Vikram Patel

Mechanistic Model‐Informed Proarrhythmic Risk Assessment of Drugs: Review of the “CiPA” Initiative and Design of a Prospective Clinical Validation Study

The Comprehensive in vitro Proarrhythmia Assay (CiPA) initiative is developing and validating a mechanistic‐based assessment of the proarrhythmic risk of drugs. CiPA proposes to assess a drugs effect on multiple ion channels and integrate the effects in a computer model of the human cardiomyocyte to predict proarrhythmic risk. Unanticipated or missed effects will be assessed with human stem cell‐derived cardiomyocytes and electrocardiogram (ECG) analysis in early phase I clinical trials. This article provides an overview of CiPA and the rationale and design of the CiPA phase I ECG validation clinical trial, which involves assessing an additional ECG biomarker (J‐Tpeak) for QT prolonging drugs. If successful, CiPA will 1) create a pathway for drugs with hERG block / QT prolongation to advance without intensive ECG monitoring in phase III trials if they have low proarrhythmic risk; and 2) enable updating drug labels to be more informative about proarrhythmic risk, not just QT prolongation.

Development and validation of a liquid-chromatography tandem mass spectrometry method to determine in vitro and in vivo histamine release

Histamine is an important biogenic amine involved in regulating numerous physiological and pathophysiological processes in humans and animals. To date, there have been very few studies focused on developing and validating sensitive liquid-chromatography-tandem mass spectrometric (LC-MS/MS) assays capable of quantitative trace level histamine analysis in biological matrices. In the present study, a rapid and sensitive LC-MS/MS assay, amenable to high throughput analysis was developed and validated to characterize in vitro and in vivo histamine release. The LC-MS/MS procedure incorporating deuterium labeled internal standards provides rapid resolution of histamine with excellent sensitivity, precision, and accuracy. Histamine eluted at 1.5 min and was well separated from endogenous plasma peaks. The total run time of the assay was 8.0 min. A linear (r(2) ≥ 0.99) instrument response over the entire concentration range of 1.0-1000 ng/mL was observed. Excellent accuracy (error ± 3.4%) and precision (CV ± 10%) of the assay was demonstrated, with the lower limit of quantitation (LLOQ) at 15.6 ng/mL. The validated LC-MS/MS assay was applied to determine histamine release in both in vitro and in vivo models. Peritoneal mast cells treated with prototypical degranulating agents (Compound 48/80 and Teicoplanin) showed that the two chemicals caused approximately 40% histamine release. In rats, using this assay, basal histamine plasma levels were typically under 100 ng/mL. Treatment with an agent suspected of causing anaphylactic type reactions resulted in plasma histamine levels to increase above 3000 ng/mL. The LC-MS/MS assay presented in this study can be applied to further characterize the physiological and pathophysiological role of histamine release in complex in vitro and in vivo models. Importantly, the LC-MS/MS assay may be useful in assessing active pharmaceutical ingredient-mediated degranulation and anaphylaxis as part of either a pre-market or a post-market assessment of drug products.

The Effect of Oseltamivir on the Disease Progression of Lethal Influenza A Virus Infection: Plasma Cytokine and miRNA Responses in a Mouse Model

Lethal influenza A virus infection leads to acute lung injury and possibly lethal complications. There has been a continuous effort to identify the possible predictors of disease severity. Unlike earlier studies, where biomarkers were analyzed on certain time points or days after infection, in this study biomarkers were evaluated over the entire course of infection. Circulating proinflammatory cytokines and/or miRNAs that track with the onset and progression of lethal A/Puerto Rico/8/34 (PR8) influenza A virus infection and their response to oseltamivir treatment were investigated up to 10 days after infection. Changes in plasma cytokines (IL-1β, IL-10, IL-12p70, IL-6, KC, TNF-α, and IFN-γ) and several candidate miRNAs were profiled. Among the cytokines analyzed, IL-6 and KC/GRO cytokines appeared to correlate with peak viral titer. Over the selected 48 miRNAs profiled, certain miRNAs were up- or downregulated in a manner that was dependent on the oseltamivir treatment and disease severity. Our findings suggest that IL-6 and KC/GRO cytokines can be a potential disease severity biomarker and/or marker for the progression/remission of infection. Further studies to explore other cytokines, miRNAs, and lung injury proteins in serum with different subtypes of influenza A viruses with varying disease severity may provide new insight into other unique biomarkers.

Pharmacodynamic Evaluation of the Activities of Six Parenteral Vancomycin Products Available in the United States

ABSTRACT A recent report found that generic parenteral vancomycin products may not have in vivo efficacies equivalent to those of the innovator in a neutropenic murine thigh infection model despite having similar in vitro microbiological activities and murine serum pharmacokinetics. We compared the in vitro and in vivo activities of six of the parenteral vancomycin products available in the United States. The in vitro assessments for the potencies of the vancomycin products included MIC/minimal bactericidal concentration (MBC) determinations, quantifying the impact of human and murine serum on the MIC values, and time-kill studies. Also, the potencies of the vancomycin products were quantified with a biological assay, and the human and mouse serum protein binding rates for the vancomycin products were measured. The in vivo studies included dose-ranging experiments with the 6 vancomycin products for three isolates of Staphylococcus aureus in a neutropenic mouse thigh infection model. The pharmacokinetics of the vancomycin products were assessed in infected mice by population pharmacokinetic modeling. No differences were seen across the vancomycin products with regard to any in vitro evaluation. Inhibitory sigmoid maximal bacterial kill (Emax) modeling of the relationship between vancomycin dosage and the killing of the bacteria in mice in vivo yielded similar Emax and EC50 (drug exposure driving one-half Emax) values for bacterial killing. Further, there were no differences in the pharmacokinetic clearances of the 6 vancomycin products from infected mice. There were no important pharmacodynamic differences in the in vitro or in vivo activities among the six vancomycin products evaluated.

More methemoglobin is produced by benzocaine treatment than lidocaine treatment in human in vitro systems.

The clinical use of local anesthetic products to anesthetize mucous membranes has been associated with methemoglobinemia (MetHba), a serious condition in which the blood has reduced capacity to carry oxygen. An evaluation of spontaneous adverse event reporting of MetHba submitted to FDA through 2013 identified 375 reports associated with benzocaine and 16 reports associated with lidocaine. The current study was performed to determine the relative ability of benzocaine and lidocaine to produce methemoglobin (MetHb) in vitro. Incubation of 500μM benzocaine with whole human blood and pooled human liver S9 over 5h resulted in MetHb levels equaling 39.8±1.2% of the total hemoglobin. No MetHb formation was detected for 500μM lidocaine under the same conditions. Because liver S9 does not readily form lidocaine hydrolytic metabolites based on xylidine, a primary metabolic pathway, 500μM xylidine was directly incubated with whole blood and S9. Under these conditions MetHb levels of 4.4±0.4% were reached by 5h. Studies with recombinant cytochrome P450 revealed benzocaine to be extensively metabolized by CYP 1A2, with 2B6, 2C19, 2D6, and 2E1 also having activity. We conclude that benzocaine produces much more MetHb in in vitro systems than lidocaine or xylidine and that benzocaine should be more likely to cause MetHba in vivo as well.

Comparative Evaluation of U.S. Brand and Generic Intravenous Sodium Ferric Gluconate Complex in Sucrose Injection: Biodistribution after Intravenous Dosing in Rats

Relative biodistribution of FDA-approved innovator and generic sodium ferric gluconate (SFG) drug products was investigated to identify differences in tissue distribution of iron after intravenous dosing to rats. Three equal cohorts of 42 male Sprague-Dawley rats were created with each cohort receiving one of three treatments: (1) the innovator SFG product dosed intravenously at a concentration of 40 mg/kg; (2) the generic SFG product dosed intravenously at a concentration of 40 mg/kg; (3) saline dosed intravenously at equivalent volume to SFG products. Sampling time points were 15 min, 1 h, 8 h, 1 week, two weeks, four weeks, and six weeks post-treatment. Six rats from each group were sacrificed at each time point. Serum, femoral bone marrow, lungs, brain, heart, kidneys, liver, and spleen were harvested and evaluated for total iron concentration by ICP-MS. The ICP-MS analytical method was validated with linearity, range, accuracy, and precision. Results were determined for mean iron concentrations (µg/g) and mean total iron (whole tissue) content (µg/tissue) for each tissue of all groups at each time point. A percent of total distribution to each tissue was calculated for both products. At any given time point, the overall percent iron concentration distribution did not vary between the two SFG drugs by more than 7% in any tissue. Overall, this study demonstrated similar tissue biodistribution for the two SFG products in the examined tissues.

Translating New Science Into the Drug Review Process: The US FDA’s Division of Applied Regulatory Science

In 2011, the US Food and drug Administration (FDA) developed a strategic plan for regulatory science that focuses on developing new tools, standards, and approaches to assess the safety, efficacy, quality, and performance of FDA-regulated products. In line with this, the Division of Applied Regulatory Science was created to move new science into the Center for Drug Evaluation and Research (CDER) review process and close the gap between scientific innovation and drug review. The Division, located in the Office of Clinical Pharmacology, is unique in that it performs mission-critical applied research and review across the translational research spectrum including in vitro and in vivo laboratory research, in silico computational modeling and informatics, and integrated clinical research covering clinical pharmacology, experimental medicine, and postmarket analyses. The Division collaborates with Offices throughout CDER, across the FDA, other government agencies, academia, and industry. The Division is able to rapidly form interdisciplinary teams of pharmacologists, biologists, chemists, computational scientists, and clinicians to respond to challenging regulatory questions for specific review issues and for longer-range projects requiring the development of predictive models, tools, and biomarkers to speed the development and regulatory evaluation of safe and effective drugs. This article reviews the Division’s recent work and future directions, highlighting development and validation of biomarkers; novel humanized animal models; translational predictive safety combining in vitro, in silico, and in vivo clinical biomarkers; chemical and biomedical informatics tools for safety predictions; novel approaches to speed the development of complex generic drugs, biosimilars, and antibiotics; and precision medicine.In 2011, the US Food and drug Administration (FDA) developed a strategic plan for regulatory science that focuses on developing new tools, standards, and approaches to assess the safety, efficacy, quality, and performance of FDA-regulated products. In line with this, the Division of Applied Regulatory Science was created to move new science into the Center for Drug Evaluation and Research (CDER) review process and close the gap between scientific innovation and drug review. The Division, located in the Office of Clinical Pharmacology, is unique in that it performs mission-critical applied research and review across the translational research spectrum including in vitro and in vivo laboratory research, in silico computational modeling and informatics, and integrated clinical research covering clinical pharmacology, experimental medicine, and postmarket analyses. The Division collaborates with Offices throughout CDER, across the FDA, other government agencies, academia, and industry. The Division is able to rapidly form interdisciplinary teams of pharmacologists, biologists, chemists, computational scientists, and clinicians to respond to challenging regulatory questions for specific review issues and for longer-range projects requiring the development of predictive models, tools, and biomarkers to speed the development and regulatory evaluation of safe and effective drugs. This article reviews the Divisions recent work and future directions, highlighting development and validation of biomarkers; novel humanized animal models; translational predictive safety combining in vitro, in silico, and in vivo clinical biomarkers; chemical and biomedical informatics tools for safety predictions; novel approaches to speed the development of complex generic drugs, biosimilars, and antibiotics; and precision medicine.

Comparative Evaluation of U.S. Brand and Generic Intravenous Sodium Ferric Gluconate Complex in Sucrose Injection: Physicochemical Characterization

The objective of this study was to evaluate physicochemical equivalence between brand (i.e., Ferrlecit) and generic sodium ferric gluconate (SFG) in sucrose injection by conducting a series of comparative in vitro characterizations using advanced analytical techniques. The elemental iron and carbon content, thermal properties, viscosity, particle size, zeta potential, sedimentation coefficient, and molecular weight were determined. There was no noticeable difference between brand and generic SFG in sucrose injection for the above physical parameters evaluated, except for the sedimentation coefficient determined by sedimentation velocity analytical ultracentrifugation (SV-AUC) and molecular weight by asymmetric field flow fractionation-multi-angle light scattering (AFFF-MALS). In addition, brand and generic SFG complex products showed comparable molecular weight distributions when determined by gel permeation chromatography (GPC). The observed minor differences between brand and generic SFG, such as sedimentation coefficient, do not impact their biological activities in separate studies of in vitro cellular uptake and rat biodistribution. Coupled with the ongoing clinical study comparing the labile iron level in healthy volunteers, the FDA-funded post-market studies intended to illustrate comprehensive surveillance efforts ensuring safety and efficacy profiles of generic SFG complex in sucrose injection, and also to shed new light on the approval standards on generic parenteral iron colloidal products.

LC-MS/MS based quantitation of ciprofloxacin and its application to antimicrobial resistance study in Balb/c mouse plasma, urine, bladder and kidneys

The authors proposed a simple, high throughput liquid chromatography-tandem mass spectrometric (LC-MS/MS) method for the determination of ciprofloxacin in small sample volumes of plasma, urine, bladder and kidneys of mice. d8-Ciprofloxacin was used as an internal standard (IS) which efficiently tracked the matrix effect in different matrices. The method employed 20 μL of plasma/30 μL of urine or tissue homogenate for sample processing using a simple protein precipitation technique. The processed samples were chromatographed on a C18 column by using a mixture of 5 mM ammonium formate (0.1% formic acid)–acetonitrile as the mobile phase at a flow rate of 0.6 mL min−1 with a gradient elution for 3 min. The calibration curve obtained was linear over the concentration range of 100–5000 ng mL−1 with r2 ≥ 0.99 in all matrices. Method validation was performed as per FDA guidelines and the results met the acceptance criteria. The multiple reaction-monitoring mode (MRM) was used for the quantification of ion transitions at m/z 332.1/230.8 and 340.1/296.1 for the analyte and the IS, respectively. The validated method was used for preclinical studies of antimicrobial resistance in mice.

A sensitive UPLC-APCI-MS/MS method for the determination of dexamethasone and its application in an ocular tissue distribution study in rabbits following topical administration

A sensitive, specific and rapid ultra-performance liquid chromatography-atmospheric pressure chemical ionization tandem mass spectrometry (UPLC-APCI-MS/MS) method has been developed and validated for the determination of dexamethasone (DEX) in ocular tissues and biofluids of rabbits. Dexamethasone-d5 (DEX-d5) was used as an internal standard. Protein precipitation was used to extract the drug from ocular tissue homogenates, biofluids and plasma. Chromatographic separation was achieved on an Acquity C18 column (100 × 2.1 mm, 1.7 μm) using a gradient run with a mobile phase composed of methanol and 5 mM ammonium formate in 0.1% formic acid in water buffer at a flow rate of 0.5 mL min−1. A detailed method validation in two matrices (bovine serum albumin and phosphate buffer) was performed as per the FDA guidelines and the calibration curve obtained was linear (r2 = 999) over the concentration range 200–100 000 pg mL−1. A Sciex 6500+ coupled with a Waters Acquity UPLC (Ultra Performance Liquid Chromatography) system was operated in the multiple monitoring mode (MRM) during the analysis. The proposed method was applied to a rabbit ocular tissue distribution study after topical ophthalmic administration of dexamethasone and tobramycin suspension.