Comparative nonclinical assessments of the biosimilar PF-06410293 and originator adalimumab.

Adalimumab, a recombinant fully human monoclonal antibody targeting tumor necrosis factor (TNF), is approved in the United States and Europe to treat various inflammatory and autoimmune indications. Biosimilars are approved biologics highly similar, but not identical, to approved biotherapeutics. To support clinical development of PF-06410293, an adalimumab biosimilar, nonclinical studies evaluated the structural, functional, toxicologic, and toxicokinetic similarity to originator adalimumab sourced from the United States (adalimumab-US) and European Union (adalimumab-EU). Structural similarity was assessed by peptide mapping. Biologic activity was measured via inhibition of TNF-induced apoptosis and Fc-based functionality assessments. In vivo nonclinical similarity was evaluated in a toxicity study in cynomolgus monkeys administered subcutaneous PF-06410293 or adalimumab-EU (0 or 157 mg/kg/week). Peptide mapping demonstrated PF-06410293, adalimumab-US, and adalimumab-EU had identical amino acid sequences. Comparative functional and binding assessments were similar. Effects of PF-06410293 and adalimumab-EU were similar and limited to pharmacologically mediated decreased cellularity of lymphoid follicles and germinal centers in spleen. Toxicokinetics were similar; maximum plasma concentration and area-under-the-concentration-time curve ratio of PF-06410293:adalimumab-EU ranged from 1.0 to 1.2. These studies supported PF-06410293 entry into clinical development. Many regulatory agencies now only request nonclinical in vivo testing if there is residual uncertainty regarding biosimilarity after in vitro analytical studies.

Deuterium isotope effects on drug pharmacokinetics. I. System-dependent effects of specific deuteration with aldehyde oxidase cleared drugs.

The pharmacokinetic properties of drugs may be altered by kinetic deuterium isotope effects. With specifically deuterated model substrates and drugs metabolized by aldehyde oxidase, we demonstrate how knowledge of the enzyme's reaction mechanism, species differences in the role played by other enzymes in a drug's metabolic clearance, and differences in systemic clearance mechanisms are critically important for the pharmacokinetic application of deuterium isotope effects. Ex vivo methods to project the in vivo outcome using deuterated carbazeran and zoniporide with hepatic systems demonstrate the importance of establishing the extent to which other metabolic enzymes contribute to the metabolic clearance mechanism. Differences in pharmacokinetic outcomes in guinea pig and rat, with the same metabolic clearance mechanism, show how species differences in the systemic clearance mechanism can affect the in vivo outcome. Overall, to gain from the application of deuteration as a strategy to alter drug pharmacokinetics, these studies demonstrate the importance of understanding the systemic clearance mechanism and knowing the identity of the metabolic enzymes involved, the extent to which they contribute to metabolic clearance, and the extent to which metabolism contributes to the systemic clearance.

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.

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.

Modulation of receptor phosphorylation contributes to activation of peroxisome proliferator activated receptor alpha by dehydroepiandrosterone and other peroxisome proliferators.

Dehydroepiandrosterone (DHEA), a C19 human adrenal steroid, activates peroxisome proliferator-activated receptor alpha (PPARalpha) in vivo but does not ligand-activate PPARalpha in transient transfection experiments. We demonstrate that DHEA regulates PPARalpha action by altering both the levels and phosphorylation status of the receptor. Human hepatoma cells (HepG2) were transiently transfected with the expression plasmid encoding PPARalpha 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 PPARalpha 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 PPARalpha mRNA and protein levels as well as increased PPARalpha transcriptional activity through decreasing receptor phosphorylation at serines in the AF1 region.

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).

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 8 mg dose of moxidectin and 3 single 7.5 mg doses of midazolam 3 days before, and 7 and 89 days after the moxidectin. Blood samples were taken for 24 hours 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, 8 mg moxidectin does not induce CYP3A4 activity and other CYP3A4 substrates are unlikely to be affected by moxidectin co-administration.

DHEA metabolites activate estrogen receptors alpha and beta.

Dehydroepiandrosterone (DHEA) levels were reported to associate with increased breast cancer risk in postmenopausal women, but some carcinogen-induced rat mammary tumor studies question this claim. The purpose of this study was to determine how DHEA and its metabolites affect estrogen receptors α or ß (ERα or ERß)-regulated gene transcription and cell proliferation. In transiently transfected HEK-293 cells, androstenediol, DHEA, and DHEA-S activated ERα. In ERß transfected HepG2 cells, androstenedione, DHEA, androstenediol, and 7-oxo DHEA stimulated reporter activity. ER antagonists ICI 182,780 (fulvestrant) and 4-hydroxytamoxifen, general P450 inhibitor miconazole, and aromatase inhibitor exemestane inhibited activation by DHEA or metabolites in transfected cells. ERß-selective antagonist R,R-THC (R,R-cis-diethyl tetrahydrochrysene) inhibited DHEA and DHEA metabolite transcriptional activity in ERß-transfected cells. Expression of endogenous estrogen-regulated genes: pS2, progesterone receptor, cathepsin D1, and nuclear respiratory factor-1 was increased by DHEA and its metabolites in an ER-subtype, gene, and cell-specific manner. DHEA metabolites, but not DHEA, competed with 17ß-estradiol for ERα and ERß binding and stimulated MCF-7 cell proliferation, demonstrating that DHEA metabolites interact directly with ERα and ERßin vitro, modulating estrogen target genes in vivo.

Evaluation of models for predicting drug-drug interactions due to induction.

IMPORTANCE OF THE FIELD: Drug-drug interactions caused by induction of metabolizing enzymes, particularly CYP3A, can impact the efficacy and safety of co-administered drugs. It is, therefore, important to understand a new compound's potential for enzyme induction and to understand how to use the induction data generated in vitro to predict potential for drug-drug interactions in vivo. AREAS COVERED IN THIS REVIEW: Recent advances in methods for using in vitro data to predict potential for CYP3A induction in vivo are reviewed. WHAT THE READER WILL GAIN: The reader will gain a comprehensive understanding of the advantages and disadvantages of various prediction methods for induction and be able to directly compare different methods using a common in vitro data set. TAKE HOME MESSAGE: The various methods for predicting clinical CYP3A induction from in vitro induction data all have demonstrated utility; it is the authors' opinion that the correlation-based approaches offer as good or better predictivity and have simpler input requirements than more complex approaches. Of the different correlation approaches, the relatively simple unbound C(max)/EC(50) or AUC/EC(50) approaches are the simplest and yet show the best correlation to the observed clinical data. While the approaches discussed herein represent an improvement in our understanding of the predictive value of in vitro induction data, it is important to recognize that there is still room for improvement in quantitative prediction of magnitude of drug interactions due to induction.

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.

A rational chemical intervention strategy to circumvent bioactivation liabilities associated with a nonpeptidyl thrombopoietin receptor agonist containing a 2-amino-4-arylthiazole motif.

The current study examined the bioactivation potential of a nonpeptidyl thrombopoietin receptor agonist, 1-(3-chloro-5-((4-(4-fluoro-3-(trifluoromethyl)phenyl)thiazol-2-yl)carbamoyl)pyridine-2-yl)piperidine-4-carboxylic acid (1), containing a 2-carboxamido-4-arylthiazole moiety in the core structure. Toxicological risks arising from P450-catalyzed C4-C5 thiazole ring opening in 1 via the epoxidation-->diol sequence were alleviated, since mass spectrometric analysis of human liver microsome and/or hepatocyte incubations of 1 did not reveal the formation of reactive acylthiourea and/or glyoxal metabolites, which are prototypic products derived from thiazole ring scission. However, 4-(4-fluoro-3-(trifluoromethyl)phenyl)thiazol-2-amine (2), the product of hydrolysis of 1 in human liver microsomes, hepatocytes, and plasma, underwent oxidative bioactivation in human liver microsomes, since trapping studies with glutathione led to the formation of two conjugates derived from the addition of the thiol nucleophile to 2 and a thiazole- S-oxide metabolite of 2. Mass spectral fragmentation and NMR analysis indicated that the site of attachment of the glutathionyl moiety in both conjugates was the C5 position in the thiazole ring. Based on the structures of the glutathione conjugates, two bioactivation pathways are proposed, one involving beta-elimination of an initially formed hydroxylamine metabolite and the other involving direct two-electron oxidation of the electron-rich 2-aminothiazole system to electrophilic intermediates. This mechanistic insight into the bioactivation process allowed the development of a rational chemical intervention strategy that involved blocking the C5 position with a fluorine atom or replacing the thiazole ring with a 1,2,4-thiadiazole group. These structural changes not only abrogated the bioactivation liability associated with 1 but also resulted in compounds that retained the attractive pharmacological and pharmacokinetic attributes of the prototype agent.

Pyrimidine benzamide-based thrombopoietin receptor agonists.

A series of pyrimidine benzamide-based thrombopoietin receptor agonists is described. The lead molecule contains a 2-amino-5-unsubstituted thiazole, a group that has been associated with idiosyncratic toxicity. The potential for metabolic oxidation at C-5 of the thiazole, the likely source of toxic metabolites, was removed by substitution at C-5 or by replacing the thiazole with a thiadiazole. Potency in the series was improved by modifying the substituents on the pyrimidine and/or on the thiazole or thiadiazole pendant aryl ring. In vivo examination revealed that compounds from the series are not highly bioavailable. This is attributed to low solubility and poor permeability.

Oxidative metabolism of seleno-L-methionine to L-methionine selenoxide by flavin-containing monooxygenases.

The roles of flavin-containing monooxygenases (FMOs) in the oxidation of seleno-l-methionine (SeMet) to l-methionine selenoxide (MetSeO) were investigated using cDNA-expressed human FMOs, purified rat liver FMOs, and rat liver microsomes. MetSeO and the N-2,4-dinitrophenyl-derivatives of SeMet and MetSeO were synthesized and characterized by 1H-NMR and ESI/MS. These reference compounds were then used to develop a sensitive HPLC assay to monitor SeMet oxidation to MetSeO. The formation of MetSeO in rat liver microsomes was time-, protein concentration-, SeMet concentration-, and NADPH-dependent. The microsomal activity exhibited a SeMet Km value (mean +/- S.D.; n = 4) of 0.91 +/- 0.29 mM and a Vmax value of 44 +/- 8.0 nmol MetSeO/mg protein/min. The inclusion of 1-benzylimidazole, superoxide dismutase, or deferoxamine caused no inhibition of the rat liver microsomal activity. Because these results suggested the involvement of FMOs in the oxidation of SeMet in rat liver microsomes, the formation of MetSeO was also examined using cDNA-expressed human and purified rat FMOs. The results showed that both rat and human FMO1 and FMO3 but not FMO5 can catalyze the reaction. The SeMet kinetic constants were obtained with purified rat liver FMO3 (Km = 0.11 mM, Vmax = 280 nmol/mg protein/min) and rat liver FMO1 (Km = 7.8 mM, Vmax = 1200 nmol/mg protein/min). Because SeMet has anti-cancer, chemopreventive, and toxic properties, the kinetic results suggest that FMO3 is likely to play a role in the biological activities of SeMet at low exposure conditions.

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 (E(max) and EC(50) 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 (r(2) 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.

Glucocorticoids inhibit interconversion of 7-hydroxy and 7-oxo metabolites of dehydroepiandrosterone: a role for 11beta-hydroxysteroid dehydrogenases?

The cytochrome p450-dependent formation and subsequent interconversion of dehydroepiandrosterone (DHEA) metabolites 7 alpha-hydroxy-DHEA (7 alpha-OH-DHEA), 7 beta-hydroxy-DHEA (7 beta-OH-DHEA), and 7-oxo-DHEA was observed in human, pig, and rat liver microsomal fractions. Rat liver mitochondria and nuclei also converted DHEA to 7 alpha-OH-DHEA and 7-oxo-DHEA, but at a lower rate. With NADP(+), and less so with NAD(+), rat, pig, and human liver microsomes and rat liver mitochondria and nuclei converted 7 alpha-OH-DHEA to 7-oxo-DHEA. This reaction was inhibited by corticosterone and the 11 beta-hydroxysteroid dehydrogenase (11 betaHSD) inhibitor carbenoxolone (CBX). The conversion of 7 alpha-OH-DHEA to 7-oxo-DHEA by rat kidney occurred at higher rates with NAD(+) than with NADP(+) and was inhibited by corticosterone. With NADPH, 7-oxo-DHEA was converted to unidentified hydroxylated metabolites and low levels of 7 alpha-OH-DHEA by rat liver microsomes. In contrast, pig liver microsomal fractions reduced 7-oxo-DHEA to nearly equal amounts of 7 alpha- and 7 beta-OH-DHEA, while human fractions produced mainly 7 beta-OH-DHEA. Dehydrocorticosterone inhibited the reduction to both isomers by pig liver microsomes, but only to 7 alpha-OH-DHEA by human microsomes; CBX inhibited both reactions. Rat kidney did not reduce 7-oxo-DHEA with either NADPH or NADH. These results demonstrate that DHEA is first converted in liver to 7 alpha-OH-DHEA, which is subsequently oxidized to 7-oxo-DHEA in both liver and kidney. In liver, interconversion of 7-oxo-DHEA and 7-OH-DHEA isomers is largely catalyzed by 11 betaHSD1, while in kidney 11 betaHSD2 (NAD(+)-dependent) and 11 betaHSD3 (NADP(+)-dependent) likely catalyze the unidirectional oxidation of 7 alpha-hydroxy-DHEA to 7-oxo-DHEA. Distinct species-specific routes of metabolism of DHEA and the interconversion of its metabolites obviate extrapolation of animal studies to humans.

Dehydroepiandrosterone affects the expression of multiple genes in rat liver including 11 beta-hydroxysteroid dehydrogenase type 1: a cDNA array analysis.

Dehydroepiandrosterone (DHEA) is a C-19 adrenal steroid precursor to the gonadal steroids. In humans, circulating levels of DHEA, as its sulfated conjugate, are high at puberty and throughout early adulthood but decline with age. Dietary supplementation to maintain high levels of DHEA purportedly has beneficial effects on cognitive memory, the immune system, and fat and carbohydrate metabolism. In rodents, DHEA is a peroxisome proliferator that induces genes for the classical peroxisomal and microsomal enzymes associated with this response. These effects are mediated through activation of peroxisome proliferator-activated receptor alpha (PPAR alpha). However, DHEA can affect the expression of genes independently of PPAR alpha, including the gene for the major inducible drug and xenobiotic metabolizing enzyme, cytochrome P450 3A23. To elucidate the biochemistry associated with DHEA treatment, we employed a cDNA gene expression array using liver RNA from rats treated with DHEA or the classic peroxisome proliferator nafenopin. Principal components analysis identified 30 to 35 genes whose expression was affected by DHEA and/or nafenopin. Some were genes previously identified as PPAR-responsive genes. Changes in expression of several affected genes were verified by quantitative reverse transcriptase-polymerase chain reaction. These included aquaporin 3, which was induced by DHEA and to a lesser extent nafenopin, nuclear tyrosine phosphatase, which was induced by both agents, and 11 beta-hydroxysteroid dehydrogenase 1, which was decreased by treatment with DHEA in a dose-dependent fashion. Regulation of 11 beta-hydroxysteroid dehydrogenase 1 expression is important since the enzyme is believed to amplify local glucocorticoid signaling, and its repression may cause some of the metabolic effects associated with DHEA.