Cytochrome P450 enzymes: Biochemistry, Pharmacology and Health Implications
Cytochrome P450 (CYP) is a super family of phase I enzyme in the biotransformation of xenobiotics and medications. Most medications undergo deactivation by CYP, and then are eliminated through either bile or kidneys from the body. CYP isozymes play a crucial role in drug interactions that may result in enhanced toxicity, reduced efficacy or onset of adverse reactions. On the other hand, many agents affecting CYP expression and activity may alter metabolic rate of different medications co-administrated. Therefore, the molecular basis, regulation by inducers or inhibitors, and pharmacologic reaction of specific CYP isozymes are the key issues of biochemical mechanisms, pharmaceutical development and safe use of various medications. This book is to meet the needs from basic molecular biochemists, pharmacologists, pharmacists, medical students, clinical practitioners and scientists, as well as broad readers who wish to understand how an herbal extract, medication or natural supplement is metabolized or transformed in the liver or other sites for deactivation and elimination. Special focuses are paid to herbal extracts and medications in the treatment of neuro-psychiatric or cardiovascular disorders, diabetes and viral hepatitis. Detailed dissection of drug interactions in a particular field intends to provide rationales for useful guidance of safe drug use in daily practice. The contributing authors are basic scientists, pharmacists, pharmacologists and on-service physicians in cardiovascular, neuro-psychiatric, gastroenterologic and hepatologic fields from Europe (Germany, France, Portugal), Australia, the US and China. Thus, the book is the collection of master pieces by well-known experts from various regions of the world, and represents the current understanding of CYP enzyme reaction and a contemporary coverage of possible drug interactions in involved fields. The featured chapters are scientific elucidation of basic biochemistry, pharmacology and clinical investigations in the interest of drug metabolism, interaction and safe use guidance in the single focus of this microsomal enzyme with multi-facet metabolic function.
... Cytochrome P450 monooxygenase Cytochrome P450 monooxygenase (Cyt-P450, EC 22.214.171.124), is a superfamily of intracellular heme-thiolate-containing monooxygenase enzymes found in several living species, including bacteria, algae, fungi and animals (Wu, 2014;Hatakeyama et al., 2016). Moktali et al. (2012) reported that the fungal genomes encode more than 300 cytochromes. ...
... Moktali et al. (2012) reported that the fungal genomes encode more than 300 cytochromes. Cyt-P450 is the most important, of the first phase of the enzymatic metabolism involved in the biodegradation of lignin and a wide range of xenobiotic metabolic reactions (Moktali et al., 2012;Wu, 2014;Hatakeyama et al., 2016). As significant components, Cyt-P450 use several substrates in stereo-selective and regioselective protocols and bind molecular oxygen. ...
Kingdom fungi include microscopic as well as macroscopic forms in all possible known habitats. Fungi are sensitive to climate perturbations and present climate itself is getting influenced by fungi. Environmental change is perceived differently by different ecosystems and is not homogeneous around the globe. Fungi inhabiting extreme environments that are prone to climate change can go locally extinct or adapt to cope up with the changed environment. To date, only certain number of fungi is classified, however advanced sequencing approach has broadened the scope globally to describe the uncultivable forms, dwelling in extreme environments. Fungi place itself separately from other microbial domain in terms of highest biomass, DNA and metabolic potential; therefore, it deserves special attention in the scenario of climate change. However, fungi inhabiting ecosystems that are less prone to climate change could be harnessed for their excellent metabolic potential due to the peculiar make up of their genome. This chapter is an effort to discuss extreme habitats in context of fungal diversity, their adaptability and mitigation options associated with climate change affecting fungi and vice versa.
Background. Patients coinfected with human immunodeficiency virus (HIV) and hepatitis C virus (HCV) are likely to use both HIV and HCV treatment. Drug–drug interactions have been demonstrated between boceprevir, an HCV protease inhibitor, and frequently prescribed antiretroviral drugs, such as efavirenz and boosted HIV protease inhibitors. Concomitant administration of boceprevir with these drugs should be avoided. This study was designed to investigate the absence of a drug-drug interaction between boceprevir and raltegravir, an HIV integrase inhibitor. Methods. This was an open-label, randomized, 2-period, crossover phase 1 trial in 24 healthy volunteers. All subjects were randomly assigned to receive boceprevir 800 mg every 8 hours for 9 days plus a single dose of raltegravir 400 mg on day 10 followed by a washout period and a single dose of raltegravir 400 mg on day 38, or the same medication in reverse order. Blood samples for pharmacokinetics were collected and pharmacokinetic parameters were calculated. Results. The geometric mean (GM) of raltegravir area under the concentration-time curve (AUC)0–12h and maximum plasma concentration (Cmax) for raltegravir + boceprevir vs raltegravir alone were 4.27 (95% confidence interval [CI], 3.22–5.66) vs 4.04 (95% CI, 3.09–5.28) mg * hour/L and 1.06 (95% CI, .76–1.49) vs 0.93 (95% CI, .70–1.23) mg/L, respectively. GM ratio estimates of raltegravir AUC0–12h and Cmax for raltegravir + boceprevir vs raltegravir alone were 1.04 (90% CI, .88–1.22) and 1.11 (90% CI, .91–1.36), respectively. The GM of boceprevir AUC0–8h, Cmax, and C8h were 5.45 (95% CI, 5.11–5.81) mg * hour/L, 1.88 (95% CI, 1.72–2.06) mg/L, and 0.09 (95% CI, .07–.11) mg/L, respectively. These data are comparable to those from historical controls. Conclusions. Due to the absence of a clinically significant drug interaction, raltegravir can be recommended for combined HIV/HCV treatment including boceprevir. Clinical Trials Registration NCT01288417.
In psychiatry, around 130 drugs are available which have been detected and developed during the last 60 years. These drugs are effective and essential for the treatment of many psychiatric disorders and symptoms. Therapeutic outcomes, however, are still far from satisfactory for many patients. One major reason underlying insufficient response or problems of tolerability or toxicity is considerable interindividual variability in the pharmacokinetic properties of the patients. At the exact same dose, a more than 20-fold interindividual variation in steady state concentrations of the drugs in the body may result. Patients differ in their ability to absorb, distribute, metabolize and excrete drugs due to concurrent disease, age, concomitant medication or genetic peculiarities. A valuable tool for tailoring the dosage of the prescribed medication(s) to the individual characteristics of a patient is therapeutic drug monitoring (TDM). For a considerable number of psychopharmacologic compounds, TDM has become a clinical routine for dose adjustment. The benefits of TDM regarding optimization of psychopharmacotherapy can be further enhanced by genotyping of cytochrome P450 enzymes (CYP). These enzymes are highly polymorphic and involved in the metabolism of most psychotherapeutic drugs. Genotyping related information may be supportive especially for drugs that are predominantly metabolized by CYP2C19 or CYP2D6. The in vivo activity of CYP isoenzymes can be determined using probe drug assays. When used appropriately, phenotyping and genotyping methods are most helpful for problem solving and improvement of the outcomes of psychopharmacotherapy for many patients.
Since the first approval of interferon for the treatment of chronic hepatitis B virus (HBV) infection in 1992, six additional antivirals have been developed: pegylated interferon-alfa2a, and the oral antivirals lamivudine, adefovir, telbivudine, entecavir and tenofovir. The availability of animal models for HBV infection and hepatocyte cell culture led to the discovery and development of oral antivirals targeted at HBV polymerase and reverse transcriptase, which inhibit viral replication. The discovery and development of entecavir, the first oral anti-HBV drug with both potent antiviral activity and a high genetic barrier to resistance, took more than 10 years before it was first approved in the USA. Since then, multiple real-life studies have provided data consistent with the findings of the registration trials and the long-term rollover study in terms of efficacy, resistance, and safety. Data from the long-term follow-up of patients enrolled in the registration studies showed that treatment with entecavir can lead to significant improvements in liver histopathology, and recent cohort studies have demonstrated that treatment with entecavir may reduce disease progression and the development of hepatocellular carcinoma (HCC) in patients with chronic hepatitis B. In addition, real-life studies suggest that entecavir may reduce HCC recurrence and increase survival rates in patients with HBV-related HCC post-surgical resection.
Introduction: Genetic polymorphisms of cytochrome P450 (CYP) may predict the treatment response or occurrence of side effects of antipsychotic drugs. Aim: We studied the association of response to clozapine treatment in schizophrenic patients in relation to polymorphisms in the CYP1A2 gene. Methods: The degree of psychosis of the patients (n=55) was assessed using the Brief Psychiatric Rating Scale (BPRS), the Scale for the Assessment of Positive Symptoms (SAPS), the Scale for the Assessment of Negative Symptoms (SANS) and routine biochemistry. The patients were monitored for 18 weeks and the scales were applied before starting the treatment and at the end of the follow up period. Clozapine was used at doses of 200 to 600 mg/day. A positive response was defined as a 20% decrease in pre- and post-treatment scores of one of the BPRS, SANS, or SAPS scores. In addition, 45 patients, who were already on clozapine treatment, were assessed retrospectively. Results: As assessed at the 18(th) week after start of therapy, lack of response to clozapine treatment was 2.4 fold higher in the patients carrying the CYP1A2*1F*1F genotype (p=0.02) compared to patients carrying at least one wild type allele (i.e. *1/*1 or *1/*1F). Smoking decreased the response rate by about 15% (p=0.014). Conclusion: The results of our study suggest that the CYP1A2*1F/*1F genotype may be a risk factor for lack of response to clozapine treatment in psychotic patients, especially in cigarette smokers.
Use of the nonsteroidal anti-inflammatory drug diclofenac (DF) is associated with serious idiosyncratic hepatotoxicity. Covalent binding of reactive intermediates of DF to proteins is considered to initiate the process leading to this severe side-effect. The aim of this study was to characterize the nature of covalent protein modifications by reactive metabolites of DF which result from bioactivation by cytochrome P450. DF and its major monohydroxylated metabolites 4′-hydroxydiclofenac (4′-OH-DF) and 5-hydroxydiclofenac (5-OH-DF) were bioactivated using a highly active P450 BM3 mutant (CYP102A1M11H) in the presence of the model target protein human glutathione-S-transferase P1-1 (hGST P1-1). Protein-adducts were subsequently identified by LC-MS/MS analysis of tryptic digests of hGST P1-1. In total, 10 different peptide adducts were observed which result from modifications of Cys-47 and Cys-14 of hGST P1-1. The majority of the protein thiol modifications appeared to be derived from 5-OH-DF, which produced seven different peptide adducts with mass increments of 289.0, 309.0, and 339.0 Da. Remarkably, no peptide adducts were observed upon the bioactivation of 4′-OH-DF. Incubations of P450 BM3 with DF also showed the peptide adducts derived from 5-OH-DF and peptide adducts that are not derived from quinone imine. A peptide adduct with a mass increment of 249.0 Da most likely results from the o-imine methide formed by oxidative decarboxylation of DF. In addition, a peptide adduct was observed with a mass increment of 259.0 Da, which corresponds to the substitution of one of the chlorine atoms of DF by protein thiol. A corresponding GSH-conjugate with a similar mass increment was only observed if incubations of DF with P450 and GSH were supplemented by human GST P1-1. The results of this study not only confirm the importance of 5-OH-DF in covalent protein-binding but also suggest that the nature of protein adduction is not necessarily reflected by chemical conjugation with GSH.
Despite the high burden, there is a dearth of (long-term) outcome data of hepatitis B virus (HBV) and hepatitis C virus (HCV) co-infected patients receiving antiretroviral treatment (ART) in a clinical setting in resource-constrained settings, particularly from Asia. We conducted a retrospective cohort study including all adults initiating standard ART (non-tenofovir-based) between 03/2003 and 09/2012. HBV infection was diagnosed by HBV surface antigen detection. HCV diagnosis relied on antibody detection. The independent effect of HBV and HCV on long-term (≥5 years) ART response in terms of mortality (using Cox regression), severe livertoxicity (using logistic regression) and CD4 count increase (using mixed-effects modelling) was determined. A total of 3089 adults were included (median age: 35 years (interquartile range 30-41); 46% male), of whom 341 (11.0%) were co-infected with HBV and 163 (5.3%) with HCV. Over a median ART follow-up time of 4.3 years, 240 individuals died. Mortality was 1.6 higher for HBV co-infection in adjusted analysis (P = 0.010). After the first year of ART, the independent mortality risk was 3-fold increased in HCV co-infection (P = 0.002). A total of 180 (5.8%) individuals discontinued efavirenz or nevirapine due to severe livertoxicity, with an independently increased risk for HBV (hazard ratio (HR) 2.3; P<0.001) and HCV (HR 2.8; P<0.001). CD4 recovery was lower in both HBV and HCV co-infection but only statistically significant for HBV (P<0.001). HBV and HCV co-infection was associated with worse ART outcomes. The effect of early ART initiation and providing effective treatment for hepatitis co-infection should be explored.
As the number of prescribed drugs is constantly rising, drug–drug interactions are an important issue. The simultaneous administration of several drugs can cause severe adverse effects based on interactions with the same metabolizing enzyme(s). The Transformer database (http://bioinformatics.charite.de/transformer) contains integrated information on the three phases of biotransformation (modification, conjugation and excretion) of 3000 drugs and >350 relevant food ingredients (e.g. grapefruit juice) and herbs, which are catalyzed by 400 proteins. A total of 100 000 interactions were found through text mining and manual validation. The 3D structures of 200 relevant proteins are included. The database enables users to search for drugs with a visual display of known interactions with phase I (Cytochrome P450) and phase II enzymes, transporters, food and herbs. For each interaction, PubMed references are given. To detect mutual impairments of drugs, the drug-cocktail tool displays interactions between selected drugs. By choosing the indication for a drug, the tool offers suggestions for alternative medications to avoid metabolic conflicts. Drug interactions can also be visualized in an interactive network view. Additionally, prodrugs, including their mechanisms of activation, and further information on enzymes of biotransformation, including 3D models, can be viewed.
Studies of boceprevir and telaprevir based antiviral therapy in liver transplant (LT) recipients with hepatitis C genotype 1 infection have demonstrated dramatic increases in tacrolimus, cyclosporine, and mTOR inhibitor exposure. In addition to empiric dose reductions, daily monitoring of immunosuppressant blood levels is required when initiating as well as discontinuing the protease inhibitors to maximize patient safety. Although improved suppression of HCV replication is anticipated, 20 to 40% of treated subjects have required early treatment discontinuation due to various adverse events including anemia (100%), infection (30%), nephrotoxicity (20%) and rejection (5 to 10%). Simeprevir and faldeprevir will likely have improved efficacy and safety profiles but potential drug interactions with other OATP1B1 substrates and unconjugated hyperbilirubinemia are expected. In contrast, sofosbuvir and daclatasvir based antiviral therapy are not expected to lead to clinically significant drug-drug interactions in LT recipients but confirmatory studies are needed. Liver transplant recipients may also be at increased risk of developing drug induced liver injury (DILI). Establishing a diagnosis of DILI in the transplant setting is very difficult with the variable latency, laboratory features and histopathological manifestations of hepatotoxicity associated with a given drug, the need to exclude competing causes of allograft injury, and the lack of an objective and verifiable confirmatory test. Nonetheless, a heightened awareness of the possibility of DILI is warranted in light of the large number of medications used in LT recipients and the potential adverse impact that DILI may have on patient outcomes.
Cytochrome P450 2S1 (CYP2S1) is an orphan P450 with an unknown biological function. Data from our laboratory and others suggest that CYP2S1 may have an important physiological role in modulating the synthesis and metabolism of bioactive lipids including prostaglandins and retinoids. CYP2S1 expression is elevated in multiple epithelial-derived cancers as well as in the chronic hyperproliferative disease psoriasis. Whether CYP2S1 expression in proliferative disease is protective, detrimental, or neutral to disease progression remains to be determined. Two human bronchial epithelial cells (BEAS-2B) were constructed to represent chronic depletion of CYP2S1 using short-hairpin RNA (shRNA) silencing directed toward the 3'UTR (759) and exon 3 (984) of the CYP2S1 gene and compared with a non-targeting shRNA control (SCRAM). Both CYP2S1 mRNA and protein were depleted by approximately 75% in stable cell lines derived from both targeted shRNA constructs (759 and 984). To elucidate the biological significance of CYP2S1, we analyzed transcriptome alterations in response to CYP2S1 depletion in human lung cells. RNA-sequencing (RNA-seq) analysis was performed to compare the transcriptome of the control (SCRAM) and the CYP2S1-depleted (759) BEAS-2B cell lines. Transcriptomes of the replicates from the two cell lines were found to be distinct populations as determined using Principal Component Analysis and hierarchical clustering. Approximately 1000 genes were differentially expressed in response to CYP2S1 depletion. Consistent with our previous phenotypes, DAVID analysis revealed altered regulation in key pathways implicated in cell proliferation and migration. Transcriptomic profiles were also consistent with the metabolism of proposed endogenous substrates. Pathway analysis also revealed significant expression changes within mTOR signaling, a critical pathway in cell growth. To determine whether these changes manifest as altered cell size, cell diameter and volume were calculated, revealing that CYP2S1 depletion promotes cell growth in BEAS-2B cells. These data suggest that pathway analysis of sequence-based gene expression estimates is a powerful method to identify pathways and phenotypic alterations in response to changes in orphan enzyme expression. Our results suggest a novel role for CYP2S1-mediated metabolism in modulating BEAS-2B cell size. These findings warrant further studies on CYP2S1 regulated pathways to elucidate potential substrates of CYP2S1.
Management of recurrent Hepatitis C virus (HCV) infection following liver transplantation remains a major challenge. In non-transplanted HCV genotype 1 patients, the introduction of protease inhibitor-based regimens has significantly increased the rate of sustained virological response. In this follow-up study, on the first published cohort of post-liver transplant patients treated with telaprevir-based triple therapy, we investigated both efficacy and safety data in follow-up to 24 weeks (SVR 24) after end of treatment (EOT). SVR 24 efficacy and safety data from 9 liver transplant HCV patients being treated with telaprevir, pegylated interferon, and ribavirin, showed 5 of the transplanted patients accomplished the full duration of the 48 week triple therapy. Notable were the 4 patients found to be HCV RNA-negative at week 4, and 8 patients at week 12. Upon EOT, at week 48, 6 patients were HCV RNA-negative. Importantly, at follow-up (24 weeks after EOT), a favorable sustained virological response rate was observed in 5 of these patients with HCV RNA remaining negative, including in one patient who discontinued treatment prematurely. Due to side effects, 2 patients discontinued, 2 suffered from virological breakthrough after the telaprevir treatment phase, and 1 patient had a relapse after EOT. Two thirds of patients exhibited hematological side effects requiring ribavirin dose reductions, administration of erythropoetin, or even blood transfusions. This retrospective analysis provides evidence that - with respect to SVR 24 - liver transplant patients suffering from HCV genotype 1 recurrence may benefit from a telaprevir-based triple therapy as this new regimen showed acceptable antiviral efficacy in this small cohort of mostly pre-treated patients. Management of drug-drug interactions is challenging, but feasible. In part severe side effects are frequent during treatment and require therapeutic interventions.
Aims: Antidepressant response varies between patients, possibly due to differences in the rate cytochrome P450 enzymes metabolise antidepressants into inactive compounds. Drug metabolism rates are influenced by common variants in the genes encoding these enzymes. However, it remains unclear whether treatment outcomes can be predicted by either CYP450 genotype or antidepressant serum concentration. Methods: In GENDEP (a pharmacogenetic study of depressed individuals treated with either escitalopram or nortriptyline), serum concentrations of antidepressants and their primary metabolite were measured after eight weeks treatment and variants in CYP2D6 and CYP2C19 were genotyped. Results: Amongst patients taking escitalopram (n=223), the genotype CYP2C19 was significantly associated with escitalopram serum concentrations and desmethylescitalopram:escitalopram ratio. For those taking nortriptyline (n=161), the CYP2D6 genotype was significantly associated with nortriptyline and 10-hydroxynortriptyline serum concentrations and 10-hydroxynortriptyline:nortrip-tyline ratio. CYP450 genotypes conferring greater enzyme activity were linked to lower drug serum concentrations and higher metabolite:drug ratios. Nonetheless, no significant association was found between either CYP450 genotype or antidepressant serum concentration and treatment response. Conclusions: While there is a significant relationship between the CYP450 genotype and serum concentrations of escitalopram and nortriptyline, the genotypes are not predictive of differences in treatment response for either drug. Furthermore, differences in antidepressant serum concentrations are not associated with variability in treatment response.
Opportunities to treat infection with hepatitis C virus (HCV) are evolving rapidly. From the introduction of interferon-α monotherapy in 1992 to the approval of telaprevir- and boceprevir-based triple therapies with pegylated interferon-α and ribavirin in 2011, the chances of curing patients infected with HCV genotype 1 have improved from <10% to approximately 70%. Significant further improvements are on the horizon, which may well cure virtually all hepatitis C patients with an all-oral, interferon-free regimen in the very near future. These exciting developments are reviewed in the present article.
Sustained suppression of serum HBV DNA levels with nucleos(t)ide analogues is the most important success obtained in the treatment of chronic HBV infection today. Tenofovir and entecavir provide more robust viral suppression. The aim of this study is to compare tenofovir and entecavir in terms of viral kinetics, side effects and virological response in patients with chronic HBV infection. Patients with chronic hepatitis B treated with tenofovir or entecavir were included in this retrospective study. Using survey analysis, we evaluated independent variables reflecting virological response to treatment and determined whether use of tenofovir or entecavir was one of them. We compared the decline in serum HBV DNA levels at the 3rd, 6th, 12th, 18th and 24th months of treatment between two groups. We also compared entecavir and tenofovir in terms of side effect rates. 117 patients [average age: 44 (20-73), 65 males (55.6%), 30 HBeAg positive (25.6%)] were enrolled in the study. Sixty-six patients (56.4%) used tenofovir and 51 (43.6%) patients used entecavir. Virological response was better in patients using tenofovir (Odd's ratio of 1.796 and p = 0.014) and having high fibrosis score (Odd's ratio of 0.182 and p = 0.018). Entecavir was more effective in reducing serum HBV DNA levels at the 3rd month of treatment (serum HBV DNA decline of 4.45 and 3.96 log10 units for entecavir and tenofovir respectively, p = 0.031), but decline rates were similar at other months. There was no difference between patient groups in terms of side effects and discontinuation of treatment due to side effects. Patients with chronic HBV infection using tenofovir have better virological response than those using entecavir.
With a better understanding of the etiology of breast cancer, molecularly targeted drugs have been developed and are being testing for the treatment and prevention of breast cancer. Targeted drugs that inhibit the estrogen receptor (ER) or estrogen-activated pathways include the selective ER modulators (tamoxifen, raloxifene, and lasofoxifene) and aromatase inhibitors (AIs) (anastrozole, letrozole, and exemestane) have been tested in preclinical and clinical studies. Tamoxifen and raloxifene have been shown to reduce the risk of breast cancer and promising results of AIs in breast cancer trials, suggest that AIs might be even more effective in the prevention of ER-positive breast cancer. However, these agents only prevent ER-positive breast cancer. Therefore, current research is focused on identifying preventive therapies for other forms of breast cancer such as human epidermal growth factor receptor 2 (HER2)-positive and triple-negative breast cancer (TNBC, breast cancer that does express ER, progesterone receptor, or HER2). HER2-positive breast cancers are currently treated with anti-HER2 therapies including trastuzumab and lapatinib, and preclinical and clinical studies are now being conducted to test these drugs for the prevention of HER2-positive breast cancers. Several promising agents currently being tested in cancer prevention trials for the prevention of TNBC include poly(ADP-ribose) polymerase inhibitors, vitamin D, and rexinoids, both of which activate nuclear hormone receptors (the vitamin D and retinoid X receptors). This review discusses currently used breast cancer preventive drugs, and describes the progress of research striving to identify and develop more effective preventive agents for all forms of breast cancer.
Nevirapine is a non-nucleoside reverse transcriptase (RT) inhibitor used for the treatment of AIDS and the prevention of mother-to-child transmission of HIV-1. Despite its therapeutic benefits, treatment with nevirapine has been associated with significant incidences of liver and dermal toxicity. The present study examined the effects of nevirapine on cell growth and death in human hepatocyte HepG2 cells and THLE2 cells and the possible pathways involved in these effects. The concentrations of nevirapine inhibiting 50% cell growth were similar for both cell lines. Nevirapine (0-250 µM) treatment caused a slight increase in the amount of lactate dehydrogenase released into the medium. Apoptotic cell death did not contribute to the decrease in viable cells. Exposing of HepG2 cells to nevirapine caused G2/M phase arrest, and the activity of senescence-associated β-galactosidase was not altered. In THLE2 cells, the percentage of cells in G1/G0 phase was increased and cellular senescence was induced in a concentration-dependent manner. Endogenous non-telomeric RT activity was not detected in either cell line. Western blot analysis indicated lower levels of p53 and phospho-p53 (ser15) in HepG2 cells as compared to THLE2 cells; no significant changes in p53 or phospho-p53 (ser15) were noted with nevirapine treatment. These data demonstrate that nevirapine inhibits cell growth, induces cell cycle arrest at different phases, and has different effects on cellular senescence in HepG2 cells and THLE2 cells. The differential responses appear to be related to differences in the basal levels of p53 in the HepG2 cells and THLE2 cells.
Immunologic derangements in rheumatoid arthritis (RA) patients likely contribute to premature atherosclerotic cardiovascular disease (CVD). Traditional CVD risk factors do not reliably identify at-risk RA patients, probably because disease-associated mechanisms are not taken into account. The purpose of this study was to determine whether plasma from subjects with RA exhibits atheroma-promoting properties leading to disruption of cholesterol homeostasis in human monocytes/macrophages. Twenty-one healthy controls (HC) and 22 RA patients were enrolled in an IRB approved study at Winthrop University Hospital. Naïve THP-1 macrophages were exposed to plasma from each HC and RA patient. Following incubation, RNA and protein were isolated. QRT-PCR and Western blotting techniques were then used to measure expression of proteins responsible for cholesterol efflux (ATP binding cassette transporter (ABC)A1, ABCG1, 27-hydroxylase) and cholesterol uptake (CD36, ScR-A1, lectin oxidized low density lipoprotein receptor (LOX)-1, CXCL16). To confirm the pro-atherogenic effects of RA plasma on macrophages, foam cell formation was quantified. Results showed that RA plasma downregulates cholesterol efflux proteins and upregulates scavenger receptors CD36, LOX1 and CXCL16. These pro-atherogenic changes in gene expression in the presence of RA plasma are associated with augmented lipid accumulation and foam cell formation by THP-1 macrophages. RA plasma induces macrophage cholesterol overload. Demonstration of disrupted cholesterol homeostasis mediated by RA plasma provides further evidence of the involvement of the immune system in atherogenesis. Our data suggest that chronic exposure to RA plasma adversely affects the capacity of monocytes/macrophages in the arterial wall to metabolize cholesterol and maintain lipid homeostasis, thereby contributing to the development of premature atherosclerosis.
We live in an age when a divine vision is dismissed as an hallucination, and desire to experience a direct communication with god is often interpreted as a sign of mental illness. Nevertheless, some scholars and scientists assert that such visions and communications are fundamentally derived from an ancient and ongoing cultural tradition. The hypothesis presented here suggests that humans have a very ancient tradition involving the use of mind-altering experiences to produce profound, more or less spiritual and cultural understanding.
CYP27A1 (sterol 27-hydroxylase) catalyses an important sterol elimination pathway in the human macrophage, and consequently may protect against atherosclerosis. We studied the expression and regulation of CYP27A1 in a human macrophage-like cell-line, THP-1, and primary HMDMs (human monocyte-derived macrophages). In both macrophage cell types, we found that CYP27A1 expression is independent of cellular cholesterol levels and of LXR (liver X receptor) -dependent control of transcription. However, the FXR (retinoid X receptor) ligand, 9-cis-retinoic acid, upregulates CYP27A1 expression. Of the RXR heterodimeric partners tested PPAR (peroxisome -proliferator-activated receptor) gamma ligands significantly increased CYP27A1 mRNA levels. Its reversal by a PPARgamma antagonist demonstrated the specificity of this effect. Interestingly, HMDMs express markedly higher levels of CYP27A1 than THP-1 macrophages, and this difference was reflected in both protein levels and enzyme activities between the two cell types. In conclusion, stimulation of CYP27AI by PPARgamma may represent a key previously unrecognized mechanism by which PPARgamma protects against atherosclerosis.
The investigation of cytochrome P450 (CVP) mediated metabolism reactions by determination of enzyme kinetic parameters, Michaelis constant (K-m) maximum reaction velocity (V-max), and intrinsic clearance (CLint) is important aspects in discovery and development of drugs. The kinetic parameters can be used to predict the clearance prior to human administration and for better understanding the mechanism of clearance in vivo. In this study, the metabolic activities of three major hepatic CYP isoforms (2C19, 2D6, and 3A4) were investigated on structurally different central nervous system (CNS) acting drugs, amitriptyline, fluphenazine, and dothiepin. By using our novel in vitro evaluation system, we could compare the kinetic parameters for the metabolism of fluphenazine and dothiepin for the first time. Comparing CLint values thus obtained, we concluded that 2C19 could be predominant for metabolic activity on tricyclic antidepressants as expected, but not on phenothiazine-related antipsychotic drugs. Since the metabolism of CNS drugs is susceptible to single nucleotide polymorphisms of human gene, our results suggest that phenothiazine could be an alternative to clinical application of CNS drugs.
There is a relationship between the hepatic production and the plasma level of HDL cholesterol. The findings also suggest that hepatic cytochrome P-450 reflects liver capacity for both HDL cholesterol production and drug hydroxylation.
Historically, clinicians have had few resources beyond empiric tools derived from population-based treatment algorithms and patient/family interviews to inform the "best choice" for psychopharmacologic intervention. Previously unappreciated interindividual variance in activity of cytochrome P450 enzymatic activity can lead to abnormal metabolism of many psychotropics and poor outcomes. Fortunately, advances in our understanding and application of psychiatric pharmacogenomic information have the potential to improve the quality of medical care for children at the level of the individual prescription.
The nonsteroidal antiinflammatory drug diclofenac causes rare but significant cases of serious hepatotoxicity, typically with a delayed onset (>1-3 months). Because there is no simple dose relationship and because liver injury cannot be reproduced in current animal models, individual patient-specific susceptibility factors have been evoked to account for the increased risk. While these patient factors have remained undefined, a number of molecular hazards have been characterized. Among these are metabolic factors (bioactivation by hCYP2C9 or hCYP3A4 to thiol-reactive quinone imines, activation by hUGT2B7 to protein-reactive acyl glucuronides and iso-glucuronides, and 4'-hydroxylation secondary to diclofenac glucuronidation), as well as kinetic factors (Mrp2-mediated concentrative transport of diclofenac metabolites into bile). From the toxicodynamic view, both oxidative stress (caused by putative diclofenac cation radicals or nitroxide and quinone imine-related redox cycling) and mitochondrial injury (protonophoretic activity and opening of the permeability transition pore) alone or in combination have been implicated in diclofenac toxicity. In some cases, immune-mediated liver injury is involved, inferred from inadvertent rechallenge data and from a number of experiments demonstrating T cell sensitization. Why certain underlying diseases (e.g., osteoarthritis) also increase the susceptibility to diclofenac hepatotoxicity is not clear. To date, cumulative damage to mitochondrial targets seems a plausible putative mechanism to explain the delayed onset of liver failure, perhaps even superimposed on an underlying silent mitochondrial abnormality. Increased efforts to identify both patient-specific risk factors and disease-related factors will help to define patient subsets at risk as well as increase the predictability of unexpected hepatotoxicity in drug development.
The human CYP3A subfamily plays a dominant role in the metabolic elimination of more drugs than any other biotransformation enzyme. CYP3A enzyme is localized in the liver and small intestine and thus contributes to first-pass and systemic metabolism. CYP3A expression varies as much as 40-fold in liver and small intestine donor tissues. CYP3A-dependent in vivo drug clearance appears to be unimodally distributed which suggests multi-genic or complex gene-environment causes of variability. Interindividual differences in enzyme expression may be due to several factors including: variable homeostatic control mechanisms, disease states that alter homeostasis, up- or down-regulation by environmental stimuli (such as smoking, drug intake, or diet), and genetic mutations. This review summarizes the current understanding and implications of genetic variation in the CYP3A enzymes. Unlike other human P450s (CYP2D6, CYP2C19) there is no evidence of a 'null' allele for CYP3A4. More than 30 SNPs (single nucleotide polymorphisms) have been identified in the CYP3A4 gene. Generally, variants in the coding regions of CYP3A4 occur at allele frequencies <. 5% and appear as heterozygous with the wild-type allele. These coding variants may contribute to but are not likely to be the major cause of inter-individual differences in CYP3A-dependent clearance, because of the low allele frequencies and limited alterations in enzyme expression or catalytic function. The most common variant, CYP3A4*1B, is an A-392G transition in the 5'-flanking region with an allele frequency ranging from 0% (Chinese and Japanese) to 45% (African-Americans). Studies have not linked CYP3A4*1B with alterations in CYP3A substrate metabolism. In contrast, there are several reports about its association with various disease states including prostate cancer, secondary leukemias, and early puberty. Linkage disequilibrium between CYP3A4*1B and another CYP3A allele (CYP3A5*1) may be the true cause of the clinical phenotype. CYP3A5 is polymorphically expressed in adults with readily detectable expression in about 10-20% in Caucasians, 33% in Japanese and 55% in African-Americans. The primary causal mutation for its polymorphic expression (CYP3A5*3) confers low CYP3A5 protein expression as a result of improper mRNA splicing and reduced translation of a functional protein. The CYP3A5*3 allele frequency varies from approximately 50% in African-Americans to 90% in Caucasians. Functionally, microsomes from a CYP3A5*3/*3 liver contain very low CYP3A5 protein and display on average reduced catalytic activity towards midazolam. Additional intronic or exonic mutations (CYP3A5*5, *6, and *7) may alter splicing and result in premature stop codons or exon deletion. Several CYP3A5 coding variants have been described, but occur at relatively low allelic frequencies and their functional significance has not been established. As CYP3A5 is the primary extrahepatic CYP3A isoform, its polymorphic expression may be implicated in disease risk and the metabolism of endogenous steroids or xenobiotics in these tissues (e.g., lung, kidney, prostate, breast, leukocytes). CYP3A7 is considered to be the major fetal liver CYP3A enzyme. Although hepatic CYP3A7 expression appears to be significantly down-regulated after birth, protein and mRNA have been detected in adults. Recently, increased CYP3A7 mRNA expression has been associated with the replacement of a 60-bp segment of the CYP3A7 promoter with a homologous segment in the CYP3A4 promoter (CYP3A7*1C allele). This mutational swap confers increased gene transcription due to an enhanced interaction between activated PXR:RXRα complex and its cognate response element (ER-6). The genetic basis for polymorphic expression of CYP3A5 and CYP3A7 has now been established. Moreover, the substrate specificity and product regioselectivity of these isoforms can differ from that of CYP3A4, such that the impact of CYP3A5 and CYP3A7 polymorphic expression on drug disposition will be drug dependent. In addition to genetic variation, other factors that may also affect CYP3A expression include: tissue-specific splicing (as reported for prostate CYP3A5), variable control of gene transcription by endogenous molecules (circulating hormones) and exogenous molecules (diet or environment), and genetic variations in proteins that may regulate constitutive and inducible CYP3A expression (nuclear hormone receptors). Thus, the complex regulatory pathways, environmentally susceptible milieu of the CYP3A enzymes, and as yet undetermined genetic haplotypes, may confound evaluation of the effect of individual CYP3A genetic variations on drug disposition, efficacy and safety.
Cytochrome P450 enzymes (P450s) are important in drug metabolism and have been linked to adverse drug reactions. P450s display broad substrate reactivity, and prediction of metabolites is complex. QM/MM studies of P450 reactivity have provided insight into important details of the reaction mechanisms and have the potential to make predictions of metabolite formation. Here we present a comprehensive study of the oxidation of three widely used pharmaceutical compounds (S-ibuprofen, diclofenac, and S-warfarin) by one of the major drug-metabolizing P450 isoforms, CYP2C9. The reaction barriers to substrate oxidation by the iron-oxo species (Compound I) have been calculated at the B3LYP-D/CHARMM27 level for different possible metabolism sites for each drug, on multiple pathways. In the cases of ibuprofen and warfarin, the process with the lowest activation energy is consistent with the experimentally preferred metabolite. For diclofenac, the pathway leading to the experimentally observed metabolite is not the one with the lowest activation energy. This apparent inconsistency with experiment might be explained by the two very different binding modes involved in oxidation at the two competing positions. The carboxylate of diclofenac interacts strongly with the CYP2C9 Arg108 side chain in the transition state for formation of the observed metabolite—but not in that for the competing pathway. We compare reaction barriers calculated both in the presence and in the absence of the protein and observe a marked improvement in selectivity prediction ability upon inclusion of the protein for all of the substrates studied. The barriers calculated with the protein are generally higher than those calculated in the gas phase. This suggests that active-site residues surrounding the substrate play an important role in controlling selectivity in CYP2C9. The results show that inclusion of sampling (particularly) and dispersion effects is important in making accurate predictions of drug metabolism selectivity of P450s using QM/MM methods.
Telbivudine, one of the oral anti-hepatitis B virus (HBV) nucleoside analogues, has been used for more than 5 years to treat HBeAg-positive and -negative chronic hepatitis B (CHB) patients. This includes not only global Phase II and III trials, but also Chinese Phase III trials and several real-world clinical practice studies from China. The present review will first focus on 1-, 2- and 3-year data of telbivudine therapy on HBV DNA suppression, alanine aminotransferase (ALT) normalization, hepatitis B e antigen (HBeAg) seroconversion, viral resistance and safety in HBeAg-positive and -negative CHB patients. Second, telbivudine treatment predictors, including HBV DNA undetectability, ALT level and other immune-related markers at 12 and 24 weeks will be summarized to optimize therapy. Besides several retrospective studies, the Chinese EFFORT prospective study adapted from telbivudine virological response at 24 weeks has shown very promising results. Finally, the Chinese experience of using telbivudine in the second and third trimesters of pregnancy to prevent perinatal transmission of HBV infection will be touched on citing the latest studies.
Introduction: The effects of escitalopram (10 mg/d) coadministration on plasma concentrations of aripiprazole and its active metabolite, dehydroaripiprazole, were studied in 13 Japanese psychiatric patients and compared with those of paroxetine (10 mg/d) coadministration. Methods: The patients had received 6-24 mg/d of aripiprazole for at least 2 weeks. Patients were randomly allocated to one of 2 treatment sequences: paroxetine-escitalopram (n=6) or escitalopram-paroxetine (n=7). Each sequence consisted of two 2-week phases. Plasma concentrations of aripiprazole and dehydroaripiprazole were measured using liquid chromatography with mass spectrometric detection. Results: Plasma concentrations of aripiprazole and the sum of aripiprazole and dehydroaripiprazole during paroxetine coadministration were 1.7-fold (95% confidence intervals [CI], 1.3-2.1, p<0.001) and 1.5-fold (95% CI 1.2-1.9, p<0.01) higher than those values before the coadministration. These values were not influenced by escitalopram coadministration (1.3-fold, 95% CI 1.1-1.5 and 1.3-fold, 95% CI 1.0-1.5). Plasma dehydroaripiprazole concentrations remained constant during the study. Conclusion: The present study suggests that low doses of escitalopram can be safely coadministered with aripiprazole, at least from a pharmacokinetic point of view.
Chronic hepatitis C is a global health problem. To prevent or reduce complications, the hepatitis C virus (HCV) infection needs to be eradicated. There have been several developments in treating these patients since the discovery of the virus. As of 1 January 2014, the drugs that are approved for treatment of chronic HCV infection are peginterferon-α, ribavirin, boceprevir, telaprevir, simeprevir and sofosbuvir. In this review we provide an overview of the clinical pharmacokinetic characteristics of these agents by describing their absorption, distribution, metabolism and excretion. In the pharmacodynamic part we summarize what is known about the relationships between the pharmacokinetics of each drug and efficacy or toxicity. We briefly discuss the pharmacokinetics and pharmacodynamics of chronic hepatitis C treatment in special patient populations, such as patients with liver cirrhosis, renal insufficiency or HCV/HIV coinfection, and children. With this knowledge, physicians, pharmacists, nurse practitioners, etc. should be educated to safely and effectively treat HCV-infected patients.
Background/AimsRecurrence of hepatitis C virus (HCV) infection after orthotopic liver transplantation (OLT) is common and associated with reduced graft and patient survival. The protease inhibitor telaprevir may enhance virologic response rates in patients after OLT in combination with pegylated-interferon-alfa and ribavirin. Pharmacokinetic studies have shown significant drug-drug interactions between telaprevir and immunosuppression (IS), but telaprevir pharmacokinetics in OLT-patients with IS are unknown. Aim of the present study was to analyze telaprevir plasma concentrations in patients with HCV genotype 1 infection after OLT in comparison to patients without OLT and IS.Methods Five patients with HCV genotype 1 infection after OLT and 37 HCV genotype 1 infected patients without prior OLT were treated with telaprevir 2250 mg daily, ribavirin 1000/1200 mg daily, and pegylated-interferon-alfa-2a 180 μg once weekly (triple therapy). Telaprevir plasma concentrations were analyzed by liquid chromatography electrospray-ionization-tandem mass spectrometry. HCV RNA was assessed by automatized reverse-transcription polymerase-chain-reaction.ResultsMedian (range) telaprevir plasma concentrations of TW 4, 8, and 12 were 3970 (1980-4430) ng/mL and 2520 (1870-8730) ng/mL in patients after OLT and ciclosporin or tacrolimus based IS, respectively, as compared to 2790 (1870-3140) in non-OLT patients (p=0.3). In one patient with tacrolimus based IS, telaprevir dose had to be adjusted to achieve virologic response. Telaprevir plasma concentrations were steady at treatment weeks 4, 8, and 12 in patients with and without IS.Conclusions Telaprevir drug monitoring may be necessary in patients with tacrolimus based IS in patients with HCV graft infection after OLT.This article is protected by copyright. All rights reserved.
St John's wort (SJW; Hypericum perforatum) induces CYP3A4 that is involved in the metabolism of the hepatitis C virus (HCV) protease inhibitor boceprevir. Reduced boceprevir exposure and efficacy would contribute to therapeutic failure and increase the risk for resistance development. Boceprevir is co-administered with interferon/ribavirin, and depression has been described frequently in patients undergoing HCV treatment. Patients may purchase over-the-counter herbals to manage depression, and knowing the interaction between SJW and boceprevir is desirable. This Phase I, open-label, three-period, cross-over pharmacokinetic study enrolled healthy males and females who, following consent and screening procedures, were randomized to receive SJW on days 1-14, SJW plus boceprevir (SJW on days 22-35 and together on days 31-35) and boceprevir on days 52-56, separated by 7 day washout periods, or the same treatment in the opposite order. Pharmacokinetic sampling was performed at the end of each phase. Seventeen (11 female) subjects completed the study and no serious adverse events were reported. Geometric mean ratios (GMRs) and 90% CIs for boceprevir (with SJW versus alone) AUC0-8, Cmax and C8 were 0.91 (0.87-0.96), 0.94 (0.82-1.07) and 1.00 (0.79-1.27), respectively. GMRs and 90% CIs for hypericin, the active component of SJW, (with boceprevir versus alone) AUC0-8, Cmax and C8 were 1.23 (1.10-1.38), 1.32 (1.16-1.52) and 1.37 (1.19-1.58), respectively. SJW did not have a clinically significant effect on boceprevir plasma concentrations (or those of its metabolite), suggesting that SJW and boceprevir can be safely co-administered.
The cytochrome P450 enzymes (CYPs) are the most important enzymes in the oxidative metabolism of hydrophobic drugs and other foreign compounds (xenobiotics). The versatility of these enzymes results in some unusual kinetic properties, stemming from the simultaneous interaction of multiple substrates with the CYP active site. Often, the CYPs display kinetics that deviate from standard hyperbolic saturation or inhibition kinetics. Non-Michaelis–Menten or “atypical” saturation kinetics include sigmoidal, biphasic, and substrate inhibition kinetics (see Chapter 3). Interactions between substrates include competitive inhibition, noncompetitive inhibition, mixed inhibition, partial inhibition, activation, and activation followed by inhibition (see Chapter 4). Models and equations that can result in these kinetic profiles will be presented and discussed.
Diazepam is often used as an adjuvant to pain therapy. Cytochrome P450 (CYP) 3A4 and 2C19 metabolize diazepam into the active metabolites: nordiazepam, temazepam and oxazepam. Owing to diazepam's side-effect profile, mortality risk and potential for drug–drug interactions with CYP 3A4 and/or CYP 2C19 inhibitors, urine drug testing (UDT) could be a helpful monitoring tool. This was a retrospective data analysis that evaluated urine specimens from pain management practices for the distribution of diazepam metabolites with and without CYP 3A4 and 2C19 inhibitors. Intersubject nordiazepam, temazepam and oxazepam geometric mean fractions were 0.16, 0.34 and 0.47, respectively. Intrasubject geometric mean fractions were 0.157, 0.311 and 0.494, respectively. Sex, but not age or urinary pH, had an effect on metabolite fractions. Methadone significantly increased temazepam and oxazepam urinary fractions via CYP3A4 inhibition, whereas fluoxetine and esomeprazole increased nordiazepam fractions via CYP2C19 inhibition. Although more studies are needed, these results suggest the viability of UDT for increased monitoring for therapy and possible drug–drug interactions.
Fluoxetine (FLX) is one of the most widely prescribed selective serotonin reuptake inhibitors. Although FLX is used as racemate in the clinic, the clinical pharmacokinetics of FLX and its N-demethylation metabolite norfluoxetine (NFLX) show obvious cytochrome P450 (CYP) polymorphism dependency and exhibit marked stereoselectivity. However, the kinetic profiles of CYP variants to FLX remain unclear. In the present study, some variants of human CYP2C8, CYP2C9, and CYP2D6 were first expressed in insect cells, and their catalytic roles with respect to FLX enantiomers were then investigated. CYP2C8.4 and CYP2C9.10 showed significantly lower activity and CYP2C8.3 showed significantly higher activity toward both R- and S-FLX compared with the wildtype, while CYP2C9.3, CYP2C9.13, and CYP2C9.16 showed significantly lower activity only toward R-FLX. Five CYP2C9 variants and CYP2D6.1 exhibited significantly stereoselective kinetic profiles prior to R-FLX, and CYP2C8.3 showed a slight stereoselectivity. Interestingly, obvious substrate inhibition was observed in the CYP2C9 wildtype and its three variants only in the case of R-FLX. Together, these findings suggest that CYP2C9 and CYP2D6 polymorphism may play an important role in the clearance of FLX and also in the stereoselective kinetic profiles of FLX enantiomers. Chirality 00:000-000, 2014. © 2014 Wiley Periodicals, Inc.
Hepatitis C virus (HCV) infection is one of the leading causes of end-stage liver disease and the main indication for liver transplantation (LT) in most countries. All patients who undergo LT with detectable serum HCV RNA experience graft reinfection. Between 20 and 30% of patients have developed cirrhosis at 5 years post-LT. The outcome of transplant patients with cirrhosis on the graft is severe, with a rate of decompensation at 1 year of approximately 40%. To date, retransplantation is the only option in patients with decompensated liver disease. Until 2011, standard antiviral therapy with pegylated interferon (PEG-IFN) and ribavirin (RBV), was the only effective therapy. Obtaining a sustained virological response (SVR) in patients with LT greatly improves overall and graft survival but this only occurs in 30% of transplanted patients. Direct acting antivirals (DAAs) such as protease inhibitors (PI), polymerase or other non-structural proteins inhibitors represent a new era in HCV associated liver disease. Although their use in the field of LT will certainly be essential there are some limitations because of safety and tolerance. One limitation is the potential interaction with calcineurin inhibitors. We describe the results of triple therapy with boceprevir (BOC) or telaprevir (TVR) for efficacy and safety and comment on future therapeutic strategies in liver transplant recipients.
Daclatasvir is a highly selective NS5A replication complex inhibitor currently in development for the treatment of chronic hepatitis C infection. Daclatasvir is active at picomolar concentrations and demonstrates in vitro activity against a broad range of HCV genotypes. The primary objective of this study was to assess the effect of daclatasvir on the pharmacokinetics of a combined oral contraceptive containing ethinyl estradiol and norgestimate (Ortho Tri-Cyclen(®)). In this open-label, single-sequence study, 20 healthy female subjects received ethinyl estradiol and norgestimate for three cycles, with coadministration of daclatasvir in cycle 3. Pharmacokinetics of ethinyl estradiol and the active metabolites of norgestimate (norelgestromin and norgestrel) were assessed in cycles 2 and 3. Adjusted ratios of geometric means and 90% confidence intervals were estimated for Cmax (ethinyl estradiol, 1.11 [1.02, 1.20]; norelgestromin, 1.06 [0.99, 1.14]; norgestrel, 1.07 [0.99, 1.16]) and AUCtau (ethinyl estradiol, 1.01 [0.95, 1.07]; norelgestromin, 1.12 [1.06, 1.17]; norgestrel, 1.12 [1.02, 1.23]). Coadministration of daclatasvir resulted in no clinically relevant effects on exposure to ethinyl estradiol, norelgestromin, or norgestrel.
Cytochrome P450 2D6 intermediate metabolizer phenotype (CYP2D6 IM) comprises various genotype subgroups. The aim of this study was to evaluate serum concentrations of the CYP2D6 substrates risperidone and aripiprazole in psychiatric patients with various CYP2D6 genotypes encoding IM phenotype. The study was based on therapeutic drug monitoring data from CYP2D6-genotyped patients (mainly of white origin) treated with orally administered risperidone (n = 190) or aripiprazole (n = 266). Patients were divided into 3 genotype subgroups encoding IM phenotype: (1) heterozygous carriers of fully functional and nonfunctional variant alleles (*1/def), (2) homozygous carriers of reduced-function variant alleles (red/red), and (3) heterozygous carriers of reduced-function and nonfunctional variant alleles (def/red). Dose-adjusted serum concentrations of risperidone and aripiprazole were compared between the genotype subgroups using *1/def, the most frequent CYP2D6 genotype among these subgroups, as the reference group. Median serum concentrations were 4.5- and 1.6-fold higher in the def/red genotype than the *1/def genotype for risperidone and aripiprazole, respectively (P < 0.01 for both). Correspondingly, the serum concentrations were 3.4- and 1.8-fold higher in the red/red subgroup compared with the reference group (P < 0.05 for both). In conclusion, this study revealed substantial variability in serum concentrations of risperidone and aripiprazole between CYP2D6 genotypes associated with IM phenotype. A considerable phenotypical difference was observed between patients carrying 1 and 2 variant alleles.
This paper reports the characterization of an electrochemical biosensor for the continuous monitoring of Naproxen based on cytochrome P450. The electrochemical biosensor is based on the drop-casting of multi-walled carbon-nanotubes (MWCNTs) and microsomal cytochrome P4501A2 (msCYP1A2) on a graphite screen-printed electrode (SPE). The proposed biosensor was employed to monitor Naproxen (NAP), a well-known anti-inflammatory compound, through cyclic voltammetry. The dynamic linear range for the amperometric detection of NAP had an upper limit of 300µM with a corresponding limit of detection (LOD) of 16±1µM (S/N=3), which is included in NAP physiological range (9-300µM). The MWCNT/msCYP1A2-SPE sensor was also calibrated for NAP detection in mouse serum that was previously extracted from mice, showing a slightly higher LOD (33±18µM). The stability of the msCYP1A2-based biosensor was assessed by longtime continuous cyclic voltammetric measurements. The ability of the sensor to monitor drug delivery was investigated by using a commercial micro-osmotic pump. Results show that the MWCNT/msCYP1A2-SPE sensor is capable of precisely monitoring the real-time delivery of NAP for 16h. This work proves that the proposed electrochemical sensor might represent an innovative point-of-care solution for the personalization of drug therapies, as well as for pharmacokinetic studies in both animals and humans.
Venlafaxine (VEN) is a serotonin–norepinephrine reuptake inhibitor marketed for the treatment of depression disorders. The molecular targets of VEN are the solute carrier family 6 (neurotransmitter transporter, serotonin), member 4 (SLC6A4), and solute carrier family 6 (neurotransmitter transporter, noradrenalin), member 2 (SLC6A2), resulting in an inhibition of serotonin and noradrenaline reuptake from the synaptic cleft. VEN also slightly inhibits dopamine reuptake. Therefore, those substances are available prolonged in the synaptic cleft for serotonergic and noradrenergic neurotransmission. VEN consists of a racemic mixture of R(+) and S(−) enantiomer. The (R) enantiomer has been shown to show greater serotonin reuptake inhibition property, whereas the (S) enantiomer inhibits the reuptake of both monoamines [1,2].
With the licensing of the direct acting antivirals telaprevir and boceprevir the topic of drug-drug interactions has come to the forefront. These first generation hepatitis C virus protease inhibitors are metabolized by and inhibit the key drug metabolizing enzyme CYP3A4, which means that knowledge of drug-drug interactions has become an essential component of the evaluation of a patient starting triple therapy. The number of potential co-medications means that many drugs will be used in hepatitis C virus patients where there are no pharmacokinetic study data. Here we have to use the data that are available and seek to extrapolate to unstudied drugs using key principles of clinical pharmacology (disposition characteristics, concentration-effect relationships, therapeutic window) in order to give some guidance for management of patients. This is a rapidly moving area in hepatitis C therapy, both in terms of understanding the drug interaction profile of telaprevir and boceprevir, interaction mechanisms that sometimes appear counterintuitive and that may involve enzymes other than CYP3A4 or transporters, but then seeking to understand the interaction potential of the next wave of drugs that will soon be with us.
There is a close relationship between chronic hepatitis B virus (HBV) infection and chronic renal disease. We analyzed changes in renal function using different markers of glomerular filtration rate (GFR) in multiple studies of telbivudine treatment of patients with chronic HBV infection. We used serum creatinine-based equations (Cockcroft-Gault, modification of diet in renal disease [MDRD], and chronic kidney disease epidemiology collaboration [CKP-EPI]) to estimate GFR (eGFRs) in adults with chronic HBV infection and compensated liver disease who participated in a phase III randomized double-blind study comparing the efficacy and safety of telbivudine (600 mg/day) and lamivudine (100 mg/day) for 2 y (the GLOBE study) and in long-term extension studies (4-6 years), as well as in patients with decompensated cirrhosis (2 y). eGFRs calculated using the Cockcroft-Gault, MDRD, and CKP-EPI equations were concordant, indicating improved renal function in telbivudine-treated patients during the 2 y GLOBE study (there was an 8.5% increase in mean eGFR, based on the MDRD equation). Improved renal function was maintained for 4-6 y. Increased eGFR with telbivudine treatment was also observed in patients at increased risk for renal impairment: patients with baseline eGFRs of 60-89 mL/min/1.73 m(2) (+17.2%), older than 50 y (+11.4%), and with liver fibrosis/cirrhosis, (+7.2% for patients with Ishak fibrosis score at 5-6). In decompensated patients with high renal risk, eGFR was also improved on telbivudine (+2.0%). In global trials of patients with compensated and decompensated cirrhosis, long-term telbivudine therapy was associated with a sustained improvement of renal function-particularly among patients with increased risk of renal impairment. The mechanisms of this renal protective effect remain to be determined.
Electrochemical biosensors consisting of cytochrome P450 enzyme modified electrodes have been developed to provide a simple method for screening the metabolism of a drug and its inhibitor. Here, we report a very simple electrochemically driven biosensor for detecting drug metabolism and its inhibition based on cytochrome P450 3A4 (CYP3A4) and a carbon nanofiber (CNF) modified film electrode without any other modified layers such as mediator films. Direct electron transfer (DET) between CYP3A4 and CNFs was observed at a formal potential of -0.302 V. The electrocatalytic reduction current increased with the addition of drugs including testosterone and quinidine. In contrast, the reduction current was greatly suppressed in the presence of ketoconazole, which is a CYP3A4 inhibitor. CNFs with high conductivity, a large surface area and sufficient edge planes provide a suitable microenvironment for achieving excellent DET and biocatalysis properties, which could not be observed when we used other carbon materials such as carbon nanotube (CNT) and carbon black (CB) modified electrodes, indicating that our system is promising as a new bioelectronic platform for electrochemical biosensing.
Although a basic understanding of the chemical and biological events leading to idiosyncratic drug toxicity is still lacking, it appears that drug-independent risk factors that increase reactive metabolite formation or alter cellular stress and immune response may be critical determinants in the response to an otherwise non-toxic drug. Thus, we were interested to determine the impact of various drug-independent stress factors - lipopolysaccharide (LPS), poly I:C (PIC) or glutathione depletion via buthionine sulfoximine (BSO) - on the toxicity of diclofenac (Dcl), a model drug associated with rare but significant cases of serious hepatotoxicity, and to understand if enhanced toxicity occurs through alterations of drug metabolism and/or modulation of stress response pathways. Co-treatment of rats repeatedly given therapeutic doses of Dcl for 7 days with a single dose of LPS 2 h before the last Dcl dose resulted in severe liver toxicity. Neither LPS nor diclofenac alone or in combination with PIC or BSO had such an effect. While it is thought that bioactivation to reactive Dcl acyl glucuronides (AG) and subsequent protein adduct formation contribute to Dcl induced liver injury, LC-MS/MS analyses did not reveal increased formation of 4'- and 5-hydroxy-Dcl, Dcl-AG or Dcl-AG dependent protein adducts in animals treated with LPS/Dcl. Hepatic gene expression analysis suggested enhanced activation of NFκB and MAPK pathways and up-regulation of co-stimulatory molecules (IL-1β, TNF-α, CINC-1) by LPS/Dcl and PIC/Dcl, while protective factors (HSPs, SOD2) were down-regulated. LPS/Dcl led to extensive release of pro-inflammatory cytokines (IL-1β, IL-6, IFN-γ, TNF-α) and factors thought to constitute danger signals (HMGB1, CINC-1) into plasma. Taken together, our results show that Dcl enhanced the inflammatory response induced by LPS - and to a lesser extent by PIC - through up-regulation of pro-inflammatory molecules and down-regulation of protective factors. This suggests sensitization of cells to cellular stress mediated by non-drug-related risk factors by therapeutic doses of Dcl, rather than potentiation of Dcl toxicity by the stress factors.
Clozapine, an often-prescribed antipsychotic drug, is implicated in severe adverse drug reactions (ADRs). Formation of reactive intermediates by cytochrome P450s (CYPs) has been proposed as a possible explanation for these ADRs. Moreover, a protective role for human glutathione S-transferases (hGSTs) was recently shown using purified enzymes. We investigated the interplay between CYP bioactivation and GST detoxification in a reconstituted cellular context using recombinant yeast expressing a bacterial CYP BM3 mutant (M11), mimicking the drug-metabolizing potential of human CYPs, combined with hGSTA1-1, M1-1 or P1-1. Clozapine and the N-desmethylclozapine metabolite caused comparable growth inhibition and reactive oxygen species (ROS) formation, whereas the clozapine-N-oxide metabolite was clearly less toxic. Clozapine metabolism by BM3 M11 and the hGSTs in yeast was confirmed by identification of stable clozapine metabolites and hGST isoform-specific glutathione-conjugates. Oxidative metabolism of clozapine by BM3 M11 increased ROS formation and growth inhibition. Co-expression of hGSTP1-1 protected yeast from BM3 M11 induced growth inhibition in presence of clozapine, whereas similar expression levels of hGSTA1-1 and hGSTM1-1 did not. ROS formation was not lowered by hGSTP1-1 co-expression and was unrelated to mitochondrial electron transport chain (mETC) activity. We present a novel cellular model to study the effect of CYP and GST interplay in drug toxicity.
The direct electrochemistry of house fly cytochrome P4506A1 (CYP6A1) confined in dioctadecyl dimethyl ammonium bromide (DDAB) film was achieved. The immobilized CYP6A1 displayed a pair of redox peaks with a formal potential of −0.36mV in pH 7.0 O2-free phosphate buffers at scan rate of 1Vs−1 and the direct electron transfer of CYP6A1 was characterized by voltammetry. The CYP6A1 in the DDAB film retained its bioactivity and could catalyze the reduction of dissolved oxygen. Upon addition of its substrate aldrin or heptachlor to the air-saturated solution, the reduction peak current of dissolved oxygen increased, which indicates the catalytic behavior of CYP6A1 to its substrates. By amperometry a calibration linear range was obtained to be 9.08×10−6–4.54×10−5molL−1 with a sensitivity of 80μAmM−1 for aldrin or 8.91×10−6–4.46×10−5molL−1 with a sensitivity of 66μAmM−1 for heptachlor. The apparent Michaelis–Menten constant for the electrocatalytic activity of CYP6A1 was found to be 7.468×10−5molL−1 for aldrin and 4.316×10−5molL−1 for heptachlor. The bioelectrocatalytic products were analysed using gas chromatography (GC) and electron ionization–mass spectrometry (EI–MS). The results confirmed that epoxidation was the main pathways of CYP6A1-mediated organochlorine pesticides oxidation.
ABSTRACT Telaprevir (TVR) effects on P-glycloprotein and cytochrome P450 (CYP) may significantly elevate serum levels of morphine and methadone. Recent literature points to major interactions when combining TVR with warfarin or rifampin. Opioid interactions are especially dangerous in hepatitis C patients, as coinfection with human immunodeficiency virus (HIV) and hepatitis C virus (HCV) occurs in 50-90% of HIV-infected drug users that are prescribed opioids for chronic pain and/or methadone for maintenance. TVR has been shown to significantly inhibit the active transport enzyme pGP and may therefore increase intestinal morphine absorption. TVR also inhibits hepatic CYP3A4 that are responsible for metabolizing methadone. Patients requiring opioid analgesics must be carefully monitored because of potential for elevated opioid levels and overdose risk. Current recommendations minimize potential drug interactions between telaprevir and opioids, especially methadone, based on a single 7-day trial. We outline the various pharmacokinetic mechanisms involved when combining TVR with methadone or morphine and recommend that current data are not sufficiently robust to minimize the potentially significant interaction with opioids, especially methadone. Clinicians must be mindful of these understated interactions, know that the opioid dose may need to be significantly increased or reduced, and use caution during upward titration of opioids affected by these enzyme systems.