RESUMO
Aim: Mass-selective quantitation is a powerful attribute of LC-MS as a platform for bioanalysis. Here, a sensitive LC-MS approach has been validated for an oligonucleotide having chemical modifications (e.g., N-acetylgalactosamine [GalNAc] conjugated), to distinguish between the conjugated and unconjugated forms of the oligonucleotide, thereby enabling a nuanced view of the pharmacokinetic profile. Results: A high-sensitivity methodology for mass-specific measurement of AZD8233, a GalNAc-conjugated 16-mer oligonucleotide, using LLE-SPE with optimized LC conditions and detection of a low-mass fragment ion was successfully validated in the range of 0.20-100 ng/ml in human plasma. Conclusion: The AZD8233 LC-MS methodology adds valuable insight on the GalNAc linker's in vivo stability to the program and should be broadly applicable to oligonucleotides requiring high sensitivity and mass-selective measurement for quantitative discrimination from metabolites and endogenous interferences.
Assuntos
Cromatografia Líquida/métodos , Oligonucleotídeos/análise , Espectrometria de Massas em Tandem/métodos , HumanosRESUMO
PURPOSE: Training emergency department (ED) personnel in the care of victims of mass-casualty incidents (MCIs) is a highly challenging task requiring unique and innovative approaches. The purpose of this study was to retrospectively explore the value of high-fidelity simulators in an exercise that incorporates time and resource limitation as an optimal method of training health care personnel in mass-casualty care. METHODS: Mass-casualty injury patterns from an explosive blast event were simulated for 12 victims using high-fidelity computerized simulators (HFCS). Programmed outcomes, based on the nature of injuries and conduct of participants, ranged from successful resuscitation and survival to death. The training exercise was conducted five times with different teams of health care personnel (n = 42). The exercise involved limited time and resources such as blood, ventilators, and imaging capability. Medical team performance was observed and recorded. Following the exercise, participants completed a survey regarding their training satisfaction, quality of the exercise, and their prior experiences with MCI simulations. The Likert scale responses from the survey were evaluated using mean with 95% confidence interval, as well as median and inter-quartile range. For the categorical responses, the frequency, proportions, and associated 95% confidence interval were calculated. RESULTS: The mean rating on the quality of experiences related trainee survey questions (n = 42) was between 4.1 and 4.6 on a scale of 5.0. The mean ratings on a scale of 10.0 for quality, usefulness, and pertinence of the program were 9.2, 9.5, and 9.5, respectfully. One hundred percent of respondents believed that this type of exercise should be required for MCI training and would recommend this exercise to colleagues. The five medical team (n = 5) performances resulted in the number of deaths ranging from two (including the expectant victims) to six. Eighty percent of medical teams attempted to resuscitate the "expectant" infant and exhausted the O- blood supply. Sixty percent of medical teams depleted the supply of ventilators. Forty percent of medical teams treated "delayed" victims too early. CONCLUSION: A training exercise using HFCS for mass casualties and employing limited time and resources is described. This exercise is a preferred method of training among participating health care personnel.
Assuntos
Planejamento em Desastres , Incidentes com Feridos em Massa , Serviço Hospitalar de Emergência , Humanos , Lactente , Simulação de Paciente , Estudos RetrospectivosRESUMO
Aim: A method has been developed and validated for quantitation of selumetinib in human whole blood collected using a Mitra™ volumetric absorptive microsampling device. This device is patient-friendly, affording less-invasive sampling with broad applicability to clinical and diagnostic applications - specifically in pediatric populations. Materials & methods: In this method, drug is extracted from the Mitra device via sonication in methanol: Ammonium hydroxide, then analyzed by LC-MS/MS. The linear range for selumetinib analysis is 2.00-2000 ng/ml. Results: All validation parameters met acceptance criteria established in agreement with current regulatory guidance for bioanalytical method validation. The stability of selumetinib in Mitra tips was established at both ambient and frozen conditions. Conclusion: A simple method has been developed and validated for determination of selumetinib from human whole blood, collected using volumetric absorptive microsampling and analyzed by LC-MS/MS.
Assuntos
Benzimidazóis/sangue , Análise Química do Sangue/métodos , Coleta de Amostras Sanguíneas/instrumentação , Cromatografia Líquida/métodos , Microtecnologia/instrumentação , Espectrometria de Massas em Tandem/métodos , Calibragem , Humanos , Limite de DetecçãoRESUMO
In this paper, experiences and learnings are shared from the 10-year application of incurred sample reanalysis (ISR) in support of the AstraZeneca small molecule portfolio. The conclusions from including ISR in every clinical bioanalysis study for a period of 5 years, generating ISR data from 550 studies, are shared. Our preclinical ISR approach is described and data generated using capillary microsampling demonstrate confidence in its routine application. The data demonstrate that ISR failures are very rare and the assessment can and should therefore be limited. Dialogue between the bioanalytical teams internally, as well as with the partner contract research organizations, is however critical for a successful bioanalytical method validation and to avoid any ISR failures.
Assuntos
Coleta de Amostras Sanguíneas/métodos , Desenvolvimento de Medicamentos/métodos , Indústria Farmacêutica/métodos , Preparações Farmacêuticas/sangue , Reprodutibilidade dos Testes , Animais , Coleta de Amostras Sanguíneas/normas , Cromatografia Líquida/métodos , Cromatografia Líquida/normas , Desenvolvimento de Medicamentos/normas , Indústria Farmacêutica/normas , Humanos , Controle de Qualidade , Bibliotecas de Moléculas Pequenas/farmacocinética , Espectrometria de Massas em Tandem/métodos , Espectrometria de Massas em Tandem/normas , Estudos de Validação como AssuntoRESUMO
The AstraZeneca and Covance Laboratories Clinical Bioanalysis Alliance (CBioA) was launched in 2011 after a period of global economic recession. In this challenging environment, AstraZeneca elected to move to a full and centralized outsourcing model that could optimize the number of people supporting bioanalytical work and reduce the analytical cost. This paper describes the key aspects of CBioA, the innovative operational model implemented, and our ways of ensuring this was much more than simply a cost reduction exercise. As we have recently passed the first 5-year cycle, this paper also summarizes some of the concluding benefits, wins and lessons learned, and how we now plan to extend and develop the relationship even further moving into a new clinical laboratory partnership.
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Serviços de Laboratório Clínico/economia , Indústria Farmacêutica/economia , Serviços Terceirizados , Serviços de Laboratório Clínico/organização & administração , Serviços de Laboratório Clínico/normas , Comunicação , Avaliação de Resultados em Cuidados de Saúde , Padrões de ReferênciaRESUMO
AIM: Naloxegol is an oral peripherally acting µ-opioid receptor antagonist approved for the treatment of opioid-induced constipation. Sensitive, robust, bioanalytical methods were required to quantitate naloxegol in human biological matrices as part of the clinical development program. METHODOLOGY/RESULTS: Analytical plasma samples were prepared using Solid Phase Extraction (SPE) coupled with concentration. The method's linearity was established at 0.1-50 ng/ml with up to 100-fold dilution. Urine samples were analyzed directly postdilution; dialysate samples were extracted by supported liquid extraction. Sensitive liquid chromatography/mass spectrometry (LC-MS/MS) assays were developed and validated, and demonstrated acceptable precision, accuracy and selectivity for naloxegol in the appropriate matrices. CONCLUSION: Methods for quantifying naloxegol in human biological matrices have been successfully validated.
Assuntos
Cromatografia Líquida/métodos , Morfinanos/sangue , Morfinanos/urina , Antagonistas de Entorpecentes/sangue , Antagonistas de Entorpecentes/urina , Extração em Fase Sólida/métodos , Espectrometria de Massas em Tandem/métodos , Analgésicos Opioides/efeitos adversos , Constipação Intestinal/induzido quimicamente , Constipação Intestinal/tratamento farmacológico , Humanos , Limite de Detecção , Polietilenoglicóis , Receptores Opioides mu/antagonistas & inibidoresRESUMO
AIM: AZD3293 is a novel BACE1 inhibitor in Phase III development for Alzheimer's disease. Sensitive and robust bioanalytical methods were required to quantitate AZD3293 and its metabolite AZ13569724 in human biological matrices. METHODOLOGY/RESULTS: Human plasma was prepared by protein precipitation. Linearity for both analytes was in the range of 0.5-500 ng/ml with up to 100-fold dilution. Plasma ultrafiltrate samples were prepared using Centrifree® ultrafiltration device. Urine and CSF samples were analyzed directly after dilution. A 27% decrease in AZD3293 concentrations in the CSF collection apparati was found due to nonspecific binding. Incurred sample reanalysis was acceptable. CONCLUSION: Methods for simultaneous quantitation of AZD3293 and its metabolite AZ13569724 in human biological matrices have been validated and successfully applied to clinical studies.
Assuntos
Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Ácido Aspártico Endopeptidases/antagonistas & inibidores , Análise Química do Sangue/métodos , Cromatografia Líquida/métodos , Imidazóis/sangue , Imidazóis/metabolismo , Compostos de Espiro/sangue , Compostos de Espiro/metabolismo , Espectrometria de Massas em Tandem/métodos , Inibidores Enzimáticos/sangue , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacologia , Humanos , Imidazóis/farmacologia , Limite de Detecção , Compostos de Espiro/farmacologia , Fatores de TempoRESUMO
PURPOSE: Two phase I, open-label trials in healthy subjects assessed whether co-administration with CYP3A4/CYP2C19 inhibitors, itraconazole/fluconazole (study A), or CYP3A4 inducer, rifampicin (study B), affects the exposure, safety/tolerability and pharmacokinetics of selumetinib and its metabolite N-desmethyl selumetinib. METHODS: In study A (n = 26), subjects received a single dose of selumetinib 25 mg and, after 4 days of washout, were randomized to treatment 1 (itraconazole 200 mg twice daily on days 1-11) or treatment 2 (fluconazole 400 mg on day 1 then 200 mg/day on days 2-11) plus co-administration of single-dose selumetinib 25 mg on day 8 (selumetinib staggered 4 h after itraconazole/fluconazole dose); Twenty-one days after discharge/washout, subjects received the alternate treatment. In study B (n = 22), subjects received a single dose of selumetinib 75 mg (day 1) then rifampicin 600 mg/day (days 4-14) plus a single dose of selumetinib 75 mg on day 12. Pharmacokinetic analysis and safety assessments were performed. RESULTS: Selumetinib co-administered with itraconazole, fluconazole (selumetinib staggered 4 h after itraconazole/fluconazole dose), or rifampicin was well tolerated. Selumetinib exposure was higher when co-administered with itraconazole or fluconazole (area under the plasma concentration-time curve (AUC) increased by 49 and 53%, respectively; maximum plasma concentration (C max) increased by 19 and 26%, respectively) but lower when co-dosed with rifampicin (AUC and C max decreased by 51 and 26%, respectively) versus selumetinib dosed alone. Co-administration with itraconazole or rifampicin decreased N-desmethyl selumetinib AUC(0-t) (11 and 55%, respectively), and C max (25 and 18%, respectively), with fluconazole, AUC(0-t) increased by 40%, but there was no effect on C max. CONCLUSIONS: Co-administration of CYP3A4/CYP2C19 inhibitors will likely increase exposure to selumetinib, while CYP3A4 inducers will likely reduce its exposure.
Assuntos
Benzimidazóis/farmacocinética , Inibidores do Citocromo P-450 CYP2C19/farmacologia , Inibidores do Citocromo P-450 CYP3A/farmacologia , Adolescente , Adulto , Benzimidazóis/sangue , Estudos Cross-Over , Citocromo P-450 CYP2C19/metabolismo , Citocromo P-450 CYP3A/metabolismo , Indutores das Enzimas do Citocromo P-450/farmacologia , Feminino , Fluconazol/farmacologia , Voluntários Saudáveis , Humanos , Itraconazol/farmacologia , MAP Quinase Quinase Quinase 1/antagonistas & inibidores , MAP Quinase Quinase Quinase 2/antagonistas & inibidores , Masculino , Rifampina/farmacologia , Adulto JovemRESUMO
Two phase I open-label studies were conducted to investigate the pharmacokinetics (PK), safety, and tolerability of single oral doses of selumetinib in subjects with end-stage renal disease (ESRD) undergoing hemodialysis and subjects with varying degrees of hepatic impairment; both studies included a matched control group comprised of healthy individuals. In the renal impairment study, subjects received single doses of selumetinib 50 mg; those with ESRD received selumetinib before and after dialysis (with a between-treatment washout period of ≥7 days). In the hepatic impairment study, subjects received varying single doses of selumetinib (20-50 mg) depending on liver dysfunction (mild, moderate, or severe as per Child-Pugh classification). PK, safety, and tolerability data were collected from both studies. Overall, 24 subjects were included in the renal impairment study (ESRD, N = 12; healthy subjects, N = 12). Selumetinib exposure (AUC and Cmax ) was not increased in the ESRD group vs healthy subjects. Selumetinib exposure was lower when selumetinib was dosed before vs after dialysis, although individual exposure was variable. Overall, 32 subjects were included in the hepatic impairment study (mild, moderate, and severe impairment, N = 8 per group; healthy subjects, N = 8). Generally, dose-normalized total selumetinib exposure was increased by 25% to 59% in subjects with moderate and severe hepatic impairment compared with healthy subjects. Increasing Child-Pugh score, decreasing serum albumin, and increasing prothrombin time correlated with increasing unbound selumetinib exposure. In both studies, selumetinib was well tolerated with no new safety concerns. These studies will inform dose adjustment considerations in patients.
Assuntos
Benzimidazóis/farmacocinética , Falência Renal Crônica/sangue , Hepatopatias/sangue , Administração Oral , Benzimidazóis/administração & dosagem , Benzimidazóis/efeitos adversos , Benzimidazóis/sangue , Feminino , Humanos , MAP Quinase Quinase Quinases/antagonistas & inibidores , Masculino , Pessoa de Meia-Idade , Diálise RenalRESUMO
PURPOSE: Selumetinib (AZD6244, ARRY-142886) is an oral, potent, and selective allosteric mitogen-activated protein kinase 1/2 inhibitor with a short t1/2. The purpose of this study was to characterize the effect of selumetinib on cardiac repolarization and a potential exposure-QT effect relationship. METHODS: A double-blind (selumetinib), randomized, 3-period crossover study was conducted to assess the effects of a single oral dose of selumetinib (75 mg) on the QTc interval compared with placebo, using moxifloxacin as an open-label positive control, in healthy male subjects aged 18 to 45 years. QT intervals were evaluated by using the Fridericia formula (QTcF) and the Bazett formula. Further analysis was conducted by using nonlinear mixed effects modeling to characterize any relationship between selumetinib exposure and QTc and was used to predict the effect if selumetinib 150 mg was administered. All adverse events were characterized and recorded. FINDINGS: A total of 54 healthy male subjects were enrolled, and 48 completed all treatments. Mean age was 27 years; four subjects were of Hispanic or Latino ethnicity, and 53.7% were White and 46.3% were Black. The BMI of subjects ranged from 19.4 to 29.6 kg/m2. After a single oral dose of selumetinib 75 mg, the highest upper bound of the 2-sided 90% CI for placebo-corrected, baseline-adjusted QTcF (ΔΔQTcF) over the 24-hour postdose measurement interval was 2.5 milliseconds, which was well below the 10-millisecond upper bound for concluding no effect. The relationship between ΔΔQTcF and selumetinib concentrations was adequately described by using a nonlinear mixed effect model. The mean estimated ∆∆QTcF interval prolongation based on the geometric mean Cmax of 75 mg selumetinib was 2.38 milliseconds (90% CI, 1.25 to 3.52), which was in good agreement with the statistical analysis results. The model also predicted mean ∆∆QTcF interval prolongations of 4.70 milliseconds (90% CI, 2.46 to 6.95) after a single supratherapeutic dose of selumetinib 150 mg, indicating the upper bound of 2-sided 90% CIs for ΔΔQTcF are predicted to be <10 milliseconds. Selumetinib, administered as a single 75 mg oral dose, was generally safe and well tolerated. IMPLICATIONS: Selumetinib 75 mg did not cause any QT/QTc interval prolongation in these healthy subjects, and selumetinib is not expected to have a clinically relevant effect on cardiac repolarization in patients at the anticipated therapeutic dose of 75 mg. The model also demonstrated the low potential for any QTc effects of selumetinib at doses higher than the standard therapeutic dose.
Assuntos
Antineoplásicos/farmacologia , Benzimidazóis/farmacologia , Coração/efeitos dos fármacos , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Adolescente , Adulto , Antineoplásicos/sangue , Benzimidazóis/sangue , Estudos Cross-Over , Método Duplo-Cego , Eletrocardiografia , Feminino , Fluoroquinolonas/farmacologia , Coração/fisiologia , Frequência Cardíaca/efeitos dos fármacos , Humanos , Masculino , Moxifloxacina , Adulto JovemRESUMO
AIM: Selumetinib is an inhibitor of MEK1/2 in Phase III development that has activity in multiple tumor types. Validated bioanalytical methods were required to quantitate selumetinib and its N-desmethyl and amide metabolites in a variety of human biological matrices. Methodology & results: LC-MS/MS assays were developed and validated that demonstrated acceptable precision, accuracy and selectivity for selumetinib and the two metabolites in human plasma, urine, blood dialysate and plasma ultrafiltrate. Incurred sample re-analysis was acceptable and issues observed in plasma with the amide metabolite, due to potential instability, were addressed. CONCLUSION: Robust and sensitive LC-MS/MS assays for the quantification of selumetinib and two of its metabolites were validated in human biological matrices and are being used to support the clinical development program.
Assuntos
Benzimidazóis/sangue , Benzimidazóis/urina , Inibidores de Proteínas Quinases/sangue , Inibidores de Proteínas Quinases/urina , Espectrometria de Massas em Tandem/métodos , Amidas/sangue , Amidas/metabolismo , Amidas/urina , Benzimidazóis/metabolismo , Cromatografia Líquida/métodos , Monitoramento de Medicamentos/métodos , Humanos , Limite de Detecção , MAP Quinase Quinase 1/antagonistas & inibidores , MAP Quinase Quinase 2/antagonistas & inibidores , Inibidores de Proteínas Quinases/metabolismoRESUMO
The use of medications plays a pivotal role in the management of children with heart diseases. Most children with increased pulmonary blood flow require chronic use of anticongestive heart failure medications until more definitive interventional or surgical procedures are performed. The use of such medications, particularly inotropic agents and diuretics, is even more amplified during the postoperative period. Currently, children are undergoing surgical intervention at an ever younger age with excellent results aided by advanced anesthetic and postoperative care. The most significant of these advanced measures includes invasive and noninvasive monitoring as well as a wide array of pharmacologic agents. This review update provides a medication guide for medical practitioners involved in care of children with heart diseases.
Assuntos
Fármacos Cardiovasculares/uso terapêutico , Cardiopatias/tratamento farmacológico , Pediatria , Farmacopeias como Assunto , Cardiologia , Criança , Gerenciamento Clínico , HumanosRESUMO
OBJECTIVE: To determine whether endotoxin-induced hyperlactatemia in hemodynamically stable animals is due to increased lactate production or decreased lactate clearance by measuring lactate turnover rate in the vascular compartment (LTRvc). DESIGN: Prospective, controlled trial. SETTING: Research laboratory in a university hospital. SUBJECTS: Male Sprague-Dawley rats weighing 275-425 g with chronic vascular catheters. INTERVENTIONS: Chronically catheterized rats were treated with 6 microg/kg endotoxin or saline. LTRvc was determined from the specific activity of carbon-14 [14C]lactate in aortic blood during a constant infusion of [14C]lactate into the inferior vena cava. The role of the splanchnic organs in lipopolysaccharide-induced alterations in LTRvc was determined from the splanchnic first-pass clearance of [14C]lactate infused into the superior mesenteric artery and direct measurements of blood lactate concentration gradients across the splanchnic organs. MEASUREMENTS AND MAIN RESULTS: Despite a 260% increase in lactate concentrations after lipopolysaccharide treatment, the specific activity of [14C]lactate and the LTRvc did not change, indicating that lipopolysaccharide-induced hyperlactatemia is caused by decreased lactate clearance from the vascular compartment rather than increased lactate flux into the vascular compartment. In contrast, lactate clearance by the splanchnic system was increased. The specific activity of [14C]lactate in aortic blood decreased 33% after lipopolysaccharide treatment when the [14C]lactate was infused into the superior mesenteric artery, indicating increased first-pass clearance of [14C]lactate by the splanchnic organs. Furthermore, the hepatic venous-aortic concentration gradient of lactate became increasingly negative after lipopolysaccharide treatment, indicating increased vascular extraction of lactate by the splanchnic system (0.07 +/- 0.07 micromol/mL vs. -0.34 +/- 0.14 micromol/mL). CONCLUSIONS: Lipopolysaccharide-induced hyperlactatemia in hemodynamically stable rats is caused by a net decrease in lactate clearance from the vascular compartment despite the fact that the clearance of lactate by the splanchnic system remains intact.
Assuntos
Acidose Láctica/fisiopatologia , Lactatos/sangue , Sepse/fisiopatologia , Acidose Láctica/sangue , Acidose Láctica/etiologia , Análise de Variância , Animais , Hemodinâmica , Lipopolissacarídeos , Fígado/metabolismo , Masculino , Estudos Prospectivos , Ratos , Ratos Sprague-Dawley , Sepse/sangue , Sepse/complicações , Circulação Esplâncnica/fisiologiaRESUMO
OBJECTIVES: The aim of this study was to test a possible influence of atorvastatin on the production of vascular endothelial growth factor (VEGF) in patients with coronary artery disease (CAD) and in vitro. BACKGROUND: Vascular endothelial growth factor is suggested to be involved in the growth of atherosclerotic plaque by inducing its neovascularization. Hepatic hydroxymethyl glutaryl-coenzyme A reductase inhibitors (statins) are known to have atheroprotective effects beyond lipid lowering. METHODS: Blood was collected from 14 male hypercholesterolemic patients with angiographically confirmed CAD at baseline and after two months of atorvastatin therapy (20 mg/d) and from eight male control patients. In an ex vivo assay, human coronary artery smooth muscle cells (HCASMC) were incubated with the patient plasma collected before and after atorvastatin therapy. To test the direct effect of atorvastatin on VEGF synthesis in vitro, HCASMC were treated with atorvastatin (1, 3 and 10 microM). The VEGF concentration was measured by enzyme-linked immunosorbent assay. RESULTS: Atorvastatin therapy reduced VEGF plasma levels in CAD patients (from 31.1 +/- 6.1 to 19.0 +/- 3.6 pg/ml; p < 0.05). The VEGF plasma concentration tended to be higher in CAD patients before treatment compared to control patients (31.1 +/- 6.1 vs. 23.4 +/- 3.6 pg/ml; p = NS). Plasma collected before therapy induced significantly more VEGF in HCASMC compared to the plasma collected after treatment and compared to control cells. In vitro, atorvastatin decreased both the basal and the interleukin-1beta-induced VEGF release in HCASMC. CONCLUSIONS: These data suggest that atorvastatin may lower the plasma level of VEGF in CAD patients, which could represent a novel beneficial effect of this and perhaps other statins.