A metagenomic analysis for combination therapy of multiple classes of antibiotics on the prevention of the spread of antibiotic-resistant genes.

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.

A Bioanalytical Method for Quantification of N-nitrosodimethylamine (NDMA) in Human Plasma and Urine with Different Meals and following Administration of Ranitidine.

Control of N-nitrosoamine impurities is important for ensuring the safety of drug products. Findings of nitrosamine impurities in some drug products led FDA to develop new guidance providing recommendations for manufacturers towards prevention and detection of nitrosamine impurities in pharmaceutical products. One of these products, ranitidine, also had a published in vivo study, which has since been retracted by its authors, suggesting a potential for in vivo conversion of ranitidine to the probable human carcinogen, N-nitrosodimethylamine (NDMA). FDA subsequently initiated a randomized, double-blind, placebo-controlled, crossover clinical investigation to assess the potential for in vivo conversion of ranitidine to NDMA with different meals. A bioanalytical method toward characterization of NDMA formation was needed as previously published methods did not address potential NDMA formation after biofluid collection. Therefore, a bioanalytical method was developed and validated as per FDA's Bioanalytical Method Validation guidance. An appropriate surrogate matrix for calibration standards and quality control sample preparation for both liquid matrices (human plasma and urine) was optimized to minimize the artifacts of assay measurements and monitor basal NDMA levels. Interconversion potential of ranitidine to NDMA was monitored during method validation by incorporating the appropriate quality control samples. The validated methods for NDMA were linear from 15.6 pg/mL to 2000 pg/mL. Low sample volumes (2 mL for urine and 1 mL for plasma) made this method suitable for clinical study samples and helped to evaluate the influence of ranitidine administration and meal types on urinary excretion of NDMA in human subjects.

Determination of five positive control drugs in hERG external solution (buffer) by LC-MS/MS to support in vitro hERG assay as recommended by ICH S7B.

ICH S7B recommends screening for hERG channel block using patch clamp recordings to assess a drug's proarrhythmic risk. Block of the hERG channel has been associated with clinical QTC prolongation as well as the rare, but potentially fatal ventricular tachyarrhythmia Torsade de Pointes (TdP). During recording, drug concentrations perfused to the cells can deviate from nominal concentrations due to molecule-specific properties (such as non-specific binding), thereby introducing error when assessing drug potency. To account for this potential source of error, both the original ICH S7B and the newly released ICH E14/S7B Q&As guidelines call for verifying drug solutions' concentrations. Dofetilide, cisapride, terfenadine, sotalol and E-4031 are hERG blockers commonly used as positive controls to illustrate hERG assay sensitivity. The first four compounds are also clinical drugs associated with high TdP risk; therefore, their safety margins may be useful comparators to better understand an investigational product's TdP risk. Having analytical methods to quantify these five compounds in the hERG external solution that will be used for patch clamp recordings is important from a regulatory science research perspective. However, a literature search revealed no analytical methods or stability information for these molecules in the high salt, serum-free matrix that constitutes the hERG external solution. This study was conducted to develop and validate LC-MS/MS methods to quantify these 5 molecules in hERG external solution. The bioanalytical methods for these positive controls were validated as per the FDA's bioanalytical method validation guidance along with various stabilities.

Effect of Paroxetine or Quetiapine Combined With Oxycodone vs Oxycodone Alone on Ventilation During Hypercapnia: A Randomized Clinical Trial.

Importance: Opioids can cause severe respiratory depression by suppressing feedback mechanisms that increase ventilation in response to hypercapnia. Following the addition of boxed warnings to benzodiazepine and opioid products about increased respiratory depression risk with simultaneous use, the US Food and Drug Administration evaluated whether other drugs that might be used in place of benzodiazepines may cause similar effects. Objective: To study whether combining paroxetine or quetiapine with oxycodone, compared with oxycodone alone, decreases the ventilatory response to hypercapnia. Design, Setting, and Participants: Randomized, double-blind, crossover clinical trial at a clinical pharmacology unit (West Bend, Wisconsin) with 25 healthy participants from January 2021 through May 25, 2021. Interventions: Oxycodone 10 mg on days 1 and 5 and the following in a randomized order for 5 days: paroxetine 40 mg daily, quetiapine twice daily (increasing daily doses from 100 mg to 400 mg), or placebo. Main Outcomes and Measures: Ventilation at end-tidal carbon dioxide of 55 mm Hg (hypercapnic ventilation) using rebreathing methodology assessed for paroxetine or quetiapine with oxycodone, compared with placebo and oxycodone, on days 1 and 5 (primary) and for paroxetine or quetiapine alone compared with placebo on day 4 (secondary). Results: Among 25 participants (median age, 35 years [IQR, 30-40 years]; 11 female [44%]), 19 (76%) completed the trial. The mean hypercapnic ventilation was significantly decreased with paroxetine plus oxycodone vs placebo plus oxycodone on day 1 (29.2 vs 34.1 L/min; mean difference [MD], -4.9 L/min [1-sided 97.5% CI, -∞ to -0.6]; P = .01) and day 5 (25.1 vs 35.3 L/min; MD, -10.2 L/min [1-sided 97.5% CI, -∞ to -6.3]; P < .001) but was not significantly decreased with quetiapine plus oxycodone vs placebo plus oxycodone on day 1 (33.0 vs 34.1 L/min; MD, -1.2 L/min [1-sided 97.5% CI, -∞ to 2.8]; P = .28) or on day 5 (34.7 vs 35.3 L/min; MD, -0.6 L/min [1-sided 97.5% CI, -∞ to 3.2]; P = .37). As a secondary outcome, mean hypercapnic ventilation was significantly decreased on day 4 with paroxetine alone vs placebo (32.4 vs 41.7 L/min; MD, -9.3 L/min [1-sided 97.5% CI, -∞ to -3.9]; P < .001), but not with quetiapine alone vs placebo (42.8 vs 41.7 L/min; MD, 1.1 L/min [1-sided 97.5% CI, -∞ to 6.4]; P = .67). No drug-related serious adverse events were reported. Conclusions and Relevance: In this preliminary study involving healthy participants, paroxetine combined with oxycodone, compared with oxycodone alone, significantly decreased the ventilatory response to hypercapnia on days 1 and 5, whereas quetiapine combined with oxycodone did not cause such an effect. Additional investigation is needed to characterize the effects after longer-term treatment and to determine the clinical relevance of these findings. Trial Registration: ClinicalTrials.gov Identifier: NCT04310579.

Calibration and Validation of a Mechanistic COVID-19 Model for Translational Quantitative Systems Pharmacology - A Proof-of-Concept Model Development for Remdesivir.

With the ongoing global pandemic of coronavirus disease 2019 (COVID-19), there is an urgent need to accelerate the traditional drug development process. Many studies identified potential COVID-19 therapies based on promising nonclinical data. However, the poor translatability from nonclinical to clinical settings has led to failures of many of these drug candidates in the clinical phase. In this study, we propose a mechanism-based, quantitative framework to translate nonclinical findings to clinical outcome. Adopting a modularized approach, this framework includes an in silico disease model for COVID-19 (virus infection and human immune responses) and a pharmacological component for COVID-19 therapies. The disease model was able to reproduce important longitudinal clinical data for patients with mild and severe COVID-19, including viral titer, key immunological cytokines, antibody responses, and time courses of lymphopenia. Using remdesivir as a proof-of-concept example of model development for the pharmacological component, we developed a pharmacological model that describes the conversion of intravenously administered remdesivir as a prodrug to its active metabolite nucleoside triphosphate through intracellular metabolism and connected it to the COVID-19 disease model. After being calibrated with the placebo arm data, our model was independently and quantitatively able to predict the primary endpoint (time to recovery) of the remdesivir clinical study, Adaptive Covid-19 Clinical Trial (ACTT). Our work demonstrates the possibility of quantitatively predicting clinical outcome based on nonclinical data and mechanistic understanding of the disease and provides a modularized framework to aid in candidate drug selection and clinical trial design for COVID-19 therapeutics.

New science, drug regulation, and emergent public health issues: The work of FDA's division of applied regulatory science.

The U.S. Food and Drug Administration (FDA) Division of Applied Regulatory Science (DARS) moves new science into the drug review process and addresses emergent regulatory and public health questions for the Agency. By forming interdisciplinary teams, DARS conducts mission-critical research to provide answers to scientific questions and solutions to regulatory challenges. Staffed by experts across the translational research spectrum, DARS forms synergies by pulling together scientists and experts from diverse backgrounds to collaborate in tackling some of the most complex challenges facing FDA. This includes (but is not limited to) assessing the systemic absorption of sunscreens, evaluating whether certain drugs can convert to carcinogens in people, studying drug interactions with opioids, optimizing opioid antagonist dosing in community settings, removing barriers to biosimilar and generic drug development, and advancing therapeutic development for rare diseases. FDA tasks DARS with wide ranging issues that encompass regulatory science; DARS, in turn, helps the Agency solve these challenges. The impact of DARS research is felt by patients, the pharmaceutical industry, and fellow regulators. This article reviews applied research projects and initiatives led by DARS and conducts a deeper dive into select examples illustrating the impactful work of the Division.

Effect of Oral Ranitidine on Urinary Excretion of N-Nitrosodimethylamine (NDMA): A Randomized Clinical Trial.

Importance: In 2019, the US Food and Drug Administration (FDA) received a citizen petition indicating that ranitidine contained the probable human carcinogen N-nitrosodimethylamine (NDMA). In addition, the petitioner proposed that ranitidine could convert to NDMA in humans; however, this was primarily based on a small clinical study that detected an increase in urinary excretion of NDMA after oral ranitidine consumption. Objective: To evaluate the 24-hour urinary excretion of NDMA after oral administration of ranitidine compared with placebo. Design, Setting, and Participants: Randomized, double-blind, placebo-controlled, crossover clinical trial at a clinical pharmacology unit (West Bend, Wisconsin) conducted in 18 healthy participants. The study began in June 2020, and the end of participant follow-up was July 1, 2020. Interventions: Participants were randomized to 1 of 4 treatment sequences and over 4 periods received ranitidine (300 mg) and placebo (randomized order) with a noncured-meats diet and then a cured-meats diet. The cured-meats diet was designed to have higher nitrites, nitrates (nitrate-reducing bacteria can convert nitrates to nitrites), and NDMA. Main Outcome and Measure: Twenty-four-hour urinary excretion of NDMA. Results: Among 18 randomized participants (median age, 33.0 [interquartile range {IQR}, 28.3 to 42.8] years; 9 women [50%]; 7 White [39%], 11 African American [61%]; and 3 Hispanic or Latino ethnicity [17%]), 17 (94%) completed the trial. The median 24-hour NDMA urinary excretion values for ranitidine and placebo were 0.6 ng (IQR, 0 to 29.7) and 10.5 ng (IQR, 0 to 17.8), respectively, with a noncured-meats diet and 11.9 ng (IQR, 5.6 to 48.6) and 23.4 ng (IQR, 8.6 to 36.7), respectively, with a cured-meats diet. There was no statistically significant difference between ranitidine and placebo in 24-hour urinary excretion of NDMA with a noncured-meats diet (median of the paired differences, 0 [IQR, -6.9 to 0] ng; P = .54) or a cured-meats diet (median of the paired differences, -1.1 [IQR, -9.1 to 11.5] ng; P = .71). No drug-related serious adverse events were reported. Conclusions and Relevance: In this trial that included 18 healthy participants, oral ranitidine (300 mg), compared with placebo, did not significantly increase 24-hour urinary excretion of NDMA when participants consumed noncured-meats or cured-meats diets. The findings do not support that ranitidine is converted to NDMA in a general, healthy population. Trial Registration: ClinicalTrials.gov Identifier: NCT04397445.

Effects of sedative psychotropic drugs combined with oxycodone on respiratory depression in the rat.

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.

Emergence of nosocomial associated opportunistic pathogens in the gut microbiome after antibiotic treatment.

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.

The effect of antibiotics on the gut microbiome: a metagenomics analysis of microbial shift and gut antibiotic resistance in antibiotic treated mice.

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.

Developing an animal model to detect drug-drug interactions impacting drug-induced respiratory depression.

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, 150 mg/kg) and with diazepam (2, 20, 200 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 150 mg/kg and diazepam 20 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.

A high-throughput bioanalytical assay to support pharmacokinetic interaction study of oxycodone and diazepam in Sprague Dawley rats.

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.

Quantitative analysis of underivatized 17 ß-estradiol using a high-throughput LC-MS/MS assay - Application to support a pharmacokinetic study in ovariectomized guinea pigs.

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 µg/kg E2 subcutaneously and blood samples collected at 0 (pre-dose), 0.25, 0.5, 1, 2, 4, 8, 12 and 24 h post-dosing. E2 was extracted using 90 µL ovariectomized guinea pig plasma by liquid-liquid extraction. The method was robust, sensitive with linear range from 3.9 to 1000 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 µg/kg dose, more than dose proportional increase in maximum E2 plasma concentration (Cmax) and AUC0-∞ and correspondingly longer half-life were observed after 40 µg/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 3 min. This assay is amenable in high-throughput settings requiring low-level E2 quantitation in basic science research and clinical settings.

Evaluation of Immunocompetent Urinary Tract Infected Balb/C Mouse Model For the Study of Antibiotic Resistance Development Using Escherichia Coli CFT073 Infection.

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.

Detection of Reg3γ by Immunohistochemistry in Cerulein-Induced Model of Acute Pancreatic Injury in Mice and Rats.

OBJECTIVE: In a continuation of previous work, Reg3γ protein was further evaluated as a biomarker of pancreatic injury using immunohistochemistry in an additional species. METHODS: Mice and rats were treated with intraperitoneal cerulein injections, creating acute pancreatic injury. Mice received 2, 4, or 6 doses, and rats received 1, 2, or 3 doses of cerulein creating low, medium, and high treatment groups. Control animals were dosed with phosphate-buffered saline at corresponding volumes and intervals. Groups of 6 animals were killed 1, 3, 6, 24, and 48 hours after final treatments. Reg3γ immunohistochemical staining and image analysis were performed on pancreatic tissue obtained 6, 24, or 48 hours after control or cerulein treatment. Staining was quantified using image analysis software to calculate area of positivity as a percentage of total tissue area. RESULTS: Percent positivity of Reg3γ in both species rose by 6 hours, peaked by 24 hours across all 3 cerulein doses, and dropped significantly by 48 hours. In high-dose rats with accompanying gene expression data, Reg3γ gene expression corresponded temporally with quantitative staining data. CONCLUSIONS: Reg3γ staining quantified through image analysis showed a time- and dose-response in cerulein-treated mice and rats.

Application of Mechanistic Ocular Absorption Modeling and Simulation to Understand the Impact of Formulation Properties on Ophthalmic Bioavailability in Rabbits: a Case Study Using Dexamethasone Suspension.

Developing mathematical models to predict changes in ocular bioavailability and pharmacokinetics due to differences in the physicochemical properties of complex topical ophthalmic suspension formulations is important in drug product development and regulatory assessment. Herein, we used published FDA clinical pharmacology review data, in-house, and literature rabbit pharmacokinetic data generated for dexamethasone ophthalmic suspensions to demonstrate how the mechanistic Ocular Compartmental Absorption and Transit model by GastroPlus™ can be used to characterize ocular drug pharmacokinetic performance in rabbits for suspension formulations. This model was used to describe the dose-dependent (0.01 to 0.1%) non-linear pharmacokinetic in ocular tissues and characterize the impact of viscosity (1.67 to 72.9 cP) and particle size (5.5 to 22 µm) on in vivo ocular drug absorption and disposition. Parameter sensitivity analysis (hypothetical suspension particle size: 1 to 10 µm, viscosity: 1 to 100 cP) demonstrated that the interplay between formulation properties and physiological clearance through drainage and tear turnover rates in the pre-corneal compartment drives the ocular drug bioavailability. The quick removal of drug suspended particles from the pre-corneal compartment renders the impact of particle size inconsequential relative to viscosity modification. The in vivo ocular absorption is (1) viscosity non-sensitive when the viscosity is high and the impact of viscosity on the pre-corneal residence time reaches the maximum physiological system capacity or (2) viscosity sensitive when the viscosity is below a certain limit. This study reinforces our understanding of the interplay between physiological factors and ophthalmic formulation physicochemical properties and their impact on in vivo ocular drug PK performance in rabbits.

An alternating polarity switching assay for quantification of oxycodone and topiramate: An application of LC-MS/MS method in support to PK/PD study in rodents.

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 5-1000 ng/mL for oxycodone and 20-5000 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 10 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.

Protocol for evaluation of topical ophthalmic drug products in different compartments of fresh eye tissues in a rabbit model.

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.

Biomarkers of drug-induced acute kidney injury: a regulatory perspective.

INTRODUCTION: Biomarkers are one of the drug development tools that are being developed through collaborative efforts among multiple stakeholder communities to enhance the drug development process. Biomarkers of acute drug-induced renal injury as used in drug development are more commonly referred to as renal safety biomarkers, the focus of this manuscript. Areas covered: This manuscript provides an overview of the history and evolution of the United States Food and Drug Administration's Center for Drug Evaluation and Research's Biomarker Qualification Program. In addition, a regulatory perspective on the potential for renal safety biomarkers to accelerate medical and pharmaceutical research is presented. The first qualification submissions (acute kidney injury biomarkers) are discussed, including how the FDA review process affected the evolution of the biomarker qualification process and the future of biomarker discovery, development, and use. This manuscript also discusses a new repository for data on novel translational safety biomarkers from drug development programs. Expert opinion: In addition to the qualification of novel biomarkers, a key achievement of the first submission for qualification was the bringing together of multiple stakeholder communities to optimize the process. Early qualification reviews provided valuable lessons that informed an overarching approach of how to develop a biomarker for regulatory use.