Sharon L. Ripp

Use of immortalized human hepatocytes to predict the magnitude of clinical drug-drug interactions caused by CYP3A4 induction.

Cytochrome P4503A4 (CYP3A4) is the principal drug-metabolizing enzyme in human liver. Drug-drug interactions (DDIs) caused by induction of CYP3A4 can result in decreased exposure to coadministered drugs, with potential loss of efficacy. Immortalized hepatocytes (Fa2N-4 cells) have been proposed as a tool to identify CYP3A4 inducers. The purpose of the current studies was to characterize the effect of known inducers on CYP3A4 in Fa2N-4 cells, and to determine whether these in vitro data could reliably project the magnitude of DDIs caused by induction. Twenty-four compounds were chosen for these studies, based on previously published data using primary human hepatocytes. Eighteen compounds had been shown to be positive for induction, and six compounds had been shown to be negative for induction. In Fa2N-4 cells, all 18 positive controls produced greater than 2-fold maximal CYP3A4 induction, and all 6 negative controls produced less than 1.5-fold maximal CYP3A4 induction. Subsequent studies were conducted to determine the relationship between in vitro induction data and in vivo induction response. The approach was to relate in vitro induction data (Emax and EC50 values) with efficacious free plasma concentrations to calculate a relative induction score. This score was then correlated with decreases in area under the plasma concentration versus time curve values for coadministered CYP3A4 object drugs (midazolam or ethinylestradiol) from previously published clinical DDI studies. Excellent correlations (r2 values >0.92) were obtained, suggesting that Fa2N-4 cells can be used for identification of inducers as well as prediction of the magnitude of clinical DDIs.

Discovery of azetidinyl ketolides for the treatment of susceptible and multidrug resistant community-acquired respiratory tract infections.

Respiratory tract bacterial strains are becoming increasingly resistant to currently marketed macrolide antibiotics. The current alternative telithromycin (1) from the newer ketolide class of macrolides addresses resistance but is hampered by serious safety concerns, hepatotoxicity in particular. We have discovered a novel series of azetidinyl ketolides that focus on mitigation of hepatotoxicity by minimizing hepatic turnover and time-dependent inactivation of CYP3A isoforms in the liver without compromising the potency and efficacy of 1.

Modulation of Receptor Phosphorylation Contributes to Activation of Peroxisome Proliferator Activated Receptor α by Dehydroepiandrosterone and Other Peroxisome Proliferators

Dehydroepiandrosterone (DHEA), a C19 human adrenal steroid, activates peroxisome proliferator-activated receptor α (PPARα) in vivo but does not ligand-activate PPARα in transient transfection experiments. We demonstrate that DHEA regulates PPARα action by altering both the levels and phosphorylation status of the receptor. Human hepatoma cells (HepG2) were transiently transfected with the expression plasmid encoding PPARα and a plasmid containing two copies of fatty acyl coenzyme oxidase (FACO) peroxisome-proliferator activated receptor responsive element consensus oligonucleotide in a luciferase reporter gene. Nafenopin treatment increased reporter gene activity in this system, whereas DHEA treatment did not. Okadaic acid significantly decreased nafenopin-induced reporter activity in a concentration-dependent manner. Okadaic acid treatment of primary rat hepatocytes decreased both DHEA- and nafenopin-induced FACO activity in primary rat hepatocytes. DHEA induced both PPARα mRNA and protein levels, as well as PP2A message in primary rat hepatocytes. Western blot analysis showed that the serines at positions 12 and 21 were rapidly dephosphorylated upon treatment with DHEA and nafenopin. Results using specific protein phosphatase inhibitors suggested that protein phosphatase 2A (PP2A) is responsible for DHEA action, and protein phosphatase 1 might be involved in nafenopin induction. Mutation of serines at position 6, 12, and 21 to an uncharged alanine residue significantly increased transcriptional activity, whereas mutation to negative charged aspartate residues (mimicking receptor phosphorylation) decreased transcriptional activity. DHEA action involves induction of PPARα mRNA and protein levels as well as increased PPARα transcriptional activity through decreasing receptor phosphorylation at serines in the AF1 region.

Juvenile toxicity assessment of anidulafungin in rats: an example of navigating case-by-case study design through scientific and regulatory challenges.

BACKGROUNDnAnidulafungin, an echinocandin antifungal marketed for adult use, is being considered for use in pediatric populations, including neonates. The evolution of the nonclinical pediatric safety strategy for anidulafungin serves as an example of case-by-case negotiation through the European Medicines Agency pediatric investigation plan process, resulting in an acceptable juvenile rat toxicity study.nnnMETHODSnStudy design challenges included animal selection, route, dose, age, and duration of dosing in relation to brain maturity, and appropriate study endpoints. The definitive study consisted of subcutaneous dosing at 0, 3, 10, and 30u2009mg/kg/day from postnatal day 4 to 62 (preterm infant to adulthood) with a 5-week recovery period. Study endpoints evaluated the potential for increased juvenile sensitivity to liver toxicity (seen in adults) and for novel toxicities in the central nervous system.nnnRESULTSnAnidulafungin-related effects included slightly reduced body weight, increased liver weight, and a mild decrease in red blood cell mass with increased reticulocyte count. There was no liver pathology and in the posttreatment phase there were no effects on neurological function. Following recovery, effects on body weight, hematology, and liver weight were reversing or reversed.nnnCONCLUSIONSnTherefore, the juvenile rat no-adverse-effect-level was 30u2009mg/kg/day. Exposures at this dose are similar to those achieved at the adult rat no-adverse-effect-level, suggesting that the juvenile rat is no more sensitive to anidulafungin than the adult rat. In conclusion, dialog and negotiation between the sponsor and the European Medicines Agency allowed for successful execution of a nonclinical safety strategy that enabled further clinical investigation of anidulafungin in pediatric populations.

Structure-activity relationships and hepatic safety risks of thiazole agonists of the thrombopoietin receptor.

5-F substitution of an aminothiazole moiety within a series of thrombopoietin receptor agonists leads to potent agents with an improved hepatic safety profile in rodent toxicology studies.

The identification of orally bioavailable thrombopoietin agonists.

Recently, we disclosed a series of potent pyrimidine benzamide-based thrombopoietin receptor agonists. Unfortunately, the structural features required for the desired activity conferred physicochemical properties that were not favorable for the development of an oral agent. The physical properties of the series were improved by replacing the aminopyrimidinyl group with a piperidine-4-carboxylic acid moiety. The resulting compounds possessed favorable in vivo pharmacokinetic properties, including good bioavailability.

Effect of moxidectin on CYP3A4 activity as evaluated by oral midazolam pharmacokinetics in healthy subjects.

In order to evaluate the potential for CYP3A4 induction by moxidectin, midazolam pharmacokinetic (PK) parameters were compared before and after moxidectin administration. Healthy subjects received a single 8u2009mg dose of moxidectin and 3 single 7.5u2009mg doses of midazolam 3 days before, and 7 and 89 days after the moxidectin. Blood samples were taken for 24u2009hours to measure midazolam and metabolites in plasma, and for 89 days to measure moxidectin in plasma after dose administration. Noncompartmental PK analyses were performed for each analyte. Analysis of variance was performed on log‐transformed midazolam parameters with treatment day as a fixed effect. Adverse events were recorded and laboratory tests, physical examinations, pulse oximetry monitoring, vital sign measurement, and electrocardiograms performed. Thirty‐nine subjects were enrolled in the study; PK data were available for 37 subjects. Moxidectin PK parameters were similar to previous studies. There were no significant changes in PK for midazolam or its metabolites 7 or 89 days after moxidectin administration. Adverse events were generally mild and there were no relevant changes in safety assessments. Thus, 8u2009mg moxidectin does not induce CYP3A4 activity and other CYP3A4 substrates are unlikely to be affected by moxidectin co‐administration.

Tissue Distribution of Anidulafungin in Neonatal Rats

Anidulafungin, an echinocandin, is currently approved for treatment of fungal infections in adults. There is a high unmet medical need for treatment of fungal infections in neonatal patients, who may be at higher risk of infections involving bone, brain, and heart tissues. This in vivo preclinical study investigated anidulafungin distribution in plasma, bone, brain, and heart tissues in neonatal rats. Postnatal day (PND) 4 and PND 8 Fischer (F344/DuCrl) rats were dosed subcutaneously once with anidulafungin (10 mg/kg) or once daily for 5 days (PND 4-8). Plasma and tissue samples were collected and anidulafungin levels were measured by liquid chromatography-tandem mass spectrometry. The mean plasma Cmax and AUC0-24 values were consistent with single-dose plasma pharmacokinetics (dose normalized) reported previously for adult rats. Observed bone concentrations were similar to plasma concentrations regardless of dosing duration, with bone-to-plasma concentration ratios of approximately 1.0. Heart concentrations were higher than plasma, with heart to plasma concentration ratios of 1.3- to 1.8-fold. Brain concentrations were low after single dose, with brain-to-plasma concentration ratio of approximately 0.23, but increased to approximately 0.71 after 5 days of dosing. Tissue concentrations were nearly identical after single-dose administration in both PND 4 and PND 8 animals, indicating that anidulafungin does not appear to differentially distribute in this period in neonatal rats. In conclusion, anidulafungin distributes to bone, brain, and heart tissues of neonatal rats; such results are supportive of further investigation of efficacy against infections involving bone, brain, and heart tissues.

Non-clinical safety assessment and toxicokinetics of voriconazole and anidulafungin in the juvenile rat: A combination study design in support of a Paediatric Investigation Plan

Anidulafungin and voriconazole are potent antifungal agents that may provide a powerful therapeutic option over current therapies when coadministered. A non-clinical combination toxicity study was required as part of the voriconazole Paediatric Investigation Plan. Rats received anidulafungin or voriconazole alone or in combination once daily from postnatal day (PND) 21-56 with a recovery period to PND 84. Doses used were based upon the approximate adult rat no observed adverse-effect level (NOAEL). Transient and reversible reductions in bodyweight, haematology, serum chemistry, liver weight and minimal liver changes were associated with anidulafungin. Voriconazole caused an increase in gamma-glutamyltransferase in female rats only. No increased toxicity was observed with the combination. Toxicokinetics were determined using a validated dual-analyte bioanalytical method. Systemic exposure at juvenile rat NOAELs was comparable to that found with adult rats in previous studies. There were no drug-drug interactions affecting exposure of either drug. Juvenile rats were not more sensitive to each drug dosed alone compared with adult rat data on the single drugs. No novel, additive or synergistic toxicities were noted with the combination in juvenile rats. This study will support future studies of the combination of voriconazole and anidulafungin in children with invasive fungal infection.

In Vitro and In Vivo Investigation of Potential for Complex CYP3A Interaction for PF-00251802 (Dagrocorat), a Novel Dissociated Agonist of the Glucocorticoid Receptor

The dissociated agonists of the glucocorticoid receptor are a novel class of agents in clinical development for rheumatoid arthritis. PF‐04171327 (fosdagrocorat) is a phosphate ester prodrug of PF‐00251802 (dagrocorat), a selective high‐affinity partial agonist of the glucocorticoid receptor, which is further metabolized to PF‐04015475. This study evaluated the cytochrome P450 (CYP)–mediated drug–drug interaction (DDI) potential of PF‐00251802 and PF‐04015475 in vitro and used model‐based prediction approaches to estimate clinical impact. PF‐00251802 is a reversible inhibitor of several CYPs, but modeling has suggested no clinically relevant interaction. PF‐00251802 and PF‐04015475 are time‐dependent inhibitors and inducers of CYP3A in vitro; PF‐00251802 is also a time‐dependent inhibitor of CYP2D6. Model‐based prediction suggested the potential for weak inhibition of CYP3A in vivo. A clinical DDI study was conducted with midazolam, a sensitive CYP3A substrate. A phase 1 open‐label, multiple‐dose study evaluated the effect of PF‐04171327 on midazolam pharmacokinetics and safety in 12 healthy volunteers. Administration of midazolam alone or concomitantly with PF‐04171327 resulted in equivalent pharmacokinetic profiles (AUCinf, 21.17 vs 20.28 ng·h/mL, respectively), indicating that PF‐04171327 had no net effect on CYP3A activity in vivo. These findings support the further development of PF‐00251802 and PF‐04171327 as potential treatments for patients with rheumatoid arthritis (NCT00987038).