Effects of major tanshinones isolated from Danshen (Salvia miltiorrhiza) on rat CYP1A2 expression and metabolism of model CYP1A2 probe substrates

This study explored the effects of Danshen on metabolism/pharmacokinetics of model CYP1A2 substrates and hepatic CYP1A2 expression in rats. The effects of Danshen and tanshinones on CYP1A2 activity was determined by metabolism of model substrates in vitro (phenacetin) and in vivo (caffeine). HPLC was used to determine model substrates/metabolites. The effect of Danshen on CYP1A2 expression was determined by Western blot. Tanshinones (1.25–50 μM) competitively inhibited phenacetin O-deethylation in vitro. Inhibition kinetics studies showed the K_i values were in the order: dihydrotanshinone (3.64 μM), cryptotanshinone (4.07 μM), tanshinone I (22.6 μM) and tanshinone IIA (23.8 μM), furafylline (35.8 μM), a CYP1A2 inhibitor. The Ki of Danshen extract (mainly tanshinones) was 72 μg/ml. Acute Danshen extract treatment (50–200 mg/kg, i.p.) decreased metabolism of caffeine to paraxanthine, with overall decrease in caffeine clearance (14–22%); increase in AUC (11–25%) and plasma T_1/2 (12–16%). Danshen treatment with (100 mg/kg/day, i.p. or 200 mg/kg/day, p.o.) for three or fourteen days showed similar pharmacokinetic changes of the CYP1A2 probe substrate without affecting CYP1A2 expression. This study demonstrated that major tanshinones competitively inhibited the metabolism of model CYP1A2 probe substrates but had no effect on rat CYP1A2 expression.     

Synthesis and CYP24A1 inhibitory activity of N-(2-(1H-imidazol-1-yl)-2-phenylethyl)arylamides

A series of N-(2-(1H-imidazol-1-yl)-2-phenylethyl)arylamides were prepared, using an efficient three- to five-step synthesis, and evaluated for their inhibitory activity against human cytochrome P450C24A1 (CYP24A1) hydroxylase. Inhibition ranged from IC₅₀ 0.3–72 μM compared with the standard ketoconazole IC₅₀ 0.52 μM, with the styryl derivative (11c) displaying enhanced activity (IC₅₀ = 0.3 μM) compared with the standard, providing a useful preliminary lead for drug development.     

Metabolic activation of phenanthrene by human and mouse cytochromes P450 and pharmacokinetics in CYP1A2 knockout mice

In the present study V79 Chinese hamster cells were genetically engineered for stable expression of the cytochromes P450 1A1, 1A2, 1B1, and 2E1 from man and mouse to investigate species-specific differences in the regioselective metabolism and toxicity of phenanthrene (Phe), the simplest polycyclic aromatic hydrocarbon (PAH) forming a bay-region. Phe is present in various environmental samples and serves as a model substrate for PAH exposure in human biomonitoring studies. For this reason we explored metabolite profiles and metabolite-dependent cytotoxic activities in vitro. The total turnover of CYP-mediated transformation of Phe was as follows: human CYP1B1 > CYP1A1 > CYP1A2 ? CYP2E1, and for mouse CYP1A2 ? CYP2E1 > CYP1A1. Striking species differences were seen as mouse CYP1B1 did not activate Phe at all, but human CYP1B1 exhibited a significant metabolic turnover comparable to CYP1A1 and CYP1A2. In vivo studies monitoring the whole blood Phe elimination in CYP1A2 knockout and wild-type mice after oral administration confirmed involvement of CYP1A2 in the bioactivation of Phe, but other processes must contribute also. Our data suggest that in humans not only CYP1A2 expressed solely in the liver plays a crucial role in Phe metabolism, but also constitutively expressed extrahepatic CYP1B1 in tissues such as lung, kidney or intestine. This finding will substantially improve the validity of human biomonitoring studies using individual Phe metabolites for the assessment of PAH exposure.     

Alkoxyresorufin (methoxy-, ethoxy-, pentoxy- and benzyloxyresorufin) O-dealkylase activities by in vitro-expressed cytochrome P450 1A4 and 1A5 from common cormorant (Phalacrocorax carbo)

Here we report the inter-paralog comparison of cytochrome P4501A (CYP1A) catalytic function in common cormorant (Phalacrocorax carbo) using the recombinant proteins synthesized by yeast-based vector system. CYP1A4 and CYP1A5 proteins from common cormorant were heterologously expressed in yeast Saccaromyces cerevisiae. Kinetic analyses revealed that among alkoxyresorufin (methoxy-, ethoxy-, pentoxy- and benzyloxyresorufin) O-dealkylase (AROD) activities V_max value for ethoxyresorufin O-deethylase (EROD) activity was the highest for both enzymes, reaching 0.91 ± 0.034 and 1.8 ± 0.043 nmol/min/nmol CYP for CYP1A4 and CYP1A5, respectively. Similar results were obtained for the catalytic efficiencies represented as the ratios of V_max to K_m (V_max/K_m). Meanwhile, distinct substrate preferences were also observed; CYP1A4 had V_max and V_max/K_m values for benzyloxyresorufin O-debenzylase (BROD) activity 12- and 46-fold greater than CYP1A5, respectively, while CYP1A5 was about 13- and 4.5-fold more efficient in methoxyresorufin O-demethylase (MROD) activity than CYP1A4. The K_m values showed no significant change among MROD, EROD, pentoxyresorufin O-depenthylase (PROD) and BROD activities for both enzymes, except for significant differences between PROD and other three activities for CYP1A4. Comparing the results in the present study with previous studies addressing chicken and rat CYP1A enzymes, it is also clear that CYP1A orthologs have different catalytic preferences for AROD activities between cormorant and rat and even between cormorant and chicken. Variations in CYP1A catalytic function between cormorant CYP1A paralogs and between CYP1A orthologs from cormorant and other species indicate that enzymatic properties should be characterized on the basis not only of a limited model species such as chicken, but also of multiple species to further understand the mechanism underlying differences in substrate selectivity and the interaction with environmental contaminants in avian species.     

CYP1AI, glutathione S-transferase gene polymorphisms and risk of Polycythemia vera

Background: Associations between polymorphisms for gene encoding enzymes involved in biotransformation of xenobiotics and susceptibility to several cancers have been shown in several studies. The aim of the present study was to evaluate the association of polymorphisms of cytochrome P450 (CYP1A1) and GST deletions with the incidence of Polycythemia vera (PV) among the Jordanian population. Methods: The study included 61 PV patients and 70 cancer-free healthy controls. CYP1A1 (m1, m2, m3, m4) and GST (T1, M1) genotypes were determined by polymerase chain reaction and restriction fragment length polymorphism. The risk of cancer associated with gene polymorphisms was estimated by calculations of odds ratio (ORs) and confidence intervals (95% CIs) using Mantel–Haenszel statistics. Results: A statistically significant difference between the PV group and the control group was observed in the case of GSTM1 null genotype with 3.38 fold increase in risk of developing PV (95% CI = 1.63–7.01, p = 0.001) while GSTT1 null genotype showed no significance (OR = 1.11; 95% CI = 0.50–2.44, p = 0.38). No significant association was found between the CYP1A1 mutant genotypes (m1, m2, m4) and PV. The m3 genotype was absent in both patients and controls. Interestingly, a substantial significant increase of PV risk for the combination of GSTM1 null genotype and CYP1A1 m1 (T6235C) genotype was observed (OR = 4.38; 95% CI = 1.15–16.73, p = .02). Furthermore, the present case–control study showed that the studied Jordanian population generally resembles Caucasian populations with respect to the frequencies of CYP1A1 polymorphisms. Conclusion: Our data suggests that GSTM1 null genotype alone and in combination with CYP1A1 m1 genotype may be predisposing risk factors for PV in the Jordanian population.     

Hepatocellular toxicity of clopidogrel: Mechanisms and risk factors

Clopidogrel is a prodrug used widely as a platelet aggregation inhibitor. After intestinal absorption, approximately 90% is converted to inactive clopidogrel carboxylate and 10% via a two-step procedure to the active metabolite containing a mercapto group. Hepatotoxicity is a rare but potentially serious adverse reaction associated with clopidogrel. The aim of this study was to find out the mechanisms and susceptibility factors for clopidogrel-associated hepatotoxicity. In primary human hepatocytes, clopidogrel (10 and 100 μM) was cytotoxic only after cytochrome P450 (CYP) induction by rifampicin. Clopidogrel (10 and 100 μM) was also toxic for HepG2 cells expressing human CYP3A4 (HepG2/CYP3A4) and HepG2 cells co-incubated with CYP3A4 supersomes (HepG2/CYP3A4 supersome), but not for wild-type HepG2 cells (HepG2/wt). Clopidogrel (100 μM) decreased the cellular glutathione content in HepG2/CYP3A4 supersome and triggered an oxidative stress reaction (10 and 100 µM) in HepG2/CYP3A4, but not in HepG2/wt. Glutathione depletion significantly increased the cytotoxicity of clopidogrel (10 and 100 µM) in HepG2/CYP3A4 supersome. Co-incubation with 1 μM ketoconazole or 10 mM glutathione almost completely prevented the cytotoxic effect of clopidogrel in HepG2/CYP3A4 and HepG2/CYP3A4 supersome. HepG2/CYP3A4 incubated with 100 μM clopidogrel showed mitochondrial damage and cytochrome c release, eventually promoting apoptosis and/or necrosis. In contrast to clopidogrel, clopidogrel carboxylate was not toxic for HepG2/wt or HepG2/CYP3A4 up to 100 µM. In conclusion, clopidogrel incubated with CYP3A4 is associated with the formation of metabolites that are toxic for hepatocytes and can be trapped by glutathione. High CYP3A4 activity and low cellular glutathione stores may be risk factors for clopidogrel-associated hepatocellular toxicity.     

Limited in vitro efficacy of CYP17A1 inhibition on human castration resistant prostate cancer

Although accumulating evidence indicates high expression of CYP17A1(P45017A1) allows castration resistant prostate cancer (CRPC) to maintain high intratumoral androgen levels, the potential P45017A1 activity has not been characterized yet. The aim of this study was to examine the potential CYP17A1 activity including 17α-hydroxylase and 17,20-lyase activities in human CRPC and the effect of a CYP17A inhibitor. We used three human CRPC cell lines: C4-2 and C4-2AT6 which was established from C4-2 under androgen ablation conditions for 6 months, and PC3. To ascertain the potential CYP17A1 activity, we cultured with the steroid precursors: ¹³C-[2,3,4]-progesterone (13C-Prog), and analyzed the sequential biosynthesis ¹³C-[2,3,4]-17-hydroxyprogesterone (13C-17OHP) and ¹³C-[2,3,4]-androstenedione(13C-Adione) by liquid chromatography/mass spectrometry (LC/MS/MS).The C4-2AT6 cells showed significantly higher CYP17A1 expression than C4-2 cells (p < 0.001). LC/MS/MS analysis enabled us to detect the 13C-17-OHP and 13C-A-dione in these cell lines. The concentration ratio of 13C-Adione/13C-17OHP (Adione–17OHP ratio), which is thought to reflect the differences between 17-hydroxylase and 17,20-lyase activities, was then determined. The Adione–17OHP ratio in C4-2AT6 cells was significantly higher than that of C4-2 cells (p < 0.001). Abiraterone were able to inhibit the CYP17A activities, although abiraterone did not have anti-proliferative effects on C4-2 and C4-2AT6 cells at clinically achievable concentrations of <1000 nM in vitro. The present study clearly demonstrates CRPC have the dual activities of CYP17A1 mediated by 17-hydroxylase activity and 17,20-lyase activity. Abiraterone doesn’t have an in vitro anti-proliferative efficacy in CRPC cells, suggesting limited efficacy in vitro.     

Metabolism of carbosulfan II. Human interindividual variability in its in vitro hepatic biotransformation and the identification of the cytochrome P450 isoforms involved

This study aims to characterize interindividual variability and individual CYP enzymes involved in the in vitro metabolism of the carbamate insecticide carbosulfan. Microsomes from ten human livers (HLM) were used to characterize the interindividual variability in carbosulfan activation. Altogether eight phase I metabolites were analyzed by LC–MS. The primary metabolic pathways were detoxification by the initial oxidation of sulfur to carbosulfan sulfinamide (‘sulfur oxidation pathway’) and activation via cleavage of the nitrogen sulfur bond (N–S) to give carbofuran and dibutylamine (‘carbofuran pathway’). Differences between maximum and minimum carbosulfan activation values with HLM indicated nearly 5.9-, 7.0, and 6.6-fold variability in the k_m, V_max and CL_int values, respectively. CYP3A5 and CYP2B6 had the greatest efficiency to form carbosulfan sulfinamide, while CYP3A4 and CYP3A5 were the most efficient in the generation of the carbofuran metabolic pathway. Based on average abundances of CYP enzymes in human liver, CYP3A4 contributed to 98% of carbosulfan activation, while CYP3A4 and CYP2B6 contributed 57 and 37% to detoxification, respectively. Significant correlations between carbosulfan activation and CYP marker activities were seen with CYP3A4 (omeprazole sulfoxidation), CYP2C19 (omeprazole 5-hydroxylation) and CYP3A4 (midazolam 1′-hydroxylation), displaying r² = 0.96, 0.87 and 0.82, respectively. Activation and detoxification pathways were inhibited by ketoconazole, a specific CYP3A4 inhibitor, by 90–97% and 47–94%, respectively. Carbosulfan inhibited relatively potently CYP3A4 and moderately CYP1A1/2 and CYP2C19 in pooled HLM. These results suggest that the carbosulfan activation pathway is more important than the detoxification pathway, and that carbosulfan activation is predominantly catalyzed in humans by CYP3A4.     

Potent inhibition of human cytochrome P450 3A isoforms by cannabidiol: role of phenolic hydroxyl groups in the resorcinol moiety

AimsIn this study, we examined the inhibitory effects of Δ⁹-tetrahydrocannabinol (Δ⁹-THC), cannabidiol (CBD), and cannabinol (CBN), the three major cannabinoids, on the activity of human cytochrome P450 (CYP) 3A enzymes. Furthermore, we investigated the kinetics and structural requirement for the inhibitory effect of CBD on the CYP3A activity.Main methodsDiltiazem N-demethylase activity of recombinant CYP3A4, CYP3A5, CYP3A7, and human liver microsomes (HLMs) in the presence of cannabinoids was determined.Key findingsAmong the three major cannabinoids, CBD most potently inhibited CYP3A4 and CYP3A5 (IC₅₀ = 11.7 and 1.65 μM, respectively). The IC₅₀ values of Δ⁹-THC and CBN for CYP3A4 and CYP3A5 were higher than 35 μM. For CYP3A7, Δ⁹-THC, CBD, and CBN inhibited the activity to a similar extent (IC₅₀ = 23–31 μM). CBD competitively inhibited the activity of CYP3A4, CYP3A5, and HLMs (K_i = 1.00, 0.195, and 6.14 μM, respectively). On the other hand, CBD inhibited the CYP3A7 activity in a mixed manner (K_i = 12.3 μM). Olivetol partially inhibited all the CYP3A isoforms tested, whereas d-limonene showed lack of inhibition. The lesser inhibitory effects of monomethyl and dimethyl ethers of CBD indicated that the ability of CYP3A inhibition by the cannabinoid attenuated with the number of methylation on the phenolic hydroxyl groups in the resorcinol moiety.SignificanceThis study indicated that CBD most potently inhibited catalytic activity of human CYP3A enzymes, especially CYP3A4 and CYP3A5. These results suggest that two phenolic hydroxyl groups in the resorcinol moiety of CBD may play an important role in the CYP3A inhibition.     

Serum androgen level is determined by autosomal dominant inheritance and regulates sex-related CYP genes in pigs

We have previously demonstrated differences between Meishan and Landrace pigs in their serum androgen levels (Meishan > Landrace) and the expression of genes encoding hepatic cytochrome P450 (CYP) 1A subfamily enzymes (Meishan < Landrace). In the present study, to clarify whether such differences are genetically controlled, we crossbred these pigs (female Meishan × male Landrace, ML; female Landrace × male Meishan, LM) and examined the expression levels of serum androgen and hepatic CYP family genes (CYP1A1, CYP1A2, CYP2A19, and CYP2E1) among ML, LM, and their parents. In sexually mature (5-month-old) male ML or LM pigs, not only the serum androgen level, but also the hepatic expression levels of all the CYPs examined were similar to those in male Meishan pigs. In addition, there were few breed differences among the females of Meishan, Landrace, ML and LM pigs in the expression of all the CYP genes examined. Furthermore, the expression levels of these CYPs in the females of Meishan and Landrace pigs could be decreased to the corresponding levels in male Meishan pigs by administration of testosterone propionate. The present findings demonstrate that serum androgen level is determined by autosomal dominant inheritance and that the level of serum androgen is one of the host factors regulating the constitutive expression of CYP1A1, CYP1A2, CYP2A19, and CYP2E1 in the pig liver.     

Simultaneous determination of tolbutamide,omeprazole, midazolam and dextromethorphan in human plasma by LC–MS/MS—A high throughput approach to evaluate drug–drug interactions

Drug–drug interactions involving cytochrome P450 (CYP450s) are an important factor for evaluation of a new chemical entity (NCE) in drug development. To evaluate the potential inhibitory effects of a NCE on the pharmacokinetics of a cocktail of representative probes of CYP enzymes (midazolam for CYP3A4, tolbutamide for CYP2C9, omeprazole for CYP2C19 and dextromethorphan for CYP2D6) and the safety and tolerability of the NCE in the presence of probe substrates, a high throughput liquid chromatography/tandem mass spectrometry (LC–MS/MS) method was developed and validated for the simultaneous determination of tolbutamide, omeprazole, midazolam and dextromethorphan in human plasma using tolbutamide-d₉, midazolam-d₄, (±)-omeprazole-d₃, and dextromethorphan-d₃ as the internal standards (ISs). Human plasma samples of 50 μL were extracted by a simple protein-precipitation procedure and analyzed using a high performance liquid chromatography electrospray tandem mass spectrometer system. Reversed-phase HPLC separation was achieved with a Hypersil GOLD AQ column (50 mm × 4.6 mm, 5 μm). MS/MS detection was set at mass transitions of 271 → 172 m/z for tolbutamide, 346 → 198 m/z for omeprazole, 326 → 291 m/z for midazolam, 272 → 171 m/z for dextromethorphan, 280 → 172 m/z for tolbutamide-d₉ (IS), 349 → 198 m/z for (±)-omeprazole-d₃ (IS), 330 → 295 m/z for midazolam-d₄ (IS), and 275 → 171 m/z for dextromethorphan-d₃ (IS) in positive mode. The high throughput LC–MS/MS method was validated for accuracy, precision, sensitivity, stability, recovery, matrix effects, and calibration range. Acceptable intra-run and inter-run assay precision (<10%) and accuracy (<10%) were achieved over a linear range of 50–50,000 ng/mL for tolbutamide, 1–1000 ng/mL for omeprazole, 0.1–100 ng/mL for midazolam and 0.05–50 ng/mL for dextromethorphan in human plasma. Method robustness was demonstrated by the 100% pass rate of 24 incurred sample analysis runs and all of the 50 clinical study samples used for incurred sample reproducibility (ISR) test having met the acceptance criterion (%Diff within 20%). The overall ISR results for all compounds showed that over 95% of the samples had a %Diff of less than 10%. The method is simple, rapid and rugged, and has been applied successfully to sample analysis in support of a drug–drug interaction study.     

CYP1A2 genetic polymorphisms and adenocarcinoma lung cancer risk in the Tunisian population

AimsIn this study, the effects of four single nucleotide polymorphisms (SNPs), ? 3860G > A, ? 2467delT, ? 739T > G and ? 163C > A, of CYP1A2 gene on lung cancer were evaluated in Tunisian population.Main methodsFour polymorphisms of CYP1A2 gene were analysed in 109 healthy smokers and in 101 lung cancer cases, including 63 with squamous cell carcinoma (SCC) and 41 with adenocarcinoma (AD). The genotyping for the SNPs ? 3860 G > A, ? 2467delT, ? 739T > G and ? 163C > A was performed by polymerase chain reaction (PCR)-restriction fragment length polymorphism analysis.Key findingsThe results showed that smokers with CYP1A2 gene polymorphisms were associated with an increased risk for the development of lung AD. There was however no significant increased risk of developing lung SCC in smokers having CYP1A2 gene polymorphisms. An increased risk of developing AD was observed in smokers who are carriers of at least one copy of ? 3680A or ? 739G giving a significant odds ratio (OR) of 6.02 (CI = 2.91–12.9) and 3.01 (CI = 1.54–5.98), respectively.SignificanceThese genotyping data are consistent with the hypothesis that tobacco-specific-N-nitrosamines (TSN) such as 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) are major contributors to the development of lung AD and that CYP1A2 gene product plays an important role in the metabolic activation of NNK. This study suggests that SNPs of CYP1A2 could be considered as promising biomarkers in the aetiology of lung AD in smokers.     

Comparative study of the oxidation of propranolol enantiomers in hepatic and small intestinal microsomes from cynomolgus and marmoset monkeys

Oxidative metabolism of propranolol (PL) enantiomers (R-PL and S-PL) to 4-hydroxypropranolol (4-OH-PL), 5-OH-PL and N-deisopropylpropranolol (NDP) was examined in hepatic microsomes from cynomolgus and marmoset monkeys and in small intestinal microsomes from monkeys and humans. In hepatic microsomes, levels of oxidation activities were similar between the two monkey species, and substrate enantioselectivity (R-PL < S-PL) was observed in the formation of 5-OH-PL and/or NDP. Kinetic experiments revealed that the formation of all metabolites was biphasic in cynomolgus monkeys, whereas only the formation of NDP was biphasic in marmosets. Inhibition experiments employing human CYP antibodies and chemical inhibitors suggested that mainly CYP2D enzymes and partially CYP1A and 2C enzymes are involved in the oxidation of PL in both monkey liver microsomes. In small intestinal microsomes, activity levels were much higher in cynomolgus monkeys than in marmosets and humans and reversed substrate enantioselectivity (R-PL > S-PL) was seen in the formation of NDP in cynomolgus monkeys and humans and in the formation of 5-OH-PL in marmosets. The formation of the three metabolites in cynomolgus monkeys and the formation of NDP in marmosets were biphasic, while the formation of 4-OH-PL in humans was monophasic. From the inhibition experiments using CYP antibodies, CYP2C9 and 2C19 were thought to be involved as N-deisopropylases and CYP2D6 and 3A4 as 4-hydroxylases in human small intestine. Furthermore, CYP1A, 2C and 3A enzymes could be involved in cynomolgus monkeys and CYP2C and 3A enzymes in marmosets. These results indicate that the oxidative profile of PL in hepatic and small intestinal microsomes differ considerably among cynomolgus monkeys, marmosets and humans.     

African American-preponderant single nucleotide polymorphisms (SNPs) and risk of breast cancer

Background: African American women more often present with more aggressive types of breast cancer than Caucasian women, but little is known whether genetic polymorphisms specific to or disproportionate in African Americans are associated with their risk of breast cancer. Methods: A population-based case-control study was conducted including 194 cases identified through the Metropolitan Detroit Cancer Surveillance System and 189 controls recruited through random digit dialing to examine polymorphisms in genes involved in estrogen metabolism and action. Results: The African American-specific CYP1A1 5639C allele was associated with an increased risk of breast cancer (odds ratio (OR) = 2.34, 95% confidence interval (CI) 1.23–4.44) and this association with the CYP1A1 5639 locus was dependent on another polymorphism in the CYP3A4 gene (P = 0.043 for the interaction). In addition, African American-predominant CYP1B1 432 Val allele was significantly more often found in the cases than in the controls overall and the HSD17B1 312 Gly allele was specifically associated with premenopausal breast cancer risk (OR = 3.00, 95%CI 1.29–6.99). Conclusion: These observations need to be confirmed in larger studies due to the limited statistical power of the study based on a small number of cases.     

Characterization of chicken cytochrome P450 1A4 and 1A5: Inter-paralog comparisons of substrate preference and inhibitor selectivity

The chicken (Gallus gallus) is one of the most economically important domestic animals and also an avian model species. Chickens have two CYP1A genes (CYP1A4 and CYP1A5) which are orthologous to mammalian CYP1A1 and CYP1A2. Although the importance of chicken CYP1As in metabolism of endogenous compounds and xenobiotics is well recognized, their enzymatic properties, substrate preference and inhibitor selectivity remain poorly understood. In this study, functional enzymes of chicken CYP1A4 and CYP1A5 were successfully produced in Escherichia coli (E. coli). The substrate preference and inhibitor specificity of the two chicken CYP1As were compared. Kinetic results showed that the enzymatic parameters (K_m, V_max, V_max/K_m) for ethoxyresorufin O-deethylase (EROD) and benzyloxyresorufin O-debenzylase (BROD) differed between CYP1A4 and CYP1A5, while no significant difference was observed for methoxyresorufin O-demethylase (MROD). Lower K_m of CYP1A4 for BROD suggests that CYP1A4 has a greater binding affinity to benzyloxyresorufin than either ethoxyresorufin or methoxyresorufin. The highest V_max/K_m ratio was seen in BROD activity for CYP1A4 and in MROD for CYP1A5 respectively. These results indicate that substrate preference of chicken CYP1As is more notably distinguished by BROD activity and CYP1A5 prefers shorter alkoxyresorufins resembling its mammalian ortholog CYP1A2. Differential patterns of MROD inhibition were observed between CYP1As and among the five CYP inhibitors (α-naphthoflavone, furafylline, piperonyl butoxide, erythromycin and ketoconazole). α-Naphthoflavone was determined to be a potent MROD inhibitor of both CYP1A4 and CYP1A5. In contrast, no or only a trace inhibitory effect (< 15%) was observed by erythromycin at a concentration of 500 μM. Stronger inhibition of MROD activity was found in CYP1A5 than CYP1A4 by relatively small molecules α-naphthoflavone, piperonyl butoxide and furafylline. AROD kinetics and inhibition profiles between chicken CYP1A4 and CYP1A5 demonstrate that the two paralogous members of the CYP1A subfamily have distinct enzymatic properties, reflecting differences in the active site geometry between CYP1A4 and CYP1A5. These findings suggest that CYP1A4 and CYP1A5 play partially overlapping but distinctly different physiological and toxicological roles in the chicken.     

Construction of recombinant Escherichia coli for enhanced bioconversion of colchicine into 3-demethylated colchicine at 70 l bioreactor level

A recombinant strain of Escherichia coli with CYP102A1 gene was developed for the demethylation of colchicine into their derivatives. The CYP102A1 gene responsible for demethylation was isolated from Bacillus megaterium ACBT03 and amplified using suitable primers. The amplified product was cloned into pET28a+ expression vector using host E. coli BL21(DE3) cells. The CYP3A4 (product of CYP102A1 gene) protein expression and other parameters like substrate toxicity, product toxicity and enzyme activity were optimized in shake flasks; and further scaled-up to 5 l bioreactor with 3 l working volume. In 5 l bioreactor, dissolved oxygen (DO) was optimized for maximum specific growth and enhanced 3-demethylated colchicine (3-DMC) production. The optimized conditions from shake flasks were scaled-up to 70 l bioreactor and resulted into ∼80% conversion of 20 mM colchicine in 48 h with a volumetric productivity of 6.62 mg l⁻¹ h⁻¹. Scale-up factors were measured as volumetric oxygen transfer coefficient (k_La) i.e., 56 h⁻¹ and impeller tip velocity (V_tip) i.e., 7.065 m s⁻¹, respectively. The kinetic parameters K_m, k_cat, and k_cat/K_m of the CYP3A4 enzyme using colchicine as the substrate were determined to be 271 ± 30 μM, 8533 ± 25 min⁻¹, and 31.49 μM min⁻¹, respectively, when IPTG induced recombinant E. coli culture was used.     

Rapid determination of tacrine and other drug metabolites in microsomal incubate by newly developed targeting algorithm on UHPLC/TOFMS

A rapid simultaneous determination method for in vitro Cytochrome P450 (CYP) activity assay of 1,2,3,4-tetrahydroacridin-9-amine (tacrine) metabolites using ultra high performance liquid chromatography (UHPLC) coupled with computer-assisted in-source collision induced dissociation (CID) monitoring was investigated. In general, enzyme inhibition assays require quantitative analysis of incubates with drugs using various concentrations of substrates/inhibitors. The assay of CYP isozyme inhibition is an important informational step in the drug discovery process and, with the many substrates listed by the FDA, high-throughput qualitative and quantitative analyses are desired. Based on sub-2-micron packing material with reversed phase chromatography combined with a single liquid chromatography/time-of-flight mass spectrometry (LC/TOFMS), a less than 1 min analysis time is presented for two additional drugs. We successfully determined seven of the eight potential isomer metabolites for the drug tacrine in 2.5 min using a 2 mm internal diameter × 100 mm length column and applying in-source CID with our newly developed chromatographic peak deconvolution technique. Although two of the peaks were heavily fused at a peak width of less than 300 ms, we could clearly identify these peaks by monitoring the chromatographic intensity difference of their fragment peaks on the mass spectrum.     

Heterologous production of caffeic acid from tyrosine in Escherichia coli

Caffeic acid is a plant secondary metabolite and its biological synthesis has attracted increased attention due to its beneficial effects on human health. In this study, Escherichia coli was engineered for the production of caffeic acid using tyrosine as the initial precursor of the pathway. The pathway design included tyrosine ammonia lyase (TAL) from Rhodotorula glutinis to convert tyrosine to p-coumaric acid and 4-coumarate 3-hydroxylase (C3H) from Saccharothrix espanaensis or cytochrome P450 CYP199A2 from Rhodopseudomonas palustris to convert p-coumaric acid to caffeic acid. The genes were codon-optimized and different combinations of plasmids were used to improve the titer of caffeic acid. TAL was able to efficiently convert 3 mM of tyrosine to p-coumaric acid with the highest production obtained being 2.62 mM (472 mg/L). CYP199A2 exhibited higher catalytic activity towards p-coumaric acid than C3H. The highest caffeic acid production obtained using TAL and CYP199A2 and TAL and C3H was 1.56 mM (280 mg/L) and 1 mM (180 mg/L), respectively. This is the first study that shows caffeic acid production using CYP199A2 and tyrosine as the initial precursor. This study suggests the possibility of further producing more complex plant secondary metabolites like flavonoids and curcuminoids.     

Cytochrome P450 binding studies of novel tacrine derivatives: Predicting the risk of hepatotoxicity

The 1,2,3,4-tetrahydroacridine derivative tacrine was the first drug approved to treat Alzheimer’s disease (AD). It is known to act as a potent cholinesterase inhibitor. However, tacrine was removed from the market due to its hepatotoxicity concerns as it undergoes metabolism to toxic quinonemethide species through the cytochrome P450 enzyme CYP1A2. Despite these challenges, tacrine serves as a useful template in the development of novel multi-targeting anti-AD agents. In this regard, we sought to evaluate the risk of hepatotoxicity in a series of C9 substituted tacrine derivatives that exhibit cholinesterase inhibition properties. The hepatotoxic potential of tacrine derivatives was evaluated using recombinant cytochrome (CYP) P450 CYP1A2 and CYP3A4 enzymes. Molecular docking studies were conducted to predict their binding modes and potential risk of forming hepatotoxic metabolites. Tacrine derivatives compound 1 (N-(3,4-dimethoxybenzyl)-1,2,3,4-tetrahydroacridin-9-amine) and 2 (6-chloro-N-(3,4-dimethoxybenzyl)-1,2,3,4-tetrahydroacridin-9-amine) which possess a C9 3,4-dimethoxybenzylamino substituent exhibited weak binding to CYP1A2 enzyme (1, IC₅₀ = 33.0 µM; 2, IC₅₀ = 8.5 µM) compared to tacrine (CYP1A2 IC₅₀ = 1.5 µM). Modeling studies show that the presence of a bulky 3,4-dimethoxybenzylamino C9 substituent prevents the orientation of the 1,2,3,4-tetrahydroacridine ring close to the heme-iron center of CYP1A2 thereby reducing the risk of forming hepatotoxic species.