Identification of Potential Lead Molecules against Dibenzo[a,l]pyrene-induced Mammary Cancer through Targeting Cytochrome P450 1A1, 1A2, and 1B1 Isozymes

Cytochrome P450 (CYP) isozymes are promising therapeutic targets against dibenzo[a,l]pyrene-induced mammary cancer. Current research aims to identify potential lead molecules against mammary cancer targetting CYP1A1, 1A2, and 1B1 using ligand-based virtual screening (LBVS), molecular interactions, MD simulation, and in vitro studies. The LBVS predicted 30,500 hits, which were reduced to 400 when sifted through Lipinski RO5, and ADMET parameters. These 400 compounds were carried forward for molecular docking with the selected CYP isozymes. The ligand CHEMBL224064 (CHEMBL1), CHEMBL2420083 (CHEMBL2), and CHEMBL61745 (CHEMBL3) depicted stronger binding respectively in CYP1A1 (-10-52 kcal/mol), 1A2 (-10.82 kcal/mol), and 1B1 (-10.78 kcal/mol) in comparison to known inhibitor alpha-naphthoflavone (ANF) (-9.13 kcal/mol, -9.66 kcal/mole, and -9.67 kcal/mol respectively in CYP1A1, 1A2, and 1B1). These compounds were found stable with their respective targets during MD studies of 50 ns duration. Furthermore, (3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide) (MTT) and enzyme inhibition assay elucidated and validated the inhibitory potential of identified ligands against mammary carcinomas. The study reveals a significant understanding of PAH-mediated mammary cancer and its prevention.

In vitro mutagenicity of selected environmental carcinogens and their metabolites in MutaMouse FE1 lung epithelial cells

Chemicals in commerce or under development must be assessed for genotoxicity; assessment is generally conducted using validated assays (e.g. Tk mouse lymphoma assay) as part of a regulatory process. Currently, the MutaMouse FE1 cell mutagenicity assay is undergoing validation for eventual use as a standard in vitro mammalian mutagenicity assay. FE1 cells have been shown to be metabolically competent with respect to some cytochrome P450 (CYP) isozymes; for instance, they can convert the human carcinogen benzo[a]pyrene into its proximate mutagenic metabolite. However, some contradictory results have been noted for other genotoxic carcinogens that require two-step metabolic activation (e.g. 2-acetylaminofluorene and 2-amino-3-methylimidazo[4,5-f]quinoxaline). Here, we examined three known or suspected human carcinogens, namely acrylamide, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and 4-aminobiphenyl (4-ABP), together with their proximate metabolites (i.e. glycidamide, N-OH-PhIP and N-OH-4-ABP), to aid in the validation of the FE1 cell mutagenicity assay. Assessments of the parent compounds were conducted both in the presence and absence of an exogenous metabolic activation mixture S9; assessments of the metabolites were in the absence of S9. The most potent compound was N-OH-PhIP -S9, which elicited a mutant frequency (MF) level 5.3-fold over background at 5 µM. There was a 4.3-fold increase for PhIP +S9 at 5 µM, a 1.7-fold increase for glycidamide −S9 at 3.5 mM and a 1.5-fold increase for acrylamide +S9 at 4 mM. Acrylamide −S9 elicited a marginal 1.4-fold MF increase at 8 mM. Treatment with PhIP −S9, 4-ABP ±S9 and N-OH-4-ABP −S9 failed to elicit significant increases in lacZ MF with any of the treatment conditions tested. Gene expression of key CYP isozymes was quantified by RT-qPCR. Cyp1a1, 1a2 and 1b1 are required to metabolise PhIP and 4-ABP. Results showed that treatment with both compounds induced expression of Cyp1a1 and Cyp1b1 but not Cyp1a2. Cyp2e1, which catalyses the bioactivation of acrylamide to glycidamide, was not induced after acrylamide treatment. Overall, our results confirm that the FE1 cell mutagenicity assay has the potential for use alongside other, more traditional in vitro mutagenicity assays.

A novel calcimimetic evocalcet for the management of secondary hyperparathyroidism with little effect on the gastrointestinal tract and cyp isozymes in vivo and in vitro

In the treatment of secondary hyperparathyroidism of end-stage chronic kidney disease, vitamin D receptor activation and allosteric modulators of the calcium-sensing receptor - inhibit glandular hyperplasia; reduce parathyroid hormone levels, impact on bone turnover and mineral density. Cinacalcet, an oral calcimimetic agent has been widely used for the management of secondary hyperparathyroidism in chronic kidney disease. Nevertheless, some patients remain refractory to the treatment, as the dose of cinacalcet cannot be sufficiently increased due to gastrointestinal symptoms and it strong inhibits of cytochrome P450 (CYP) 2D6. In order to resolve this issue, was develop a newly synthesized calcimimetic agent, evocalcet (MT-4580/KHK7580). In a rat model of chronic kidney disease induced by 5/6 nephrectomy, and in multicenter, open-label study phase 3, and in clinical practice oral administration of evocalcet efficiently suppressed the secretion of parathyroid hormone. Evocalcet also demonstrated the less induction of emesis and gastro-intestinal effects, and its pharmacological effects were observed at lower doses because of its higher bioavailability than cinacalcet. In addition, evocalcet showed no substantial direct inhibition of any CYP isozymes in in vitro. These findings suggest that evocalcet can be a better alternative to cinacalcet with a wider safety margin.

In vitro modulation of cytochrome P450 isozymes and pharmacokinetics of caffeine by extracts of Hibiscus sabdariffa Linn calyx

BackgroundHibiscus sabdariffabeverage (HSB) is widely consumed as a medicinal herb and sometimes used concomitantly with drugs. This study evaluated thein vitroinhibitory potential of the aqueous extract ofH. sabdariffacalyces (AEHS) on selected cytochrome P450 (CYP) isozymes and the effect of HSB on the pharmacokinetics of caffeinein vivo.MethodsIn vitroinhibitions of eight major CYP isozymes by AEHS were estimated by monitoring CYP-specific model reactions of 10 CYP probe substrates usingN-in-one assay method. Subsequently, an open, randomized, two-period crossover design was used to evaluate the effect of HSB on the pharmacokinetics of single-dose 200 mg caffeine in six healthy human volunteers. Blood samples were obtained at specific times over a 24 h period. Probe drugs and metabolites were analyzed in their respective matrices with ultra-performance liquid chromatography/mass spectrometer/mass spectrometer and reversed-phase high-performance liquid chromatography/ultraviolet detection.ResultsTheH. sabdariffaaqueous extract weakly inhibited the selected CYP isozymesin vitro, with IC50of >100 μgmL-1in the order of CYP1A2 > CYP2C8 > CYP2B6 >> CYP2D6 > CYP2C19 > CYP3A4 > CYP2A6 > CYP2C9. HSB decreased terminal t1/2and Tmaxof caffeine by 13.6% and 13.0%, respectively, and increased Cmaxby 10.3%. Point estimates of primary pharmacokinetic endpoints, Cmax= 1.142 (90% confidence interval (CI) = 0.882, 1.480) and AUC0–∞= 0.992 (90% CI = 0.745, 1.320), were outside the 90% CI of 0.8–1.25 bioequivalence limits.ConclusionThe aqueous extract ofH. sabdariffaweakly inhibited eight CYP isozymesin vitro, but HSB modified the exposure to caffeine in human. Caution should be exercised in administering HSB with caffeine or similar substrates of CYP1A2 until more clinical data are available.

β-Naphtoflavone and Ethanol Induce Cytochrome P450 and Protect towards MPP+ Toxicity in Human Neuroblastoma SH-SY5Y Cells

Cytochrome P450 (CYP) isozymes vary their expression depending on the brain area, the cell type, and the presence of drugs. Some isoforms are involved in detoxification and/or toxic activation of xenobiotics in central nervous system. However, their role in brain metabolism and neurodegeneration is still a subject of debate. We have studied the inducibility of CYP isozymes in human neuroblastoma SH-SY5Y cells, treated with β-naphtoflavone (β-NF) or ethanol (EtOH) as inducers, by qRT-PCR, Western blot (WB), and metabolic activity assays. Immunohistochemistry was used to localize the isoforms in mitochondria and/or endoplasmic reticulum (ER). Tetrazolium (MTT) assay was performed to study the role of CYPs during methylphenyl pyridine (MPP+) exposure. EtOH increased mRNA and protein levels of CYP2D6 by 73% and 60% respectively. Both β-NF and EtOH increased CYP2E1 mRNA (4- and 1.4-fold, respectively) and protein levels (64% both). The 7-ethoxycoumarin O-deethylation and dextromethorphan O-demethylation was greater in treatment samples than in controls. Furthermore, both treatments increased by 22% and 18%, respectively, the cell viability in MPP+-treated cells. Finally, CYP2D6 localized at mitochondria and ER. These data indicate that CYP is inducible in SH-SY5Y cells and underline this in vitro system for studying the role of CYPs in neurodegeneration.