Cytochrome P450 Isozymes Research Articles

Significant role of CYP450 genetic variants in cyclophosphamide based breast cancer treatment outcomes: a multi-analytical strategy

BackgroundCyclophosphamide (CP), an alkylating chemotherapeutic drug, is catalyzed by the Phase I cytochrome P450 (CYPs) isozymes — CYP3A4, CYP3A5, CYP2B6, CYP2C8, CYP2C9 and CYP2C19. Hence this study aimed to elucidate the influence of genetic variants in CYP450 metabolizing enzymes on breast cancer treatment outcomes, using multi-analytical approaches. MethodsTreatment response was noticed in 111 patients whereas 234 patients were followed for myelo-toxicity. Eight known functional single nucleotide polymorphisms (SNPs) in six CYP450 genes were selected for the study on the basis of CP metabolizing enzyme polymorphisms. The possible functional effects of CYP450 polymorphisms were determined by online Web servers F-SNP. Multifactor dimensionality reductions (MDR), haplotype analysis were combined with logistic regression to characterize gene–gene interaction model with treatment outcomes. ResultsHaplotype analysis revealed significant association of Grs10509681-*1rs1799853-*3rs1057910-Grs4244285 on chromosome 10 with overall toxicity (P=0.024) and grade 2–4 leucopenia (P=0.03). On MDR analysis, CYP3A5*3, CYP2C19*2, CYP2B6*5 yielded the highest testing accuracy for treatment response (0.60) and CYP2C8*3, CYP2C9*2 for overall toxicity (0.50). ConclusionMulti-analytical approaches may provide a better clinical prediction of pharmacogenetic based treatment outcomes in breast cancer patients.

Aldrin epoxidation in flathead mullet (Mugil cephalus): possible involvement of CYP1A and CYP3A.

The primary objective of this study was to determine specific cytochrome P450 isozyme(s) involved in the metabolism of aldrin to its toxic metabolite dieldrin in flathead mullet (Mugil cephalus) liver microsomes. To identify the cytochrome P450 isozyme responsible for the aldrin metabolism in mullet liver, the effects of mammalian-specific cytochrome P450 inhibitors and substrates were determined in the epoxidation reaction of aldrin. CYP3A-related inhibitors, ketoconazole, SKF-525A, and cimetidine, inhibited the metabolism of aldrin. The contribution of CYP1A to the aldrin metabolism was shown by the inhibition of 7-ethoxyresorufin-O-deethylase activity in the presence of aldrin. The results indicate that CY1A and CYP3A are the cytochrome P450s involved in aldrin epoxidase activity in mullet. In addition, the suitability of aldrin epoxidase activity for monitoring of environmental pollution was also assessed in the fish samples caught from four different locations of the West Black Sea coast of Turkey.

Deletion of 30 Murine Cytochrome P450 Genes Results In Viable Mice With Compromised Drug Metabolism

In humans, 75% of all drugs are metabolized by the cytochrome P450-dependent monooxygenase system. Enzymes encoded by the CYP2C, CYP2D, and CYP3A gene clusters account for ∼80% of this activity. There are profound species differences in the multiplicity of cytochrome P450 enzymes, and the use of mouse models to predict pathways of drug metabolism is further complicated by overlapping substrate specificity between enzymes from different gene families. To establish the role of the hepatic and extrahepatic P450 system in drug and foreign chemical disposition, drug efficacy, and toxicity, we created a unique mouse model in which 30 cytochrome P450 genes from the Cyp2c, Cyp2d, and Cyp3a gene clusters have been deleted. Remarkably, despite a wide range of putative important endogenous functions, Cyp2c/2d/3a KO mice were viable and fertile, demonstrating that these genes have evolved primarily as detoxification enzymes. Although there was no overt phenotype, detailed examination showed Cyp2c/2d/3a KO mice had a smaller body size (15%) and larger livers (20%). Changes in hepatic morphology and a decreased blood glucose (30%) were also noted. A five-drug cocktail of cytochrome P450 isozyme probe substrates were used to evaluate changes in drug pharmacokinetics; marked changes were observed in either the pharmacokinetics or metabolites formed from Cyp2c, Cyp2d, and Cyp3a substrates, whereas the metabolism of the Cyp1a substrate caffeine was unchanged. Thus, Cyp2c/2d/3a KO mice provide a powerful model to study the in vivo role of the P450 system in drug metabolism and efficacy, as well as in chemical toxicity.

RETRACTED: Corrigendum to “Preclinical metabolism of LB42908, a novel farnesyl transferase inhibitor, and its effects on the cytochrome P450 isozyme activities” [Bioorg. Med. Chem. Lett. 22 (2012), 3067–3071

RETRACTED: Corrigendum to “Preclinical metabolism of LB42908, a novel farnesyl transferase inhibitor, and its effects on the cytochrome P450 isozyme activities” [Bioorg. Med. Chem. Lett. 22 (2012), 3067–3071

The Curious Case of Benzbromarone: Insight into Super-Inhibition of Cytochrome P450

Cytochrome P450 (CYP) family of redox enzymes metabolize drugs and xenobiotics in liver microsomes. Isozyme CYP2C9 is reported to be inhibited by benzbromarone (BzBr) and this phenomenon was hitherto explained by classical active-site binding. Theoretically, it was impossible to envisage the experimentally derived sub-nM Ki for an inhibitor, when supra-nM enzyme and 10X KM substrate concentrations were employed. We set out to find a more plausible explanation for this highly intriguing “super-inhibition” phenomenon. In silico docking of various BzBr analogs with known crystal structure of CYP2C9 did not provide any evidence in support of active-site based inhibition hypothesis. Experiments tested the effects of BzBr and nine analogs on CYPs in reconstituted systems of lab-purified proteins, complex baculosomes & crude microsomal preparations. In certain setups, BzBr and its analogs could even enhance reactions, which cannot be explained by an active site hypothesis. Generally, it was seen that Ki became smaller by orders of magnitude, upon increasing the dilution order of BzBr analogs. Also, it was seen that BzBr could also inhibit other CYP isozymes like CYP3A4, CYP2D6 and CYP2E1. Further, amphipathic derivatives of vitamins C & E (scavengers of diffusible reactive oxygen species or DROS) effectively inhibited CYP2C9 reactions in different reaction setups. Therefore, the inhibition of CYP activity by BzBr analogs (which are also surface-active redox agents) is attributed to catalytic scavenging of DROS at phospholipid interface. The current work expands the scope of interpretations of inhibitions in redox enzymes and ushers in a new cellular biochemistry paradigm that small amounts of DROS may be obligatorily required in routine redox metabolism for constructive catalytic roles.

Effect of Exposure to Acute and Chronic High-Altitude Hypoxia on the Activity and Expression of CYP1A2, CYP2D6, CYP2C9, CYP2C19 and NAT2 in Rats

We investigated the effect of exposure to acute and chronic high-altitude hypoxia (AHH and CHH) on the activity and expression of CYP1A2, CYP2D6, CYP2C9, CYP2C19 and NAT2 in rats. The rats were divided into plain (400 m), acute middle-altitude hypoxia (2,800 m), chronic middle-altitude hypoxia (2,800 m), AHH (4,300 m) and CHH (4,300 m). After probe drugs had been orally administered to the rats of the 5 groups, the serum or urine concentration of the probe drug and its metabolite was determined by reversed-phase HPLC. The activity of cytochrome P450 isozyme and NAT2 was evaluated by the ratio of the metabolite to the probe drug. The ELISA and real-time PCR were used to analyze the protein and mRNA expression of cytochrome P450 isozyme and NAT2, respectively. AHH and CHH caused significant decreases in the activity and protein and mRNA expression of rat CYP1A2 in vivo. AHH downregulates the activity and mRNA expression of rat NAT2 in vivo, and CHH upregulates the activity and protein and mRNA expression of rat CYP2D6. AHH and CHH did not change the expression of CYP2C9 and CYP2C19 in rats. This study found significant changes in the activity and protein and mRNA expression of CYP1A2, CYP2D6 and NAT2 in rats in the special environment of high-altitude hypoxia.

3,4,2′-Trimethoxy-trans-stilbene – a potent CYP1B1 inhibitor

A novel series of methoxy-trans-stilbenes with 3,4-dimethoxy motifs was designed and synthesized. The inhibitory potency of 3,4-dimethoxystilbene derivatives against cytochrome P450 isozymes CYP1A1, CYP1B1 and CYP1A2 was evaluated. 3,4,2′-Trimethoxy-trans-stilbene (3,4,2′-TMS) exhibited extremely potent inhibitory action against CYP1B1 activity with an IC50 of 0.004 μM. 3,4,2′-TMS exhibited 90-fold selectivity for CYP1B1 over CYP1A1 and 830-fold selectivity for CYP1B1 over CYP1A2. However, 3,4,2′,4′-tetramethoxy-trans-stilbene appeared to be the most selective inhibitor of both CYP1B1 and CYP1A1 showing very low affinity toward CYP1A2. Complementary experimental studies and computational methods were used to explain what structural determinants decide the specific affinity of stilbene derivatives to CYP1A2 and CYP1B1 binding sites.

EPA-1471 – Polypharmacy in geriatric psychiatry: risks and management of pharmacokinetic interactions

Psychogeriatric patients frequently suffer from comorbidities such as dementia, depression, and somatic diseases. In addition, patients with dementia often display a heterogeneous group of symptoms, the Behavioral and Psychological Symptoms of Dementia (BPSD), for which both pharmacotherapy with several drugs and non-pharmacological treatments are recommended. They are frequently comedicated with different psychopharmacological agents including antidepressants, antipsychotics, anxiolytics, hypnotics, cognitive enhancers, and Li, antipychotics and anticonvulsants used as mood stabilizers. In addition, somatic drugs are co-prescribed for the treatment of other concomitant diseases. As a consequence, these patients, who are characterised by an increased sensitivity to adverse effects, by a lowered homeostatic reserve and by decreased compliance are nonetheless often exposed to complex drug regimes. Such patients are therefore vulnerable to pharmacokinetic and pharmacodynamic interactions with severe clinical consequences. Studies in very old patients (> 80 y) are often lacking. Therefore, treatment needs to be carefully and individually tailored. Therapeutic drug monitoring may be a useful tool to optimise treatment, as some ‘classical’ indications apply for this population: Lack of compliance, adverse effects despite the use of generally recommended doses, suspected drug interactions, combination treatment with a drug known for its interaction potential, patients with pharmacokinetically relevant comorbidities (hepatic or renal insufficiency, cardiovascular disease). The increasing knowledge on the role of cytochrome P-450 isozymes in the metabolism of drugs and their interaction potential has fortunately led to a situation, which allows, to some extent, predicting risks for adverse effects after introducing a polymedication.

Effects of hydroxysafflor yellow A on the activity and mRNA expression of four CYP isozymes in rats

Ethnopharmacological relevanceIn traditional therapy with Chinese medicine, hydroxysafflor yellow A (HSYA), a main active component isolated from the dried flower of Carthamus tinctorius L., is the principal efficiency ingredient of Safflor Yellow Injection. Now HSYA has been demonstrated to have good pharmacological activities of antioxidation, myocardial and cerebral protective and neuroprotective effects. The purpose of this study was to find out whether HSYA influences the effect on rat cytochrome P450 (CYP) enzymes (CYP1A2, CYP2C11, CYP2D4 and CYP3A1) by using cocktail probe drugs in vivo; the influence on the levels of CYP mRNA was also studied. Materials and methodsA cocktail solution at a dose of 5mL/kg, which contained phenacetin (20mg/kg), tolbutamide (5mg/kg), dextromethorphan (20mg/kg) and midazolam (10mg/kg), was given as oral administration to rats treated with short or long period of intravenous HSYA via the caudal vein. Blood samples were collected at a series of time-points and the concentrations of probe drugs in plasma were determined by HPLC–MS/MS. The corresponding pharmacokinetic parameters were calculated by the software of DAS 2.0. In addition, real-time RT-PCR was performed to determine the effect of HSYA on the mRNA expression of CYP1A2, CYP2C11, CYP2D4 and CYP3A1 in rat liver. ResultsHSYA had significant inhibition effects on CYP1A2 and CYP2C11 in rats as oriented from the pharmacokinetic profiles of the probe drugs. Furthermore, HSYA had no effects on rat CYP2D4. However, CYP3A1 enzyme activity was induced by HSYA. The mRNA expression results were in accordance with the pharmacokinetic results. ConclusionsHSYA can either inhibit or induce activities of CYP1A2, CYP2C11 and CYP3A1. Therefore, co-administration of some CYP substrates with HSYA may need dose adjustment to avoid an undesirable herb–drug interaction.

In vitro metabolism and metabolic effects of ajulemic acid, a synthetic cannabinoid agonist.

Ajulemic acid is a synthetic analog of Δ8-THC-11-oic acid, the terminal metabolite of Δ8-THC. Unlike Δ9-THC, the psychoactive principle of Cannabis, it shows potent anti-inflammatory action and has minimal CNS cannabimimetic activity. Its in vitro metabolism by hepatocytes from rats, dogs, cynomolgus monkeys and humans was studied and the results are reported here. Five metabolites, M1 to M5, were observed in human hepatocyte incubations. One metabolite, M5, a glucuronide, was observed in the chromatogram of canine hepatocyte incubations. In monkey hepatocyte incubations, M5 was observed in the chromatograms of both the 120 and 240 min samples, trace metabolite M1 (side-chain hydroxyl) was observed in the 120 min samples, and trace metabolite M4 (side-chain dehydrogenation) was observed in the 240 min samples. No metabolites were found in the rat hepatocyte incubations. Unchanged amounts of ajulemic acid detected after the 2-h incubation were 103%, 90%, 86%, and 83% for rat, dog, monkey, and human hepatocytes, respectively. Additional studies were done to ascertain if ajulemic acid can inhibit the activities of five principal human cytochrome P450 isozymes; CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4/5. In contrast to the phytocannabinoids Δ9-THC and CBD, no significant inhibition of cytochrome activity was observed. These data further support the conclusions reached in earlier reports on ajulemic acid's high margin of safety and suggest that it undergoes minimal metabolism and is not likely to interfere with the normal metabolism of drugs or endogenous substances.

FV-162 Is a Novel Orally Bioavailable Proteasome Inhibitor With Improved Pharmacokinetics That Displays Preclinical Efficacy In Vitro and In Vivo

Proteasome inhibitors (PIs), such as bortezomib and carfilzomib, are often associated with serious toxicities, poor pharmacokinetics (PK), and the inconvenience of intravenous administration. Therefore, there is a need for safer and more effective orally active PIs. Applying a novel fluorine-based medicinal chemistry technology, we designed, synthesized and tested novel proprietary analogs of epoxyketone-based PIs. Our initial screening identified a lead orally bioavailable PI, FV-162, which had superior potency in proteasome activity inhibition, cytotoxicity in cultured human multiple myeloma (MM) cells and metabolic stability in mouse liver microsomes, compared to an oral PI currently under clinical evaluation, ONX-0912.FV-162 inhibited the β subunits of purified proteasomes of T. acidophilum and specifically inhibited the β5 subunit of the proteasome in 10 human myeloma cell lines with an IC50 range of 12-58 nM. In comparison, IC50 values for the β1 and β2 subunits were consistently greater than 10 µM. FV-162 also disrupted the ubiquitin-proteasome pathway in the LP-1 human myeloma cell line in a time- and dose-dependent manner, inducing intracellular accumulation of ubiquitylated proteins. FV-162 was cytotoxic to 10 human MM cell lines with an IC50 range of 16-611 nM. Furthermore, in 4 MM patient samples tested, at least 80% of primary CD138+ myeloma cells were preferentially killed by FV-162 at concentrations less than or equal to 0.5 µM after 24 hours of incubation, while more than 70% of CD138- normal hematopoietic cells remained viable.FV-162 displayed permeability in murine gastrointestinal epithelial cells, consistent with excellent oral absorption. In addition, cytochrome P450 isozyme assays in pooled human liver microsomes using up to 3 μM FV-162 suggested minimal risk for drug-drug interaction.The toxicity of epoxyketone-based PIs, such as carfilzomib, appears to be related to the Cmax, while efficacy is more dependent on the AUC. We hypothesized that the novel fluorine chemistry of FV-162 would enhance the stability of the drug and thereby improve its PK profile and therapeutic index. Therefore, we measured the oral PK of FV-162 and ONX-0912 in NOD/SCID mice (25 mg/kg). FV-162 reached a peak plasma concentration (Cmax) of 21 ± 5 ng/mL (mean ± SD) after 1 h (Tmax), while ONX-0912 reached a Cmax of 38 ± 46 ng/mL after a Tmax of 5 min. Despite the lower Cmax, the AUC of FV-162 was 6-fold higher compared to ONX-0912 (3117 vs. 513 min*ng/mL). In addition, the oral bioavailability and half-life of FV-162 were 3- and 4.3-fold higher than ONX-0912, respectively. Attenuated Cmax and improved AUC may improve the tolerability profile of FV-162 compared to ONX-0912 and carfilzomib. Supporting this contention, the maximum tolerated dose of ONX-0912 in NOD/SCID mice was 30 mg/kg, while mice tolerated 200 mg/kg FV-162 for 28 days without significant toxicity.Next, we evaluated FV-162 activity in mouse models. Inhibition of β5 subunit activity reached 80% in murine red blood cells within 1 hour of oral administration of FV-162 to NOD/SCID mice (30-100 mg/kg), which was sustained for at least 24 hours. Oral dosing of 100 mg/kg FV-162 achieved similar β5 inhibition as 30 mg/kg ONX-0912. Finally, continuous daily oral administration of FV-162 at 50 or 100 mg/kg significantly inhibited tumor growth in a myeloma MM.1S mouse xenograft model without toxicity and was as effective as intravenous carfilzomib or ONX-0912 administered at their maximal doses.Taken together, our preclinical data suggest that FV-162 has superior PK, an improved safety profile, and high oral potency, identifying it as a promising orally bioavailable PI for myeloma therapy. Further clinical investigation of this novel PI in MM is therefore warranted. Disclosures:Wang:Fluorinov Pharma Inc.: partial salary/fellowship support Other. Dove:Fluorinov Pharma Inc.: Employment. Climie:Fluorinov Pharma Inc.: Employment. O'Neill:Fluorinov Pharma Inc.: Employment. Slassi:Fluorinov Pharma Inc.: Employment.

Muscle symptoms in statin users, associations with cytochrome P450, and membrane transporter inhibitor use: A subanalysis of the USAGE study

Drug interactions have been identified as a risk factor for muscle-related side effects in statin users. The aim was to assess whether use of medications that inhibit cytochrome P450 (CYP450) isozymes, organic anion transporting polypeptide 1B1 (OATP1B1), or P-glycoprotein (P-gp) are associated with muscle-related symptoms among current and former statin users. Persons (n = 10,138) from the Understanding Statin Use in America and Gaps in Education (USAGE) internet survey were categorized about whether they ever reported new or worsening muscle pain while taking a statin (n = 2935) or ever stopped a statin because of muscle pain (n = 1516). Univariate and multivariate logistic regression models were used to assess associations between use of concomitant therapies that inhibit CYP450 isozymes, OATP1B1, P-gp, or a combination and muscle-related outcomes. In multivariate analyses, concomitant use of a CYP450 inhibitor was associated with increased odds for new or worse muscle pain (odds ratio [OR] = 1.42; P < .001) or ever having stopped a statin because of muscle pain (OR = 1.28; P = .037). Concomitant use of medication known to inhibit both OATP1B1 and P-gp was also associated with increased odds (OR = 1.80; P = .030) of ever having stopped a statin because of muscle pain. Concomitant use of medication(s) that inhibit statin metabolism was associated with increased odds of new or worse muscle pain while taking a statin and having previously stopped a statin because of muscle symptoms. These data emphasize the importance of enhancing the capabilities of clinicians and health systems for identifying and reducing statin drug interactions.

Abstract C83: Nonclinical in vitro ADME, disposition, and pharmacokinetic assessment of ARQ 092, a selective AKT inhibitor.

Abstract Introduction: ARQ 092 is a potent and selective pan-AKT inhibitor currently in Phase 1 clinical studies for the treatment of advanced solid tumors. Here, we present preclinical in vitro drug-drug interaction, metabolism, disposition, and pharmacokinetic studies with ARQ 092. Methods: ARQ 092 was assessed in vitro for cross-species metabolic stability; CYP450 (cytochrome P450) and UGT (UDP-glucuronosyltransferase) reaction phenotyping; CYP450 inhibition/induction; Caco-2 absorption and efflux; and P-gp (P-glycoprotein), BCRP (Breast Cancer Resistance Protein), and MRP2 (Multidrug Resistance-associated Protein 2) mediated ATPase stimulation. Distribution of ARQ 092 to various tissues was measured after single and repeat dosing to mice as well as pharmacokinetics in mice, rats, and monkeys. Results: Cross-species NADPH-dependent metabolism studies revealed that ARQ 092 metabolism was highly species dependent with ARQ 092 being most stable in rat and human liver microsomes (t1/2 &amp;gt; 52 min) and least stable in mouse, dog, and monkey liver microsomes (t1/2 &amp;gt; 13-33 min). Reaction phenotyping with CYP450 and UGT isozymes and CYP450 inhibitors/antibodies indicated that CYP2D6, CYP3A, UGT1A4, and possibly CYP2C9 are involved in ARQ 092 metabolism. ARQ 092 inhibited CYP2D6, 2C9, and 2C19 in HLM with IC50 values of 10.2, 3.0, and 4.0 µM, respectively, but had little activity against other isozymes tested; and no induction of CYP3A, 2B6, or 1A2 was observed. Based on these in vitro results, interactions with CYP3A4, 2D6, CYP2C9, or CYP2C19 substrates and/or inhibitors in the clinic are possible. Caco-2 and ATPase transporter studies showed that ARQ 092 was highly permeable with little efflux in Caco-2 monolayers but may be an inhibitor/substrate of P-gp and mutant BCRP. Oral bioavailability values of 23, 62, and 50% were determined in mice, rats, and monkeys, respectively, with half-lives ranging widely from 1.6 to 13.6 hrs with iv dosing and 4.3 to 16.6 hrs with po dosing. In mice, ARQ 092 was highly distributed to tissues with liver, lung, and kidney having the highest concentrations. This result correlates with the high volume of distribution of the compound in mice of 24.5 L/kg. In toxicokinetic studies, increases in Cmax and AUC were generally greater than dose proportional with repeat dosing and accumulation was observed in rats but not monkeys. Conclusion: Collectively, these data indicate that ARQ 092 has sufficient oral bioavailability to advance into clinical testing. However, the potential for drug-drug interactions with ARQ 092 in the clinic will need to be investigated further. Additionally, careful selection of dosing regimens for ARQ 092 may be crucial based on the high volume of distribution and accumulation observed preclinically. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C83. Citation Format: Laurie P. Volak, Karen R. Bresciano, Terence Hall, David Vensel, Jean-Marc Lapierre, Inese Smukste, David K. McKearn, Ronald E. Savage. Nonclinical in vitro ADME, disposition, and pharmacokinetic assessment of ARQ 092, a selective AKT inhibitor. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C83.

Sucralose, A Synthetic Organochlorine Sweetener: Overview Of Biological Issues

Sucralose is a synthetic organochlorine sweetener (OC) that is a common ingredient in the world's food supply. Sucralose interacts with chemosensors in the alimentary tract that play a role in sweet taste sensation and hormone secretion. In rats, sucralose ingestion was shown to increase the expression of the efflux transporter P-glycoprotein (P-gp) and two cytochrome P-450 (CYP) isozymes in the intestine. P-gp and CYP are key components of the presystemic detoxification system involved in first-pass drug metabolism. The effect of sucralose on first-pass drug metabolism in humans, however, has not yet been determined. In rats, sucralose alters the microbial composition in the gastrointestinal tract (GIT), with relatively greater reduction in beneficial bacteria. Although early studies asserted that sucralose passes through the GIT unchanged, subsequent analysis suggested that some of the ingested sweetener is metabolized in the GIT, as indicated by multiple peaks found in thin-layer radiochromatographic profiles of methanolic fecal extracts after oral sucralose administration. The identity and safety profile of these putative sucralose metabolites are not known at this time. Sucralose and one of its hydrolysis products were found to be mutagenic at elevated concentrations in several testing methods. Cooking with sucralose at high temperatures was reported to generate chloropropanols, a potentially toxic class of compounds. Both human and rodent studies demonstrated that sucralose may alter glucose, insulin, and glucagon-like peptide 1 (GLP-1) levels. Taken together, these findings indicate that sucralose is not a biologically inert compound.

In vitro metabolism of alisol A and its metabolites’ identification using high-performance liquid chromatography–mass spectrometry

A liquid chromatography-mass spectrometry (LC-MS) method was developed and successfully applied to the study on the enzyme kinetics of alisol A in rat liver microsomes (RLM) and human liver microsomes (HLM) incubation systems, and employed for semi-quantitative determination of each metabolite of alisol A. The metabolites of alisol A in RLM, HLM and human recombinant CYP3A4 enzyme incubation systems were identified by high-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (HPLC-QTOF MS). A total of 3 and 6 oxidative metabolites were found in RLM and HLM incubation systems, respectively. 3 metabolites found in both incubation systems were identified. The exact position of hydroxylation for the metabolites M1 and M2 could not be determined. Chemical inhibitors of cytochrome P450 (CYP450) and individual human recombinant CYP450 enzyme were used to identify the CYP450 isozymes involved in the formation of each metabolite of alisol A. The result indicated that the formation of each metabolite of alisol A was mainly catalyzed by CYP3A4 enzyme.

Inhibitory Effects of Spirulina platensis on Carcinogen-Activating Cytochrome P450 Isozymes and Potential for Drug Interactions

Spirulina platensis (SP) has been considered as potential food source of 21st century due to its remarkable nutrient profile and therapeutic benefits. However, the cytochrome P450 (CYP)-mediated drug/chemical interaction potential of SP has not yet been pursued. We investigated the effects of SP on the expressions and enzymatic activities of main CYP isozymes. After the rats were orally administered with SP daily for 5 consecutive weeks, there were significant downregulations in hepatic expression levels and inhibition in enzymatic activities of CYP1A2 and CYP2E1 compared to controls. In addition, a significant decrease was observed in CYP2C6-associated enzyme activity with no remarkable changes in messenger RNA (mRNA)/protein levels. The SP application resulted in significant increases in mRNA/protein levels of both CYP2B1 and CYP3A1 without a significant change in enzyme activities. These findings partly explain the chemopreventive properties of SP toward various organ toxicities, mutagenesis, and carcinogenesis; however, its coadministration with some CYP substrates may lead to undesirable drug interactions.

Hypericum perforatum : Pharmacokinetic, Mechanism of Action, Tolerability, and Clinical Drug-Drug Interactions

Hypericum perforatum (HP) belongs to the Hypericaceae family and is one of the oldest used and most extensively investigated medicinal herbs. The medicinal form comprises the leaves and flowering tops of which the primary ingredients of interest are naphthodianthrones, xanthones, flavonoids, phloroglucinols (e.g. hyperforin), and hypericin. Although several constituents elicit pharmacological effects that are consistent with HP's antidepressant activity, no single mechanism of action underlying these effects has thus far been found. Various clinical trials have shown that HP has a comparable antidepressant efficacy as some currently used antidepressant drugs in the treatment of mild/moderate depression. Interestingly, low-hyperforin-content preparations are effective in the treatment of depression. Moreover, HP is also used to treat certain forms of anxiety. However, HP can induce various cytochrome P450s isozymes and/or P-glycoprotein, of which many drugs are substrates and which are the main origin of HP-drug interactions. Here, we analyse the existing evidence describing the clinical consequence of HP-drug interactions. Although some of the reported interactions are based on findings from in vitro studies, the clinical importance of which remain to be demonstrated, others are based on case reports where causality can, in some cases, be determined to reveal clinically significant interactions that suggest caution, consideration, and disclosure of potential interactions prior to informed use of HP.

Fluvoxamine and celecoxib may have caused adverse events in an elderly patient

FLUVOXAMINE, WHICH DOES not have anticholinergic activity, is suitable for elderly patients. Selective serotonin re-uptake inhibitors (SSRI), however, are metabolized by and inhibit isozymes of cytochrome P450 (CYP).1 Therefore, when SSRI are prescribed, drug interactions are a concern. Here we report the case of an elderly patient whose mental state had been controlled by fluvoxamine for several years, and who developed delirium following the additional prescription of celecoxib. Informed consent was obtained from her proxy. The patient was a 94-year-old woman at the time of the present episode. Six years previously she had become amnesic, and her daily life had been supported by her family. She was anxious because of the amnesia. At 4 years before the present episode she was referred to Showa University Fujigaoka Hospital. Her score on the Revised Hasegawa Dementia Scale was 25. We diagnosed her with Alzheimer's disease. Because her cognitive functions were relatively preserved, we decided to treat her anxiety first. Therefore, we prescribed fluvoxamine (25 mg) to be taken after dinner. Subsequently, her mental state was stable for 4 years. Just before the present episode she developed a swelling in her ankle due to arthritis for which celecoxib was prescribed by another physician, without knowledge of her fluvoxamine medication history. On the first night of taking both medications, she became restless and said ‘I should go to work,’ and tried to leave her home. Her family was upset and stopped both medications. However, because she became irritable and her ankle was swollen, they restarted fluvoxamine, and her physician changed her prescription from celecoxib to loxoprofen. Since then, her mental state and ankle swelling have improved. This episode was narrated to us by her proxy once she started recovering. Therefore, we cannot elucidate the true nature of the symptoms. We suggest, however, that this condition was caused by drug interaction. Because fluvoxamine is metabolized by1 and celecoxib inhibited the CYP2D6 enzyme,2 blood concentrations of fluvoxamine seem to be increased by celecoxib. The time at which maximum concentration occurs for fluvoxamine and celecoxib is approximately 5 h and 2 h after oral intake, respectively. The plasma half-life of celecoxib is 5 h. When the patient took this medication around 8 pm, the concentration of fluvoxamine may have reached a peak and the concentration of celecoxib may have reached half of its peak by midnight. Therefore, the blood concentration of fluvoxamine reached toxic levels because of inhibition of CYP2D6 by celecoxib. Moreover, loxoprofen does not inhibit the CYP2D6 enzyme and is not metabolized by the CYP1A2 enzyme. Therefore, loxoprofen and fluvoxamine do not interact. Although we have not found any reports on this issue, SSRI should be carefully prescribed when combined with other medicines, particularly in elderly patients.

Interspecies comparison of hepatic metabolism of six newly synthesized retinoid X receptor agonistic compounds possessing a 6-[N-ethyl-N-(alkoxyisopropylphenyl)amino]nicotinic acid skeleton in rat and human liver microsomes

Objective: The hepatic metabolism of six compounds newly synthesized as retinoid X receptor agonists was characterized in rat and human liver microsomes to examine the relationship between their hepatic metabolism profiles and side chain structures, considering the interspecies difference.Materials and methods: The compounds used have a 6-[N-ethyl-N-(3-alkoxy-4-isopropylphenyl)amino]nicotinic or 6-[N-ethyl-N-(4-alkoxy-3-isopropylphenyl)amino]nicotinic acid skeleton, in which the isopropoxy, isobutoxy or cyclopropylmethoxy group is employed for the alkoxy group. These compounds were incubated with the microsomes, and their Michaelis--Menten parameters were determined. The incubation study was also performed with various cytochrome P450 (CYP) inhibitors to examine their susceptibilities to the inhibitors. In addition, a molecular docking simulation was conducted to assess the compound’s spatial configuration with the CYP isoform when necessary.Results: The Michaelis--Menten parameters determined are comparable between rats and humans for the compounds having 3-isobutoxy, 4-isobutoxy, 4-isopropoxy and 4-cyclopropylmethoxy groups. However, it was indicated that all compounds except that having the 3-isobutoxy group are metabolized in a different manner between rats and humans. That is, the extent of the contribution of each CYP isozyme is different between those two species. A molecular docking simulation showed that the spatial configuration of the compound to be associated with CYP2D6 markedly changes depending on whether the isobutoxy group is situated at the 3- or 4-position.Conclusion: A slight difference in the side chain structures markedly alters the compound’s metabolic profile, which amplifies the interspecies difference regarding the profile, increasing the difficulty in characterizing the profile in humans with the structural-property relationship and interspecies extrapolation.

Disruption of biomolecule function by nanoparticles: How do gold nanoparticles affect Phase I biotransformation of persistent organic pollutants?

The potential influence of nanoparticles on cytochrome P-450 (CYP) isozyme mediated Phase I biotransformation of persistent organic pollutants (POPs) in vitro was investigated using citrate-capped gold nanoparticles (AuNPs) and 2,2′,3,5′,6-pentachlorobiphenyl (PCB 95) as the probe nanoparticle and compound, respectively. AuNPs affected the biotransformation activity of rat CYP2B1 and changed the atropisomeric composition of PCB 95, depending on the incubation time and the AuNP concentration. Electrostatic repulsion between citrate-coated AuNPs and rat CYP2B1 may influence the active conformation of the isozyme and consequently affect its activity and stereoselectivity. In addition, the effects of AuNPs on rat CYP2B1 activity also appeared to be through interference with the CYP catalytic cycle’s electron transfer chain. Incubations with AuNPs had a decline in buffer conductance and an absorbance band red shift of AuNPs, from electrostatic interactions of K+ with negatively-charged AuNP aggregates. These ionic strength changes affected the formation rate of nicotinamide adenine dinucleotide phosphate, which provides electrons for the oxidative reaction cycle, and the biotransformation activity and stereoselectivity of CYP. This study suggests that charged nanoparticles may be able to alter the functions of biomolecules directly, by electrostatic interaction, or indirectly, by changes to the surrounding ionic strength. These factors should be taken into account for further understanding and prediction of the environmental behavior and fate of POPs and nanoparticles.