-Y gastric bypass technique, classic laparoscopic surgical retractors are usually rigid, require an additional incision for its installation, or must be handled by an assistant during the surgical procedure, involving a risk of liver injury. The aim of this study was to evaluate and validate a technique of the esophagogastric junction exposure obtained by the flexible liver retractor in bariatric surgery, comparing its efficacy with the retractor classically used for this purpose. METHODS: This study was performed as a randomized, open, prospective, controlled, and comparative design in patients with medical indications of bariatric surgery. The subjects were distributed in the classic (control) and flexible (test) retractor groups. RESULTS: A total of 100 patients (n=50 control group, n=50 test group) were included. No statistically significant difference was observed in the mean duration of surgery. Regarding visibility, 100% of the patients in the flexible retractor group demonstrated an optimal visibility level, although without statistical significance concerning the classic retractor group (94%). Invariably, carrying a trocar was necessary when using the classic retractor. CONCLUSIONS: The flexible liver retractor is safe, effective, ergonomic, and inexpensive. Furthermore, it presented a satisfactory aesthetic profile, and the use of specific instruments, new adaptation curve, and training for its handling were not required.

OBJETIVOS: Os afastadores clássicos de cirurgia laparoscópica são geralmente rígidos, necessitando de uma incisão adicional para sua instalação ou de um auxiliar para manuseio durante o ato cirúrgico e ainda, podem envolvem risco de injúria hepática. Avaliar e validar uma técnica de exposição da junção esofagogástrica obtida pelo afastador flexível de fígado em cirurgia bariátrica comparando sua eficácia com a de afastador classicamente utilizado para este fim. MÉTODOS: Tratou-se de um estudo prospectivo, aberto, controlado e comparativo em pacientes com indicação de cirurgia, distribuídos de forma randomizada em dois grupos: clássico (controle) e afastador flexível (teste). RESULTADOS: Foram incluídos 100 pacientes (n=50 grupo controle, n=50 grupo teste), sem diferença estatística na distribuição por idade e por morbidades, havendo diferença estatística somente no gênero (grupo controle obteve proporção maior de homens, p=0,020). Em relação ao tempo médio de realização das operações, não foi constatada diferença estatística. No quesito visibilidade, verificou-se que 100% dos pacientes do grupo afastador flexível obteve nível de visibilidade ótima, porém sem significância estatística com relação ao grupo clássico (94%). Invariavelmente, foi necessário um portal a mais de trocarte quando do uso do afastador clássico. CONCLUSÃO: O afastador flexível de fígado demonstrou-se seguro, eficaz, ergonômico, de baixo custo, de perfil estético satisfatório, não requerendo instrumental específico para uso ou nova curva de adaptação e aprendizado para manuseio.

Polypodium leucotomos targets multiple aspects of oral carcinogenesis and it is a potential antitumor phytotherapy against tongue cancer growth.

Introduction: Oral cancer refers to malignant tumors, of which 90% are squamous cell carcinomas (OSCCs). These malignancies exhibit rapid progression, poor prognosis, and often mutilating therapeutical approaches. The determination of a prophylactic and/or therapeutic antitumor role of the polyphenolic extract Polypodium leucotomos(PL) would be relevant in developing new tools for prevention and treatment. Methods: We aimed to determine the antitumor effect of PL by treating OSCC cell lines with PL metabolites and evaluating its action during OSCC progression in vivo. Results: PL treatment successfully impaired cell cycling and proliferation, migration, and invasion, enhanced apoptosis, and modulated macrophage polarization associated with the tumoral immune-inflammatory response of tongue cancer cell lines (TSCC). PL treatment significantly decreased the expression of MMP1 (p < 0.01) and MMP2 (p < 0.001), and increased the expression of TIMP1 (p < 0.001) and TIMP2 (p < 0.0001) in these cells. The mesenchymal-epithelial transition phenotype was promoted in cells treated with PL, through upregulation of E-CAD (p < 0.001) and reduction of N-CAD (p < 0.05). PL restrained OSCC progression in vivo by inhibiting tumor volume growth and decreasing the number of severe dysplasia lesions and squamous cell carcinomas. Ki-67 was significantly higher expressed in tongue tissues of animals not treated with PL(p < 0.05), and a notable reduction in Bcl2 (p < 0.05) and Pcna (p < 0.05) cell proliferation-associated genes was found in dysplastic lesions and TSCCs of PL-treated mice. Finally, N-cad(Cdh2), Vim, and Twist were significantly reduced in tongue tissues treated with PL. Conclusion: PL significantly decreased OSCC carcinogenic processes in vitro and inhibited tumor progression in vivo. PL also appears to contribute to the modulation of immune-inflammatory oral tumor-associated responses. Taken together, these results suggest that PL plays an important antitumor role in processes associated with oral carcinogenesis and may be a potential phytotherapeutic target for the prevention and/or adjuvant treatment of TSCCs.

Prospective Prediction of Dapaconazole Clinical Drug-Drug Interactions Using an In Vitro to In Vivo Extrapolation Equation and PBPK Modeling.

This study predicted dapaconazole clinical drug−drug interactions (DDIs) over the main Cytochrome P450 (CYP) isoenzymes using static (in vitro to in vivo extrapolation equation, IVIVE) and dynamic (PBPK model) approaches. The in vitro inhibition of main CYP450 isoenzymes by dapaconazole in a human liver microsome incubation medium was evaluated. A dapaconazole PBPK model (Simcyp version 20) in dogs was developed and qualified using observed data and was scaled up for humans. Static and dynamic models to predict DDIs following current FDA guidelines were applied. The in vitro dapaconazole inhibition was observed for all isoforms investigated, including CYP1A2 (IC50 of 3.68 µM), CYP2A6 (20.7 µM), 2C8 (104.1 µM), 2C9 (0.22 µM), 2C19 (0.05 µM), 2D6 (0.87 µM), and 3A4 (0.008−0.03 µM). The dynamic (PBPK) and static DDI mechanistic model-based analyses suggest that dapaconazole is a weak inhibitor (AUCR > 1.25 and <2) of CYP1A2 and CYP2C9, a moderate inhibitor (AUCR > 2 and <5) of CYP2C8 and CYP2D6, and a strong inhibitor (AUCR ≥ 5) of CYP2C19 and CYP3A, considering a clinical scenario. The results presented may be a useful guide for future in vivo and clinical dapaconazole studies.

Quantification of 6-nitrodopamine in Krebs-Henseleit's solution by LC-MS/MS for the assessment of its basal release from Chelonoidis carbonaria aortae in vitro.

In this study, the development and validation of a method for quantification of 6-nitrodopamine in Krebs-Henseleit's solution by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) with positive ion electrospray ionization is described. Aortic rings taken from tortoise were either denuded or left with endothelium intact (15 mm, N = 6) and were incubated for 30 min in 5 mL Krebs-Henseleit's solution in an organ bath. Solid phase extraction (SPE) was performed for aliquots of 1 mL of the supernatant. The separation of 6-nitrodopamine was obtained on a 150 mm × 3 mm Shim-pack GIST-HP C18 column, using 75% of mobile phase A consisted of deionized water with 0.1% formic acid (v/v) and 25% of mobile phase B consisted of acetonitrile/deionized water (50/50, v/v) + 0.1% formic acid at a flow rate of 350 µL/min in an isocratic mode. The method was linear over the concentration range of 0.1-20 ng/mL. The method was sensitive, precise and accurate for the assessment of the basal release of 6-nitrodopamine from Chelonoidis carbonaria aortae in vitro. The mean ± SEM concentrations of 6-nitrodopamine released from endothelium-intact and endothelium-denuded aortae were 0.44 ± 0.06 ng/mL and 0.18 ± 0.05 ng/mL, respectively. These results indicate that tortoise's aortae display a basal endothelium-derived 6-nitrodopamine release.

Single-dose pharmacokinetics and pharmacodynamics assessment of oestriol and trimegestone containing vaginal rings in healthy women with childbearing potential.

PURPOSE: To evaluate the pharmacokinetics and pharmacodynamics of oestriol (E3) and trimegestone (TMG) in healthy women after application of three different vaginal rings over 21 days. The vaginal rings had a nominal delivery rate of 0.413/0.050 mg/day (Test 1), 0.311/0.090 mg/day (Test 2) and 0.209/0.137 mg/day (Test 3) E3/TMG. METHODS: Thirty-five healthy women were randomised to receive a single application of Test 1, 2 or 3 (Clinical Trial NCT03343912). The E3 and TMG plasma concentration was determined by LC-MS/MS. Oestradiol (E2) and progesterone (PG) serum concentrations, and bleeding patern were determined as pharmacodynamic parameters. Safety was assessed by evaluation of adverse events and local tolerability. RESULTS: The total and maximum exposure of E3 and TMG increased in a proportional ratio to dose. However, not in a magnitude which was expected from the dose differences for E3. During Test 2 and 3 treatment all E2 and PG values remained on a well suppressed level until end of treatment. E2 and PG serum levels increased distinctly earlier after ring removal with Test 1 compared to Test 2 and 3. Test 3 achieved 95.24% of "no bleeding" days under treatment followed by Test 1 (91.67%), and Test 2 (86.15%). CONCLUSIONS: The Test 3 formulation presented the best dose combination of E3/TMG for contraception. Moreover, all vaginal rings were well tolerated.

Simultaneous quantification of cyclosporin, tacrolimus, sirolimus and everolimus in whole blood by UHPLC-MS/MS for therapeutic drug monitoring.

The aim of this study was to develop and validate a UHPLC-MS/MS assay to quantify cyclosporin (CYC), tacrolimus (TAC), sirolimus (SIR) and everolimus (EVE) in human whole blood for therapeutic drug monitoring. Analytes were extracted from 50 µL human whole blood by protein precipitation. The separation of the drugs was performed on an Acquity UPLC BEH C18 column. Analytes were eluted with a mobile phase consisting of 2 mM ammonium acetate with 0.1% formic acid (v/v) in deionised water and 2 mM ammonium acetate with 0.1% formic acid (v/v) in methanol at a flow rate of 300 µL/min in gradient elution. The method performance was evaluated by analysing patient blood samples and/or external quality control samples [proficiency testing (PT) scheme]. The method was linear from 23.75 to 1094.0, 1.3 to 42.4, 1.3 to 47.0 and 1.2-41.6 µg/mL for CYC, TAC, SIR and EVE, respectively. The within- and between-assay reproducibility results were ˂ 11%. Results from PT and patient sample quantification were comparable to those obtained previously by an in-house validated method using protein precipitation and liquid-liquid extraction. This method showed good analytical performance for quantifying CYC, TAC, SIR and EVE in whole blood over their respective calibration ranges.

Determination of dopamine, noradrenaline, and adrenaline in Krebs-Henseleit solution by liquid chromatography coupled with tandem mass spectrometry and measurement of their basal release from Chelonoidis carbonaria aortae in vitro.

This study presented for the first time the development and validation of a sensitive method for quantification of dopamine, noradrenaline, and adrenaline in Krebs-Henseleit solution by LC-tandem mass spectrometry. Aliquots of 2.0 mL calibrators, quality controls, and samples of Krebs-Henseleit solution incubated with tortoise's aortic ring for 30 min were extracted by solid-phase extraction. Catecholamine separation was achieved on a 100 × 4.6 mm LiChrospher RP-8 column and the quantification was performed by a mass spectrometer equipped with an electrospray interface operating in positive ion mode. The run time was 4 min and the calibration curve was linear over the range of 0.1-20.0 ng/mL. The method was applied to the measurement of basal release of dopamine, noradrenaline, and adrenaline from the tortoise Chelonoidis carbonaria aortae in vitro. One aortic ring (30 mm) per tortoise (n = 5) was incubated for 30 min in a 5 mL organ bath filled with Krebs-Henseleit solution. The method demonstrated sensitivity, precision, and accuracy enough for its application in the measurement of basal release of these catecholamines from C. carbonaria aortic rings in vitro. The mean (standard deviation) concentrations of dopamine, noradrenaline, and adrenaline were 3.48 (2.55) ng/mL, 1.40 (0.57) ng/mL, and 1.87 (1.09) ng/mL, respectively.

Quantification of mycophenolic acid in human plasma by liquid chromatography with time-of-flight mass spectrometry for therapeutic drug monitoring.

This study presents, for the first time, the development and validation of a liquid chromatography and time-of-flight mass-spectrometry (LC-TOF-MS) based assay to quantify mycophenolic acid (MPA) in patient samples as part of a routine therapeutic drug monitoring service. MPA was extracted from 50 µl human plasma by protein precipitation, using sulindac as internal standard (IS). Separation was obtained on a Luna™ Omega polar C18 column kept at 40°C. The mobile phase consisted of a mixture of acetonitrile-deionized water (50:50, v/v) with 0.1% formic acid at a flow rate of 350 µl/min. Analyte and IS were monitored on a TOF-MS using a Jet-Stream™ (electrospray) interface running in positive mode. Assay performance was evaluated by analysing patient plasma (N = 69) and external quality assessment (N = 6) samples. The retention times were 2.66 and 2.18 min for MPA and IS, respectively. The lower limit of quantification of MPA was 0.1 µg/ml. The within- and between-assay reproducibility results ranged from 1.81 to 10.72%. Patient and external quality assessment sample results were comparable with those obtained previously by an in-house validated LC-MS/MS method. This method showed satisfactory analytical performance for the determination of MPA in plasma over the calibration range of 0.1-15.0 µg/ml.

Técnica de exposição da junção esofagogástrica obtida por meio de afastador flexível de fígado em cirurgia bariátrica: ensaio clínico randomizado / Technique of exposure of the esophagogastric junction obtained by the flexible liver retractor in bariatric surgery: a randomized controlled trial

RESUMO - RACIONAL: Os afastadores clássicos de cirurgia laparoscópica são geralmente rígidos, necessitando de uma incisão adicional para sua instalação ou de um auxiliar para manuseio durante o ato cirúrgico e ainda, podem envolvem risco de injúria hepática. OBJETIVOS: Avaliar e validar uma técnica de exposição da junção esofagogástrica obtida pelo afastador flexível de fígado em cirurgia bariátrica comparando sua eficácia com a de afastador classicamente utilizado para este fim. MÉTODOS: Tratou-se de um estudo prospectivo, aberto, controlado e comparativo em pacientes com indicação de cirurgia, distribuídos de forma randomizada em dois grupos: clássico (controle) e afastador flexível (teste). RESULTADOS: Foram incluídos 100 pacientes (n=50 grupo controle, n=50 grupo teste), sem diferença estatística na distribuição por idade e por morbidades, havendo diferença estatística somente no gênero (grupo controle obteve proporção maior de homens, p=0,020). Em relação ao tempo médio de realização das operações, não foi constatada diferença estatística. No quesito visibilidade, verificou-se que 100% dos pacientes do grupo afastador flexível obteve nível de visibilidade ótima, porém sem significância estatística com relação ao grupo clássico (94%). Invariavelmente, foi necessário um portal a mais de trocarte quando do uso do afastador clássico. CONCLUSÃO: O afastador flexível de fígado demonstrou-se seguro, eficaz, ergonômico, de baixo custo, de perfil estético satisfatório, não requerendo instrumental específico para uso ou nova curva de adaptação e aprendizado para manuseio.

ABSTRACT - BACKGROUND: In the Roux-en-Y gastric bypass technique, classic laparoscopic surgical retractors are usually rigid, require an additional incision for its installation, or must be handled by an assistant during the surgical procedure, involving a risk of liver injury. Aim: The aim of this study was to evaluate and validate a technique of the esophagogastric junction exposure obtained by the flexible liver retractor in bariatric surgery, comparing its efficacy with the retractor classically used for this purpose. Methods: This study was performed as a randomized, open, prospective, controlled, and comparative design in patients with medical indications of bariatric surgery. The subjects were distributed in the classic (control) and flexible (test) retractor groups. Results: A total of 100 patients (n=50 control group, n=50 test group) were included. No statistically significant difference was observed in the mean duration of surgery. Regarding visibility, 100% of the patients in the flexible retractor group demonstrated an optimal visibility level, although without statistical significance concerning the classic retractor group (94%). Invariably, carrying a trocar was necessary when using the classic retractor. Conclusions: The flexible liver retractor is safe, effective, ergonomic, and inexpensive. Furthermore, it presented a satisfactory aesthetic profile, and the use of specific instruments, new adaptation curve, and training for its handling were not required.

Pharmacodynamic evaluation of L-carnitine and piracetam in muscle injury induced by the chronic use of simvastatin.

OBJECTIVES: To evaluate the effect of L-carnitine and piracetam on the muscle injury induced by simvastatin in healthy male subjects during the therapy with oral doses of 10 mL of a solution containing L-carnitine 100 mg/mL + piracetam 80 mg/mL (test group) or placebo (control group) and 40 mg simvastatin once a day during 35 consecutive days. The effect of L-carnitine and piracetam in the reduction of myopathic symptomatology caused by exercise, as well as safety and tolerability were also evaluated. MATERIALS AND METHODS: This study was performed on two different occasions, of which 42 subjects were investigated on occasion 1 and 19 on occasion 2. Discomfort or pain was evaluated according to modified Borg scale. Serum levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), γ-glutamyl transferase (γ-GT), creatine kinase (CK), and lactic dehydrogenase (LDH) were evaluated on the 4th, 11th, 18th, 25th, and 32nd day after therapy, and before and until 4 hours after an exercise test performed on a treadmill on day 36. RESULTS: A higher incidence of pain or discomfort was observed in the control group than in the test group, mainly in occasion 1 (29% vs. 62% experienced pain or discomfort in any period, p = 0.0295). The serum levels of AST, ALT, and LDH were statistically different, with lower values in the test group compared to the control group. CONCLUSION: Concomitant use of L-carnitine and piracetam might have a muscle-protective effect and protection against simvastatin-induced myalgia. Furthermore, the formulation was safe and well tolerated by the subjects investigated in this trial.

Bioavailability study of two 81-mg coated tablet formulations of acetylsalicylic acid in fed healthy subjects
.

OBJECTIVE: To perform a comparative bioavailability study between a test (re-formulation) and a reference acetylsalicylic acid formulation (Ecasil-81, 81 mg coated tablet) in healthy subjects under fed condition. MATERIALS AND METHODS: Healthy subjects (n = 48) were included in this monocentric, open-label, randomized, two-way crossover pharmacokinetic study. They received a single 81-mg oral dose of a test or a reference formulation of acetylsalicylic acid under fed condition, with a 7-day washout period between the treatments. Blood samples were collected over a period of 36 hours. The salicylic acid plasma concentration was measured by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). Pharmacokinetic analysis was performed using WinNonlin software. RESULTS: The geometric mean and 90% confidence interval of test/reference formulation ratios were 109.32% (102.54 - 116.54%) and 106.94% (102.97 - 111.07%) for salicylic acid Cmax and AUC0-last, respectively. Food decreased the AUC and Cmax (p < 0.001) and delayed the tmax (p = 0.0077). The investigated women presented higher AUC0-∞ and Cmax values (p < 0.001) than men. The clinical and laboratory exams did not show significant alterations. CONCLUSION: The re-formulation is bioequivalent to the reference formulation regarding the absorption extent and rate in fed healthy subjects. The administration of acetylsalicylic acid with food decreased its bioavailability. Moreover, differences in salicylic acid disposition related to sex were observed. The treatments were well tolerated by the investigated subjects.

Quantification of Plasma and Urine Thymidine and 2'-Deoxyuridine by LC-MS/MS for the Pharmacodynamic Evaluation of Erythrocyte Encapsulated Thymidine Phosphorylase in Patients with Mitochondrial Neurogastrointestinal Encephalomyopathy.

Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is an ultra-rare disorder caused by mutations in TYMP, leading to a deficiency in thymidine phosphorylase and a subsequent systemic accumulation of thymidine and 2'-deoxyuridine. Erythrocyte-encapsulated thymidine phosphorylase (EE-TP) is under clinical development as an enzyme replacement therapy for MNGIE. Bioanalytical methods were developed according to regulatory guidelines for the quantification of thymidine and 2'-deoxyuridine in plasma and urine using liquid chromatography-tandem mass spectrometry (LC-MS/MS) for supporting the pharmacodynamic evaluation of EE-TP. Samples were deproteinized with 5% perchloric acid (v/v) and the supernatants analyzed using a Hypercarb column (30 × 2.1 mm, 3 µm), with mobile phases of 0.1% formic acid in methanol and 0.1% formic acid in deionized water. Detection was conducted using an ion-spray interface running in positive mode. Isotopically labelled thymidine and 2'-deoxyuridine were used as internal standards. Calibration curves for both metabolites showed linearity (r > 0.99) in the concentration ranges of 10-10,000 ng/mL for plasma, and 1-50 µg/mL for urine, with method analytical performances within the acceptable criteria for quality control samples. The plasma method was successfully applied to the diagnosis of two patients with MNGIE and the quantification of plasma metabolites in three patients treated with EE-TP.

Comparative bioavailability of two zolpidem hemitartrate formulations in healthy human Brazilian volunteers using high-performance liquid chromatography coupled to tandem mass spectrometry.

To assess the bioequivalence of two zolpidem hemitartrate formulations in 30 healthy volunteers. Plasma samples were obtained over a 24 h period. Plasma concentrations of zolpidem were analyzed by liquid chromatography coupled to tandem mass spectrometry with positive ion electrospray ionization using multiple reaction monitoring. Values of peak concentration (Cmax ), area under curve (AUC), half-life, elimination constant, volume of distribution and clearance showed statistically significant differences when comparing women (604.34 ng h/ml, 127.36 ng/ml, 4.4 h, 0.18 1/h, 50.56 L and 8.55 L/h, respectively) and men (276.1 ng h/ml, 70.9 ng/ml, 3.3 h, 0.26 1/h, 91.42 L and 24.34 L/h, respectively), receiving the same dose (5 mg), respectively. The geometric means with corresponding 90% confidence interval for Test/Reference percentage ratios were 99.73% (CI 93.69-106.16) for Cmax, 97.44% (90% CI = 91.85-103.37%) for area under curve of plasma concentration until the last concentration observed (AUClast ) and 98.30% (90% CI = 92.48-104.49) for the area under curve between the first sample (pre-dosage) and infinity (AUC0-inf ). Since the 90% CI for AUClast , AUC0-inf and Cmax ratios were within the 80-125% interval proposed by the US Food and Drug Administration, it was concluded that zolpidem hemitartrate formulation (5 mg orodispersible tablet) is bioequivalent to the zolpidem hemitartrate formulation (Patz SL 5 mg sublingual tablet) with regard to both the rate and the extent of absorption. A new formulation of zolpidem 2.5 mg may be useful in women for the same clinical benefits as the 5 mg formulation in men.

Development, Validation of LC-MS/MS Method and Determination of Pharmacokinetic Parameters of the Stroke Neuroprotectant Neurounina-1 in Beagle Dog Plasma After Intravenous Administration.

Neurounina-1 [chemical name: 7-nitro-5-phenyl-1-(pyrrolidin-1-ylmethyl)-1H-benzo[e][1,4]diazepin-2(3H)-one] is a new compound provided with relevant neuroprotective effect during stroke and in neonatal hypoxia by increasing the Na+/Ca2+ exchanger (NCX) isoforms NCX1 and NCX2 activity. This study shows for the first time, the development and validation of a sensitive and selective method for analysis of neurounina-1 in beagle dog plasma by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). The sample preparation consisted of extraction of the analyte and the internal standard (IS) (ropivacaine) from plasma (50 µL) by liquid-liquid extraction using acetonitrile (100 µL). The selected reaction monitoring mode of the positive ion was performed and the precursor to the product ion transitions of m/z 365 > 83 and m/z 275 > 126 were used to measure the derivative of neurounina-1 and ropivacaine. The chromatographic separation was achieved using a Phenomenex C18 Luna (150 mm × 4.6 mm × 5 µm) analytical column with an isocratic mobile phase composed of methanol/acetonitrile/water (50/40/10, v/v/v) + 0.1% formic acid + 1 M ammonium formate. The method was linear over a concentration range of 1-500 ng/mL. The method was applied to evaluate the pharmacokinetics of neurounina-1 after a single intravenous administration of three different doses (0.1 mg/kg, 0.3 mg/kg, and 1 mg/kg) to beagle dogs (n = 5). The mean AUC0-tlast values were 26.10, 115.81, and 257.28 ng∗h/mL following intravenous administration of 0.1, 0.3, and 1 mg/kg, respectively. Linear pharmacokinetics was observed up to 1.0 mg/kg. The neurounina-1 was rapidly eliminated, with mean CL values of 46.24, 47.57, and 69.15 L/h, Vd of 130.31, 154.15, and 210.79 L and t1/2 of 2.14, 2.54, and 2.04 h after intravenous administration of 0.1, 0.3, and 1 mg/kg, respectively. This new analytical method allows the rapid determination of the neurounina-1, a new developed compound, able to exert a remarkable neuroprotective effect in the low nanomolar range.

Quantification of estradiol cypionate in plasma by liquid chromatography coupled with tandem mass spectrometry: Application in a pharmacokinetic study in healthy female volunteers.

The combination of medroxyprogesterone acetate 25 mg + estradiol cypionate 5 mg is a highly effective, monthly injectable contraceptive. For the first time, this study presents the development and validation of a sensitive method for estradiol cypionate analysis in human plasma by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). Aliquots (500 µL) of plasma were extracted with ethyl ether (100%) and derivatized with dansyl chloride. Its separation was performed on a Jones Chromatography Genesis C8 column and the quantification was performed with a mass spectrometer equipped with an electrospray interface operating in negative ion mode. The run time was 6 min and the calibration curve was linear over the range of 0.005-0.15 ng/mL. The method was applied to evaluate the pharmacokinetics of estradiol cypionate in plasma collected up to 1008 h (42 days) after a single intramuscular administration of 25 mg/mL medroxyprogesterone acetate +5 mg/mL estradiol cypionate to healthy female volunteers (n = 12). The estradiol cypionate maximum plasma concentration (Cmax) was 0.14 ± 0.08 ng/mL reached at 16.83 ± 21.07 h and the area under the plasma concentration versus time curve (AUC0-last) was 14.07 ± 6.32 ng.h/mL. Elimination half-life (t½), apparent volume of distribution (Vd/F), apparent clearance (CL/F) and mean residence time (MRT) were 89.65 ± 76.04 h, 28038 ± 9636 L, 49.02 ± 10.62 L/h and 576.05 ± 238.32 h, respectively, showing that the estradiol cypionate release from the administration site was prolonged and there was no drug accumulation.