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.

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.

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.

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.

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.

Regulation of CYP2C11 by dehydroepiandrosterone and peroxisome proliferators: identification of the negative regulatory region of the gene.

Treatment of rats with peroxisome proliferators is known to affect gene expression, including suppression of CYP2C11. The current study examined the mechanism of negative regulation of CYP2C11, comparing the effects of a classic peroxisome proliferator, nafenopin, with those of the steroid dehydroepiandrosterone (DHEA). In vivo dose-response experiments for DHEA were carried out with rats. Only the highest dose of DHEA in the diet (0.45%), a dose previously shown to produce peroxisome proliferation, caused suppression of CYP2C11 expression. Lower doses of DHEA (0.012 to 0.20% in diet) had little effect on CYP2C11 expression. In HepG2 cells, negative regulation of a CYP2C11 reporter gene by nafenopin required coexpression of PPARalpha, whereas negative regulation by DHEA did not. Deletion analysis revealed that the responsive region for both DHEA and nafenopin was between -108 and -60 relative to the transcription start site. Mutations in several putative transcription factor binding sites in the 5'-flanking region of CYP2C11 were produced. A mutation at -121 bp significantly diminished basal expression of CYP2C11 but did not affect negative regulation by DHEA or nafenopin. A mutation at -75 bp had only a small effect on basal expression but completely abolished negative regulation by DHEA and nafenopin. Gel shift experiments indicated that PPARalpha/RXRalpha heterodimers do not bind DNA in this region. Therefore, the sequence at -75 bp of CYP2C11 is necessary for negative regulation by both DHEA and nafenopin. However, the upstream events leading to suppression at this site must differ for DHEA and nafenopin.