RESUMO
BACKGROUND: Emergence of antibiotic resistance is a global public health concern. The relationships between antibiotic use, the gut community composition, normal physiology and metabolism, and individual and public health are still being defined. Shifts in composition of bacteria, antibiotic resistance genes (ARGs) and mobile genetic elements (MGEs) after antibiotic treatment are not well-understood. METHODS: This project used next-generation sequencing, custom-built metagenomics pipeline and differential abundance analysis to study the effect of antibiotic monotherapy on resistome and taxonomic composition in the gut of Balb/c mice infected with E. coli via transurethral catheterization to investigate the evolution and emergence of antibiotic resistance. RESULTS: There is a longitudinal decrease of gut microbiota diversity after antibiotic treatment. Various ARGs are enriched within the gut microbiota despite an overall reduction of the diversity and total amount of bacteria after antibiotic treatment. Sometimes treatment with a specific class of antibiotics selected for ARGs that resist antibiotics of a completely different class (e.g. treatment of ciprofloxacin or fosfomycin selected for cepA that resists ampicillin). Relative abundance of some MGEs increased substantially after antibiotic treatment (e.g. transposases in the ciprofloxacin group). CONCLUSIONS: Antibiotic treatment caused a remarkable reduction in diversity of gut bacterial microbiota but enrichment of certain types of ARGs and MGEs. These results demonstrate an emergence of cross-resistance as well as a profound change in the gut resistome following oral treatment of antibiotics.
Assuntos
Antibacterianos/farmacologia , Metagenômica/métodos , Animais , Resistência Microbiana a Medicamentos/genética , Microbioma Gastrointestinal/efeitos dos fármacos , Microbioma Gastrointestinal/genética , Sequenciamento de Nucleotídeos em Larga Escala , Camundongos , Camundongos Endogâmicos BALB CRESUMO
BACKGROUND: As nanoparticles (NPs) become more prevalent in the pharmaceutical industry, questions have arisen from both industry and regulatory stakeholders about the long term effects of these materials. This study was designed to evaluate whether gold (10 nm), silver (50 nm), or silica (10 nm) nanoparticles administered intravenously to mice for up to 8 weeks at doses known to be sub-toxic (non-toxic at single acute or repeat dosing levels) and clinically relevant could produce significant bioaccumulation in liver and spleen macrophages. RESULTS: Repeated dosing with gold, silver, and silica nanoparticles did not saturate bioaccumulation in liver or spleen macrophages. While no toxicity was observed with gold and silver nanoparticles throughout the 8 week experiment, some effects including histopathological and serum chemistry changes were observed with silica nanoparticles starting at week 3. No major changes in the splenocyte population were observed during the study for any of the nanoparticles tested. CONCLUSIONS: The clinical impact of these changes is unclear but suggests that the mononuclear phagocytic system is able to handle repeated doses of nanoparticles.
Assuntos
Ouro/toxicidade , Fígado/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Nanopartículas , Dióxido de Silício/toxicidade , Prata/toxicidade , Baço/efeitos dos fármacos , Animais , Biomarcadores/sangue , Feminino , Ouro/administração & dosagem , Ouro/metabolismo , Injeções Intravenosas , Fígado/metabolismo , Fígado/patologia , Macrófagos/metabolismo , Macrófagos/patologia , Nanopartículas Metálicas , Camundongos Endogâmicos BALB C , Medição de Risco , Dióxido de Silício/administração & dosagem , Dióxido de Silício/metabolismo , Prata/administração & dosagem , Prata/metabolismo , Baço/metabolismo , Baço/patologia , Fatores de Tempo , Distribuição TecidualRESUMO
Attempts to characterize and formally qualify biomarkers for regulatory purposes have raised questions about how histological and histopathological methods impact the evaluation of biomarker performance. A group of pathologists was asked to analyze digitized images prepared from rodent kidney injury experiments in studies designed to investigate sources of variability in histopathology evaluations. Study A maximized variability by using samples from diverse studies and providing minimal guidance, contextual information, or opportunities for pathologist interaction. Study B was designed to limit interpathologist variability by using more uniform image sets from different locations within the same kidneys and allowing pathologist selected interactions to discuss and identify the location and injury to be evaluated but without providing a lexicon or peer review. Results from this study suggest that differences between pathologists and across models of disease are the largest sources of variability in evaluations and that blind evaluations do not generally make a significant difference. Results of this study generally align with recommendations from both industry and the U.S. Food and Drug Administration and should inform future studies examining the effects of common lexicons and scoring criteria, peer review, and blind evaluations in the context of biomarker performance assessment.
Assuntos
Moléculas de Adesão Celular/urina , Nefropatias/patologia , Nefropatias/urina , Animais , Biomarcadores/urina , Cisplatino/toxicidade , Nefropatias/induzido quimicamente , Masculino , Curva ROC , Ratos , Ratos Sprague-DawleyRESUMO
Glucagon Like Peptide-1 (GLP-1) drugs are currently used to treat type-2 diabetes. Safety concerns for increased risk of pancreatitis and pancreatic ductal metaplasia have accompanied these drugs. High fat diet (HFD) is a type-2 diabetes risk factor that may affect the response to GLP-1 drug treatment. The objective of the present study was to investigate the effects of diet and GLP-1 based drugs on the exocrine pancreas in mice. Experiments were designed in a mouse model of insulin resistance created by feeding a HFD or standard diet (STD) for 6weeks. The GLP-1 drugs, sitagliptin (SIT) and exenatide (EXE) were administered once daily for additional 6weeks in both mice fed HFD or STD. The results showed that body weight, blood glucose levels, and serum levels of pro-inflammatory cytokines (TNFα, IL-1ß, and KC) were significantly greater in HFD mice than in STD mice regardless of GLP-1 drug treatment. The semi-quantitative grading showed that pancreatic changes were significantly greater in EXE and SIT-treated mice compared to control and that HFD exacerbated spontaneous exocrine pancreatic changes seen in saline-treated mice on a standard diet. Exocrine pancreatic changes identified in this study included acinar cell injury (hypertrophy, autophagy, apoptosis, necrosis, and atrophy), vascular injury, interstitial edema and inflammation, fat necrosis, and duct changes. These findings support HFD as a risk factor to increased susceptibility/severity for acute pancreatitis and indicate that GLP-1 drugs cause pancreatic injury that can be exacerbated in a HFD environment.
Assuntos
Dieta Hiperlipídica/efeitos adversos , Hipoglicemiantes/toxicidade , Pâncreas/efeitos dos fármacos , Peptídeos/toxicidade , Pirazinas/toxicidade , Receptores de Glucagon/agonistas , Triazóis/toxicidade , Peçonhas/toxicidade , Doença Aguda , Animais , Apoptose/efeitos dos fármacos , Atrofia , Exenatida , Receptor do Peptídeo Semelhante ao Glucagon 1 , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Necrose , Pâncreas/patologia , Pancreatite/etiologia , Fosfato de SitagliptinaRESUMO
Mild injury of the exocrine pancreas is often asymptomatic and can be under- or mis-diagnosed. The pancreas-enriched microRNAs miR-216a and miR-217 were evaluated as potential serum biomarkers of exocrine pancreas injury in rodent models of acute pancreatitis induced by caerulein, l-arginine, and pancreatic duct ligation. Both microRNAs showed time- and dose- relevant responses to pancreatic injury and wider dynamic ranges of response than serum amylase or lipase. Pancreas-selective microRNAs were found to be relatively sensitive serum biomarkers of pancreatic injury in rodents with potentially greater specificity than the current standard assays.
Assuntos
MicroRNAs/sangue , Pâncreas/patologia , Pancreatite/sangue , Amilases/sangue , Animais , Arginina , Biomarcadores/sangue , Ceruletídeo , Masculino , Camundongos Endogâmicos C57BL , Pancreatite/induzido quimicamente , Curva ROC , Ratos Sprague-DawleyRESUMO
Histopathology generally represents the reference standard for performance evaluation of nonclinical biomarkers used to inform regulatory decision making. This study uses drug-induced nephrotoxicity in rats to evaluate histopathology methods utilized in biomarker performance assessments. Male Sprague-Dawley rats received a single dose of cisplatin (0.5-5.0 mg/kg, intraperitoneally) to produce mild renal injury. Animals were euthanized 72 hr postdose and perfusion fixed. Kidneys were processed for histology and stereology procedures. Kidney injury molecule-1 (Kim-1) was measured in urine and in kidney tissue. Digital slide images were generated and analyzed by pathologists after collaborating on a training set of glass slides and digital images. Image analysis identified immunohistochemistry (IHC)-defined tubular injury. Stereology methods yielded estimations of proximal tubular cell number and volume. Statistical relationships among data sets were determined using correlation coefficients. Receiver operator characteristic (ROC) analyses determined the effect of method on biomarker assessment. Urinary Kim-1 was strongly correlated with digital image analysis and secondarily to histopathology evaluations. Stereology demonstrated weak or no correlation to pathology and urinary Kim-1. In ROC analyses, semiquantitative evaluations determined higher values for urinary Kim-1 performance than did IHC-based qualitative digital analyses. Semiquantitative evaluation as used in this study was most predictive of urinary Kim-1 values.
Assuntos
Cisplatino/toxicidade , Nefropatias/induzido quimicamente , Nefropatias/patologia , Rim/efeitos dos fármacos , Rim/patologia , Testes de Toxicidade/métodos , Testes de Toxicidade/normas , Animais , Histocitoquímica , Processamento de Imagem Assistida por Computador , Masculino , Curva ROC , Ratos , Ratos Sprague-DawleyRESUMO
Previously we found that regulation of eNOS is an important part of the pathogenic process of Drug-induced vascular injury (DIVI) for PDE4i. The aims of the current study were to examine the phosphorylation of eNOS in mesentery versus aorta at known regulatory sites across DIVI-inducing drug classes and to compare changes across species. We found that phosphorylation at S615 in rats was elevated 35-fold 2 hr after the last dose of CI-1044 in mesentery versus 3-fold in aorta. Immunoprecipitation studies revealed that many of the upstream regulators of eNOS activation were associated with eNOS in 1 or more signalosome complexes. Next rats were treated with drugs from 4 other classes known to cause DIVI. Each drug was given alone and in combination with SIN-1 (NO donor) or L-NAME (eNOS inhibitor), and the level of eNOS phosphorylation in mesentery and aorta tissue was correlated with the extent of vascular injury and measured serum nitrite. Drugs or combinations produced altered serum nitrite levels as well as vascular injury score in the mesentery. The results suggested that phosphorylation of S615 may be associated with DIVI activity. Studies with the species-specific A2A adenosine agonist CI-947 in rats versus primates showed a similar pattern.
Assuntos
Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos , Óxido Nítrico Sintase Tipo III/metabolismo , Lesões do Sistema Vascular/induzido quimicamente , Lesões do Sistema Vascular/patologia , Adenosina/administração & dosagem , Adenosina/efeitos adversos , Adenosina/análogos & derivados , Animais , Aorta/metabolismo , Azepinas/administração & dosagem , Azepinas/efeitos adversos , Relação Dose-Resposta a Droga , Masculino , Niacinamida/administração & dosagem , Niacinamida/efeitos adversos , Niacinamida/análogos & derivados , Óxido Nítrico/sangue , Óxido Nítrico Sintase Tipo III/genética , Nitritos/sangue , Fosforilação , Ratos , Ratos Sprague-DawleyRESUMO
Contrast-induced nephropathy (CIN) refers to a decline in renal function following exposure to iodinated contrast media (CM). The present study was initiated to explore the role of known human risk factors (spontaneous hypertension, diabetes, protein-losing nephropathy) on CIN development in rodent models and to determine the effect of CM administration on kidney injury biomarkers in the face of preexisting kidney injury. Spontaneously hypertensive rats (hypertension), streptozotocin-treated Sprague Dawley rats (diabetes), and Dahl salt-sensitive rats (protein-losing nephropathy) were given single intravenous injections of the nonionic, low osmolar contrast medium, iohexol. Blood urea nitrogen (BUN), serum creatinine (sCr), and urinary biomarkers; albumin, lipocalin 2 (Lcn-2), osteopontin (Opn), kidney injury molecule 1 (Kim-1), renal papillary antigen 1 (Rpa-1), α-glutathione S-transferase (α-Gst), µ-glutathione S-transferase (µ-Gst), and beta-2 microglobulin (ß2m) were measured in disease models and appropriate controls to determine the response of these biomarkers to CM administration. Each disease model produced elevated biomarkers of kidney injury without CM. Preexisting histopathology was exacerbated by CM but little or no significant increases in biomarkers were observed. When 1.5-fold or greater sCr increases from pre-CM were used to define true positives, receiver-operating characteristic curve analysis of biomarker performance showed sCr was the best predictor of CIN across disease models. ß2m, Lcn-2, and BUN were the best predictors of histopathology defined kidney injury.
Assuntos
Diabetes Mellitus/sangue , Diabetes Mellitus/urina , Hipertensão/sangue , Hipertensão/urina , Nefropatias/sangue , Nefropatias/urina , Animais , Biomarcadores/sangue , Biomarcadores/urina , Nitrogênio da Ureia Sanguínea , Meios de Contraste/química , Iohexol/química , Nefropatias/induzido quimicamente , Nefropatias/patologia , Testes de Função Renal , Masculino , Curva ROC , Ratos , Ratos Endogâmicos Dahl , Ratos Endogâmicos SHR , Ratos Sprague-DawleyRESUMO
Antibiotics used systemically to treat infections may have off-target effects on the gut microbiome, potentially resulting in the emergence of drug-resistant bacteria or selection of pathogenic species. These organisms may present a risk to the host and spread to the environment with a risk of transmission in the community. To investigate the risk of emergent antibiotic resistance in the gut microbiome following systemic treatment with antibiotics, this metagenomic analysis project used next-generation sequencing, a custom-built metagenomics pipeline, and differential abundance analysis to study the effect of antibiotics (ampicillin, ciprofloxacin, and fosfomycin) in monotherapy and different combinations at high and low doses, to determine the effect on resistome and taxonomic composition in the gut of Balb/c mice. The results showed that low-dose monotherapy treatments showed little change in microbiome composition but did show an increase in expression of many antibiotic-resistant genes (ARGs) posttreatment. Dual combination treatments allowed the emergence of some conditionally pathogenic bacteria and some increase in the abundance of ARGs despite a general decrease in microbiota diversity. Triple combination treatment was the most successful in inhibiting emergence of relevant opportunistic pathogens and completely suppressed all ARGs after 72 h of treatment. The relative abundances of mobile genetic elements that can enhance transmission of antibiotic resistance either decreased or remained the same for combination therapy while increasing for low-dose monotherapy. Combination therapy prevented the emergence of ARGs and decreased bacterial diversity, while low-dose monotherapy treatment increased ARGs and did not greatly change bacterial diversity.
Assuntos
Microbioma Gastrointestinal , Microbiota , Animais , Camundongos , Antibacterianos/farmacologia , Ampicilina/farmacologia , Ciprofloxacina/farmacologia , Bactérias/genética , Genes BacterianosRESUMO
Cationic amphiphilic drugs and aminoglycoside antibiotics can induce phospholipidosis (PLD), an abnormal accumulation of phospholipids in lysosome-derived vesicles, in preclinical studies. The incidence of PLD in patients and its clinical relevance are difficult to assess without noninvasive biomarkers. Di-docosahexaenoyl bis(monoacylglycerol)phosphate (di-22:6-BMP) is a phospholipid that is enriched in lysosomal membranes and a proposed urinary biomarker of drug-induced PLD. The specificity of di-22:6-BMP for PLD was compared to other phospholipid species that can increase in urine with nephrotoxicity. Using liquid chromatography coupled to mass spectrometry, 12 phospholipids were assayed in the urine of rats treated with drugs that induced PLD or caused renal or skeletal muscle injury. In receiver operating curve analyses, urinary di-22:6-BMP was a significantly better predictor of PLD and the least predictive of tissue injury of the phospholipids assayed. The data provide evidence supporting the use of di-22:6-BMP as a urinary biomarker of PLD in rats.
Assuntos
Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos , Nefropatias/induzido quimicamente , Lisofosfolipídeos/urina , Fosfolipídeos/urina , Animais , Biomarcadores/urina , Moléculas de Adesão Celular/urina , Cisplatino/efeitos adversos , Feminino , Gentamicinas/efeitos adversos , Hexestrol/efeitos adversos , Hexestrol/análogos & derivados , Nefropatias/patologia , Nefropatias/urina , Lipocalina-2 , Lipocalinas/urina , Fígado/efeitos dos fármacos , Fígado/patologia , Pulmão/efeitos dos fármacos , Pulmão/patologia , Linfonodos/efeitos dos fármacos , Linfonodos/patologia , Masculino , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Osteopontina/urina , Ratos , Ratos Endogâmicos F344 , Ratos Sprague-Dawley , Sinvastatina/efeitos adversos , Baço/efeitos dos fármacos , Baço/patologia , Troponina I/sangueRESUMO
We designed a study to provide reversibility and comparative injury data for several candidate urinary biomarkers of kidney injury in the United States Food and Drug Administration biomarker qualification process. The nephrotoxin gentamicin was given to rats once on each of three days and the animals were killed during dosing or over the following 42 days. Between days one and three, all biomarkers except albumin were elevated, peaked at day 7, and returned to control levels by day 10 (µ- and α-glutathione S-transferases, and renal papillary antigen-1) or day 15 (kidney injury molecule-1, lipocalin-2, osteopontin, and clusterin). All biomarkers performed better during injury than during recovery except osteopontin, which performed equally well in both time periods. During the evolution of injury, kidney injury molecule-1, renal papillary antigen-1, and clusterin best mirrored the histopathologic lesions. During injury resolution, kidney injury molecule-1, osteopontin, and blood urea nitrogen best reflected recovery. Based on histopathology, necrosis, or apoptosis scoring, kidney injury molecule-1 was the best biomarker of overall renal injury. Evaluation by regeneration score showed that renal papillary antigen-1 best reflected tubular and/or collecting duct regeneration, especially during recovery. Thus, these biomarkers performed with different effectiveness when evaluated by individual pathological processes such as necrosis, apoptosis, and regeneration.
Assuntos
Injúria Renal Aguda/urina , Biomarcadores/urina , Túbulos Renais Proximais/metabolismo , Proteômica , Injúria Renal Aguda/induzido quimicamente , Injúria Renal Aguda/patologia , Injúria Renal Aguda/fisiopatologia , Animais , Apoptose , Biomarcadores/sangue , Moléculas de Adesão Celular/urina , Clusterina/urina , Gentamicinas , Glutationa Transferase/urina , Imuno-Histoquímica , Túbulos Renais Proximais/patologia , Túbulos Renais Proximais/fisiopatologia , Lipocalinas/urina , Masculino , Necrose , Osteopontina/urina , Valor Preditivo dos Testes , Proteômica/métodos , Curva ROC , Ratos , Ratos Sprague-Dawley , Kit de Reagentes para Diagnóstico , Regeneração , Fatores de TempoRESUMO
Aim: To develop and validate a fit for purpose method for the simultaneous determination of dexamethasone and its major metabolite, 6ß-hydroxydexamethasone, in rabbit plasma and ocular matrices to measure the in vivo release and distribution profile of dexamethasone from intravitreal implants. Materials & methods: An UHPLC-MS/MS system was employed to perform the bioanalysis. The method was validated according to the US FDA Bioanalytical Method Validation Guidance for Industry. Results & conclusion: The method was found to be fit-for-purpose for the described biological matrices and had a LLOQ of 0.1 ng/ml.
Assuntos
Humor Aquoso/química , Cromatografia Líquida de Alta Pressão/métodos , Dexametasona/análogos & derivados , Retina/química , Espectrometria de Massas em Tandem/métodos , Corpo Vítreo/química , Animais , Dexametasona/análise , Dexametasona/sangue , CoelhosRESUMO
INTRODUCTION: According to the Centers for Disease Control's 2015 Hospital Acquired Infection Hospital Prevalence Survey, 1 in 31 hospital patients was infected with at least one nosocomial pathogen while being treated for unrelated issues. Many studies associate antibiotic administration with nosocomial infection occurrence. However, to our knowledge, there is little to no direct evidence of antibiotic administration selecting for nosocomial opportunistic pathogens. AIM: This study aims to confirm gut microbiota shifts in an animal model of antibiotic treatment to determine whether antibiotic use favors pathogenic bacteria. METHODOLOGY: We utilized next-generation sequencing and in-house metagenomic assembly and taxonomic assignment pipelines on the fecal microbiota of a urinary tract infection mouse model with and without antibiotic treatment. RESULTS: Antibiotic therapy decreased the number of detectable species of bacteria by at least 20-fold. Furthermore, the gut microbiota of antibiotic treated mice had a significant increase of opportunistic pathogens that have been implicated in nosocomial infections, like Acinetobacter calcoaceticus/baumannii complex, Chlamydia abortus, Bacteroides fragilis, and Bacteroides thetaiotaomicron. Moreover, antibiotic treatment selected for antibiotic resistant gene enriched subpopulations for many of these opportunistic pathogens. CONCLUSIONS: Oral antibiotic therapy may select for common opportunistic pathogens responsible for nosocomial infections. In this study opportunistic pathogens present after antibiotic therapy harbored more antibiotic resistant genes than populations of opportunistic pathogens before treatment. Our results demonstrate the effects of antibiotic therapy on induced dysbiosis and expansion of opportunistic pathogen populations and antibiotic resistant subpopulations of those pathogens. Follow-up studies with larger samples sizes and potentially controlled clinical investigations should be performed to confirm our findings.
Assuntos
Antibacterianos/farmacologia , Infecção Hospitalar/microbiologia , Microbioma Gastrointestinal , Infecções Oportunistas/microbiologia , Animais , Antibacterianos/efeitos adversos , Bactérias/classificação , Disbiose/induzido quimicamente , Feminino , Camundongos , Camundongos Endogâmicos BALB CRESUMO
Following a decision to require label warnings for concurrent use of opioids and benzodiazepines and increased risk of respiratory depression and death, the US Food and Drug Administratioin (FDA) recognized that other sedative psychotropic drugs may be substituted for benzodiazepines and be used concurrently with opioids. In some cases, data on the ability of these alternatives to depress respiration alone or in conjunction with an opioid are lacking. A nonclinical in vivo model was developed that could detect worsening respiratory depression when a benzodiazepine (diazepam) was used in combination with an opioid (oxycodone) compared to the opioid alone based on an increased arterial partial pressure of carbon dioxide (pCO2 ). The current study used that model to assess the impact on respiration of non-benzodiazepine sedative psychotropic drugs representative of different drug classes (clozapine, quetiapine, risperidone, zolpidem, trazodone, carisoprodol, cyclobenzaprine, mirtazapine, topiramate, paroxetine, duloxetine, ramelteon, and suvorexant) administered alone and with oxycodone. At clinically relevant exposures, paroxetine, trazodone, and quetiapine given with oxycodone significantly increased pCO2 above the oxycodone effect. Analyses indicated that most pCO2 interaction effects were due to pharmacokinetic interactions resulting in increased oxycodone exposure. Increased pCO2 recorded with oxycodone-paroxetine co-administration exceeded expected effects from only drug exposure suggesting another mechanism for the increased pharmacodynamic response. This study identified drug-drug interaction effects depressing respiration in an animal model when quetiapine or paroxetine were co-administered with oxycodone. Clinical pharmacodynamic drug interaction studies are being conducted with these drugs to assess translatability of these findings.
Assuntos
Quimioterapia Combinada/efeitos adversos , Hipnóticos e Sedativos/efeitos adversos , Oxicodona/efeitos adversos , Psicotrópicos/efeitos adversos , Insuficiência Respiratória/induzido quimicamente , Animais , Oxicodona/administração & dosagem , Psicotrópicos/administração & dosagem , Ratos , Ratos Sprague-DawleyRESUMO
Difference in female sex hormone, ß-estradiol (E2), levels can contribute to sex differences in biological processes that underlie target tissue functions (QT interval), vulnerability to diseases (hepatitis or HIV), and response toward therapies. Accurate quantification of plasma E2 level is thus an important aspect in both basic science research examining hormone-regulated physiological mechanisms and in clinical settings to support patient care associated with altered E2 levels. Due to lack of a high-throughput high-sensitivity analytical method, we developed and validated a LC-MS/MS assay for accurate low-level quantification of E2 and demonstrated its application to a guinea pig pharmacokinetic study in which guinea pigs were treated with 10 or 40⯵g/kg E2 subcutaneously and blood samples collected at 0 (pre-dose), 0.25, 0.5, 1, 2, 4, 8, 12 and 24â¯h post-dosing. E2 was extracted using 90⯵L ovariectomized guinea pig plasma by liquid-liquid extraction. The method was robust, sensitive with linear range from 3.9 to 1000â¯pg/mL, and the assay met acceptance criteria for validation parameters listed in the current FDA Guidance on Bioanalytical Method Validation. Compared to the 10⯵g/kg dose, more than dose proportional increase in maximum E2 plasma concentration (Cmax) and AUC0-∞ and correspondingly longer half-life were observed after 40⯵g/kg dose. This assay is a significant improvement over existing E2 quantification methods in bioanalytical field, with high precision and accuracy, low sample and injection volumes, no derivatization, and short assay run time of 3â¯min. This assay is amenable in high-throughput settings requiring low-level E2 quantitation in basic science research and clinical settings.
Assuntos
Cromatografia Líquida/métodos , Estradiol/farmacocinética , Espectrometria de Massas em Tandem/métodos , Animais , Área Sob a Curva , Relação Dose-Resposta a Droga , Estradiol/administração & dosagem , Feminino , Cobaias , Meia-Vida , Ensaios de Triagem em Larga Escala , Extração Líquido-Líquido , OvariectomiaRESUMO
Benzodiazepines potentiate respiratory depression when combined with an opioid leading the U.S Food and Drug Administration (FDA) to recommend updating the labels of these products with a boxed warning for respiratory depression with co-use. Potential respiratory depression upon co-administration of opioids with some psychotropic drugs is not well understood. The FDA is currently investigating various psychotropic drug interactions with the commonly used opioid, oxycodone, in a rat model assessing respiratory depression. Pharmacokinetic and/or pharmacodynamic (PK/PD) interaction between oxycodone and diazepam was evaluated in a positive control arm of these experiments. Understanding the systemic exposure of these drugs alone and in combination exposures was used to identify PK/PD interactions. The authors developed a simple, high throughput liquid chromatography-tandem mass spectrometric (LC-MS/MS) assay for the simultaneous determination of oxycodone and diazepam in rat plasma. Sample preparation was performed in 96-well protein precipitation plates using acetonitrile. Processed samples were analyzed using a C18 column with a gradient mobile phase composed of 2 mM aqueous ammonium formate with 0.1% formic acid and acetonitrile. A Thermo TSQ Quantum Ultra AM triple quadrupole mass spectrometer with multiple reaction monitoring (MRM) mode was used to acquire data. The method was validated for selectivity, specificity, linearity, precision and accuracy, dilution integrity and stability. The validated LC-MS/MS assay was utilized for quantifying oxycodone and diazepam in concomitantly treated Sprague Dawley (SD) rats.
RESUMO
Opioids and benzodiazepines were frequently co-prescribed to patients with pain and psychiatric or neurological disorders; however, co-prescription of these drugs increased the risk for severe respiratory depression and death. Consequently, the U.S. Food and Drug Administration added boxed label warnings describing this risk for all opioids and benzodiazepines. Sedating psychotropic drugs with differing mechanisms of action (e.g., antipsychotics, antidepressants, non-benzodiazepine sedative-hypnotics, etc.) may be increasingly prescribed in place of benzodiazepines. Despite being marketed for years, many sedating psychotropic drugs have neither human nor animal data that quantify or qualify the potential for causing respiratory depression, either alone or in combination with an opioid. In this study, diazepam was selected as the benzodiazepine to detect any additive or synergistic effects on respiratory depression caused by the opioid, oxycodone. Pharmacokinetic studies were conducted at three doses with oxycodone (6.75, 60, 150â¯mg/kg) and with diazepam (2, 20, 200â¯mg/kg). Dose dependent decrease in arterial partial pressure of oxygen and increase in arterial partial pressure of carbon dioxide were observed with oxycodone. Diazepam caused similar partial pressure changes only at the highest dose. Further decreases in arterial partial pressure of oxygen and increases in arterial partial pressure of carbon dioxide consistent with exacerbated respiratory depression were observed in rats co-administered oxycodone 150â¯mg/kg and diazepam 20â¯mg/kg. These findings confirm previous literature reports of exacerbated opioid-induced respiratory depression with benzodiazepine and opioid co-administration and support the utility of this animal model for assessing opioid-induced respiratory depression and its potential exacerbation by co-administered drugs.
RESUMO
Urinary tract infections (UTI) are common worldwide and are becoming increasingly difficult to treat because of the development of antibiotic resistance. Immunocompetent murine models of human UTI have been used to study pathogenesis and treatment but not for investigating resistance development after treatment with antibiotics. In this study, intravesical inoculation of uropathogenic Escherichia coli CFT073 in immunocompetent Balb/c mice was used as a model of human UTI. The value of the model in investigating antibiotic exposure on in vivo emergence of antibiotic resistance was examined. Experimentally infected mice were treated with 20 or 200 mg/kg ampicillin, 5 or 50 mg/kg ciprofloxacin, or 100 or 1000 mg/kg of fosfomycin. Ampicillin and ciprofloxacin were given twice daily at 8 h intervals, and fosfomycin was given once daily. Antibiotic treatment began 24 h after bacterial inoculation and ended after 72 h following the initial treatment. Although minimum inhibitory concentrations (MIC) for the experimental strain of E. coli were exceeded at peak concentrations in tissues and consistently in urine, low levels of bacteria persisted in tissues in all experiments. E. coli from bladder tissue, kidney, and urine grew on plates containing 1× MIC of antibiotic, but none grew at 3× MIC. This model is not suitable for studying emergent resistance but might serve to examine bacterial persistence.
RESUMO
Topical ophthalmic drugs are the most commonly used dosage form to treat diseases of the anterior segment of the eye. Although this dosage form has the advantages of ease of application, small volume dose, and rapid action and is largely devoid of systemic adverse effects, the bioavailability is low due to pre-corneal anatomical barriers and the nature of the drug formulation itself. Some complex generic formulations (suspensions, ointments, gels) for topical ophthalmic products face impediments to rapid regulatory approval because of the complex nature of the formulations and difficulties in determining bioequivalence with the innovator product. Clinical endpoint bioequivalence studies of ophthalmic products in humans are challenging due to inaccessibility of internal compartments of eye, large inter-subject variability that reduces study sensitivity, patient safety issues, and the prohibitively high costs of these types of clinical studies. Because of its ocular anatomical similarity to human eye, rabbits are frequently used as a model in early product development. Generating appropriate animal model data can inform physiological-based pharmacokinetic (PBPK) model building that might eventually replace the need for extensive, expensive preclinical and clinical testing. Little detail was found in the existing literature on sampling and bioanalytical protocols for determining drug concentration in different compartments of fresh eye tissues. This study describes in detail a sampling protocol for evaluating dexamethasone concentration in different tissues of freshly harvested eyes using TobraDex ST topical ophthalmic drug product in a rabbit model.
Assuntos
Modelos Animais , Combinação Tobramicina e Dexametasona/administração & dosagem , Combinação Tobramicina e Dexametasona/farmacocinética , Animais , Disponibilidade Biológica , Sistemas de Liberação de Medicamentos , Olho/efeitos dos fármacos , Masculino , Soluções Oftálmicas/administração & dosagem , Soluções Oftálmicas/farmacocinética , Coelhos , Distribuição AleatóriaRESUMO
In mass spectrometry, compounds that have different ionization properties experience challenges in simultaneous analysis. In the present paper, the authors proposed a polarity switching (+ve and -ve) LC-MS/MS method to analyze oxycodone and topiramate in a single run. The developed method was validated in the range of 5-1000â¯ng/mL for oxycodone and 20-5000â¯ng/mL for topiramate as per the US FDA guidelines. The mass spectrometer was operated in multiple reaction monitoring (MRM) mode to analyze oxycodone and topiramate simultaneously using oxycodone-d6 and topiramate-d12 as internal standards, respectively. Sample preparation was performed in 96-well protein precipitation plates using acetonitrile. Processed samples were analyzed using a C18 column with a gradient mobile phase composed of 10â¯mm ammonium formate with 0.1% formic acid and acetonitrile. The method was validated for selectivity, specificity, linearity, precision and accuracy, dilution integrity and stability. After validation, this method was successfully applied to quantify oxycodone and topiramate in plasma of concomitantly treated Sprague Dawley (SD) rats.