Na Gao

Content and activity of human liver microsomal protein and prediction of individual hepatic clearance in vivo

The lack of information concerning individual variation in content and activity of human liver microsomal protein is one of the most important obstacles for designing personalized medicines. We demonstrated that the mean value of microsomal protein per gram of liver (MPPGL) was 39.46 mg/g in 128 human livers and up to 19-fold individual variations existed. Meanwhile, the metabolic activities of 10 cytochrome P450 (CYPs) were detected in microsomes and liver tissues, respectively, which showed huge individual variations (200-fold). Compared with microsomes, the activities of liver tissues were much suitable to express the individual variations of CYP activities. Furthermore, individual variations in the in vivo clearance of tolbutamide were successfully predicted with the individual parameter values. In conclusion, we offer the values for MPPGL contents in normal liver tissues and build a new method to assess the in vitro CYP activities. In addition, large individual variations exist in predicted hepatic clearance of tolbutamide. These findings provide important physiological parameters for physiologically-based pharmacokinetics models and thus, establish a solid foundation for future development of personalized medicines.

Effect of Cytochrome b5 Content on the Activity of Polymorphic CYP1A2, 2B6, and 2E1 in Human Liver Microsomes.

Human cytochrome b5 (Cyt b5) plays important roles in cytochrome P450 (CYP)-mediated drug metabolism. However, the expression level of Cyt b5 in normal human liver remains largely unknown. The effect of Cyt b5 on overall CYP activity in human liver microsomes (HLM) has rarely been reported and the relationship between Cyt b5 and the activity of polymorphic CYP has not been systematically investigated. In this study, we found that the median value of Cyt b5 protein was 270.01 pmol/mg from 123 HLM samples, and 12- and 19-fold individual variation was observed in Cyt b5 mRNA and protein levels, respectively. Gender and smoking clearly influenced Cyt b5 content. In addition, we found that Cyt b5 protein levels significantly correlated with the overall activity of CYP1A2, 2B6, and 2E1 in HLM. However, when the CYP activities were sorted by single nucleotide polymorphisms (SNP), the effect of Cyt b5 protein on the kinetic parameters varied greatly. There were significant correlations between Cyt b5 content and Vmax and CLint of CYP1A2 wild-types (3860GG, 2159GG, and 5347CC) as well as homozygous mutants (163AA and 3113GG). In contrast to Vmax and CLint, the Km of CYP2B6 516GG and 785AA genotypes was inversely associated with Cyt b5 content. Correlations between Cyt b5 content and Vmax and CLint of CYP2E1 -1293GG, -1293GC, 7632TT, 7632TA, -333TT, and -352AA genotypes were also observed. In conclusion, Cyt b5 expression levels varied considerably in the Chinese cohort from this study. Cyt b5 had significant impact on the overall activity of CYP1A2, 2B6, and 2E1 in HLM and the effects of Cyt b5 protein on polymorphic CYP1A2, 2B6, and 2E1 activity were SNP-dependent. These findings suggest that Cyt b5 plays an important role in CYP-mediated activities in HLM and may possibly be a contributing factor for the individual variation observed in CYP enzyme activities.

Physiological Content and Intrinsic Activities of 10 Cytochrome P450 Isoforms in Human Normal Liver Microsomes.

Due to a lack of physiologic cytochrome P450 (P450) isoform content, P450 activity is typically only determined at the microsomal level (per milligram of microsomal protein) and not at the isoform level (per picomole of P450 isoform), which could result in the misunderstanding of variations in P450 activity between individuals and further hinder development of personalized medicine. We found that there were large variations in protein content, mRNA levels, and intrinsic activities of the 10 P450s in 100 human liver samples, in which CYP2E1 and CYP2C9 showed the highest expression levels. P450 gene polymorphisms had different effects on activity at two levels: CYP3A5*3 and CYP2A6*9 alleles conferred increased activity at the isoform level but decreased activity at the microsomal level; CYP2C9*3 had no effect at the isoform level but decreased activity at the microsomal level. The different effects at each level stem from the different effects of each polymorphism on the resulting P450 protein. Individuals with CYP2A6*1/*4, CYP2A6*1/*9, CYP2C9*1/*3, CYP2D6 100C>T TT, CYP2E1 7632T>A AA, CYP3A5*1*3, and CYP3A5*3*3 genotypes had significantly lower protein content, whereas CYP2D6 1661G>C mutants had a higher protein content. In conclusion, we first offered the physiologic data of 10 P450 isoform contents and found that some single nucleotide polymorphisms had obvious effects on P450 expression in human normal livers. The effects of gene polymorphisms on intrinsic P450 activity at the isoform level were quite different from those at the microsomal level, which might be due to changes in P450 protein content.

Inhibition of Baicalin on Metabolism of Phenacetin, a Probe of CYP1A2, in Human Liver Microsomes and in Rats

Baicalin has been used as mainly bioactive constituent of about 100 kinds of traditional Chinese medicines in Chinese pharmacopoeia. The effect of baicalin on cytochrome P450 should be paid more attention because baicalin was used widely. The aim of this study was to investigate whether baicalin could inhibit CYP1A2 in pooled human liver microsomes (HLMs) and in rats in vivo and the gene polymorphisms could affect inter-individual variation in IC50 in 28 human livers. Phenacetin was used as probe of CYP1A2. Kinetic parameter of CYP1A2 and IC50 of baicalin on CYP1A2 to each sample were measured and the common CYP1A2 polymorphisms (−3860G>A and −163C>A) were genotyped. The results showed that baicalin exhibited a mixed-type inhibition in pooled HLMs, with a Ki value of 25.4 µM. There was substantial variation in Km, Vmax, CLint of CYP1A2 and IC50 of baicalin on CYP1A2 (3∼10-fold). The range was from 26.6 to 114.8 µM for Km, from 333 to 1330 pmol·min−1·mg−1protein for Vmax and from 3.8 to 45.3 µL·min−1·mg−1 protein for CLint in HLMs (n = 28). The Mean (range) value of IC50 in 28 HLMs was 36.3 (18.9 to 56.1) µM. The genotypes of −3860G>A and −163C>A had no significant effect on the inhibition of baicalin on CYP1A2. The animal experiment results showed that baicalin (450 mg/kg, i.v.) significantly decreased the Cmax and CL of phenacetin, and increased C60 min, t1/2, Vd and AUC (P<0.05). There were significant correlations between percentage of control in C60 min, t1/2, CL, AUC of phenacetin and Cmax of baicalin in 11 rats (P<0.05). Protein binding experiments in vitro showed that baicalin (0–2000 mg/L) increased the unbound phenacetin from 14.5% to 28.3%. In conclusion, baicalin can inhibit the activity of CYP1A2 in HLMs and exhibit large inter-individual variation that has no relationship with gene polymorphism. Baicalin can change the pharmacokinetics of phenacetin in rats.

Significant change of cytochrome P450s activities in patients with hepatocellular carcinoma

The lack of information concerning individual variation in drug-metabolizing enzymes is one of the most important obstacles for designing personalized medicine approaches for hepatocellular carcinoma (HCC) patients. To assess cytochrome P450 (CYP) in the metabolism of endogenous and exogenous molecules in an HCC setting, the activity changes of 10 major CYPs in microsomes from 105 normal and 102 HCC liver tissue samples were investigated. We found that CYP activity values expressed as intrinsic clearance (CLint) differed between HCC patients and control subjects. HCC patient samples showed increased CLint for CYP2C9, CYP2D6, and CYP2E1 compared to controls. Meanwhile, CYP1A2, CYP2C8, and CYP2C19 CLint values decreased and CYP2A6, CYP2B6, and CYP3A4/5 activity was unchanged relative to controls. For patients with HCC accompanied by fibrosis or cirrhosis, the same activity changes were seen for the CYP isoforms, except for CYP2D6 which had higher values in HCC patients with cirrhosis. Moreover, CYP2D6*10 (100C>T), CYP2C9*3 (42614 A>C), and CYP3A5*3 (6986A>G) polymorphisms had definite effects on enzyme activities. In the HCC group, the CLint of CYP2D6*10 mutant homozygote was decreased by 95% compared to wild-type samples, and the frequency of this homozygote was 2.8-fold lower than the controls. In conclusion, the activities of CYP isoforms were differentially affected in HCC patients. Genetic polymorphisms of some CYP enzymes, especially CYP2D6*10, could affect enzyme activity. CYP2D6*10 allelic frequency was significantly different between HCC patients and control subjects. These findings may be useful for personalizing the clinical treatment of HCC patients as well as predicting the risk of hepatocarcinogenesis.

Changes in cytochrome P450s-mediated drug clearance in patients with hepatocellular carcinoma in vitro and in vivo : a bottom-up approach

Hepatocellular carcinoma (HCC) accompanied by severe liver dysfunction is a serious disease, which results in altered hepatic clearance. Generally, maintenance doses depend upon drug clearance, so individual dosage regimens should be customized for HCC patients based on the condition of patients. Based on clearance of CYP isoform-specific substrates at the microsomal level (CLM), microsomal protein per gram of liver (MPPGL), liver weight, hepatic blood flow, hepatic clearance values (CLH) for 10 CYPs in HCC patients (n=102) were extrapolated using a predictive bottom-up pharmacokinetic model. Compared with controls, the CLM values for CYP2C9, 2D6, 2E1 were significantly increased in HCC patients. Additionally, CYP1A2, 2C8, 2C19 CLM values decreased while the values for CYP2A6, 2B6, 3A4/5 were unchanged. The MPPGL values in HCC tissues were significantly reduced. CLH values of HCC patients for CYP1A2, 2A6, 2B6, 2C8, 2C19, and 3A4/5 were significantly reduced, while this for CYP2E1 were markedly increased and those for CYP2C9 and 2D6 did not change. Moreover, disease (fibrosis and cirrhosis) and polymorphisms of the CYP genes have influenced the CLH for some CYPs. Prediction of the effects of HCC on drug clearance may be helpful for the design of clinical studies and the clinical management of drugs in HCC patients.

Gene polymorphisms and contents of cytochrome P450s have only limited effects on metabolic activities in human liver microsomes

Extensive inter-individual variations in pharmacokinetics are considered as a major reason for unpredictable drug responses. As the most important drug metabolic enzymes, inter-individual variations of cytochrome P450 (CYP) activities are not clear in human liver. In this paper, metabolic activities, gene polymorphisms and protein contents of 10 CYPs were determined in 105 human normal liver microsomes. The results indicated substantial inter-individual variations in CYP activities, with the greatest being CYP2C19 activity (>600-fold). Only half of 10 CYP isoforms and 26 gene polymorphism sites had limited effects on metabolic activities, such as CYP2A6, CYP2B6, CYP2C9, CYP2D6 and CYP3A4/5, others had almost no effects. Compared with their respective wild type, Km, Vmax, and CLint decreased by 51.6%, 88.7% and 70.7% in CYP2A6*1/*4 genotype, Vmax and CLint decreased by 32.8% and 60.2% in CYP2C9*1/*3 genotype, Km increased by 118.4% and CLint decreased by 65.2% in CYP2D6 100TT genotype, respectively. Moreover, there were only 4 CYP isoforms, CYP1A2, CYP2A6, CYP2E1 and CYP3A5, which had moderate or weak correlations between Vmax values and corresponding contents. In conclusions, the genotypes and contents of some CYPs have only limited effects on metabolic activities, which imply that there are other more important factors to influence inter-individual variations.

Relationships between specific serum IgE, IgG, IFN-γ level and IFN-γ, IFNR1 polymorphisms in patients with penicillin allergy

ObjectiveThe findings of numerous studies have suggested that both genetic and environmental influences are involved in the pathogenesis of allergic disease and atopy. We studied the polymorphisms in the interferon (IFN)-gamma (γ) and IFN-γ receptor 1 (IFNR1) gene with the aim of clarifying the relationships among these polymorphisms, penicillin allergy and anti-penicillin antibodies.MethodsA restriction endonuclease fragment length polymorphism (RFLP)-PCR analysis and sequencing were used to study the IFNR1 and IFN-γ polymorphisms. The presence and level of eight specific immunoglobulin (Ig)E and IgG antibodies were determined by the radioallergosorbent test (RAST) and enzyme-linked immunosorbent assay (ELISA), respectively.ResultsThe positive rates of specific IgE and IgG were 61.11 and 53.92%, respectively. There was no significant difference in the whole-allele of IFN-γ distribution between patients with a penicillin allergy and control subjects. Allele 7 (18CA repeat) was significantly less frequent in the urticaria group (3.19 vs. 11.93%) than in the controls. There was no difference in IFN-γ production among different alleles in IFN-γ. The frequency of G/A (Val/Met) in the IFNR1 gene in allergic patients was significantly less than that in the controls (P < 0.05). There were no significant differences in the positive rate of IgE among different alleles of IFN-γ. The same was true for the positive rate of IgG.ConclusionsThe Met/Val allele in IFNR1 gene may have a protective role in the non-penicillin allergic population. The allele 18CA repeat in IFN-γ gene may be associated with urticaria.

Hypersensitivity reactions to penicillins : studies in a group of patients with negative benzylpenicillin G skin test

Background:  Although skin tests are usually employed to evaluate current penicillin allergy status, a negative result does not exclude hypersensitivity. There is a need for accurate in vitro tests to exclude hypersensitivity. A radioallergosorbent test (RAST) is a potentially good supplementary approach, but there is little information on the suitability of this method to diagnose penicillin hypersensitivity in subjects with a negative skin test to benzylpenicillin.

Correlation of Cytochrome P450 Oxidoreductase Expression with the Expression of 10 Isoforms of Cytochrome P450 in Human Liver

Human cytochrome P450 oxidoreductase (POR) provides electrons for all microsomal cytochromes P450 (P450s) and plays an indispensable role in drug metabolism catalyzed by this family of enzymes. We evaluated 100 human liver samples and found that POR protein content varied 12.8-fold, from 12.59 to 160.97 pmol/mg, with a median value of 67.99 pmol/mg; POR mRNA expression varied by 26.4-fold. POR activity was less variable with a median value of 56.05 nmol/min per milligram. Cigarette smoking and alcohol consumption clearly influenced POR activity. Liver samples with a 2286822 TT genotype had significantly higher POR mRNA expression than samples with CT genotype. Homozygous carriers of POR2286822C>T, 2286823G>A, and 3823884A>C had significantly lower POR protein levels compared with the corresponding heterozygous carriers. Liver samples from individuals homozygous at 286823G>A, 1135612A>G, and 10954732G>A generally had lower POR activity levels than those from heterozygous or wild-type samples, whereas the common variant POR*28 significantly increased POR activity. There was a strong association between POR and the expression of P450 isoforms at the mRNA and protein level, whereas the relationship at the activity level, as well as the effect of POR protein content on P450 activity, was less pronounced. POR transcription was strongly correlated with both hepatocyte nuclear factor 4 alpha and pregnane X receptor mRNA levels. In conclusion, we have elucidated some potentially important correlations between POR single-nucleotide polymorphisms and POR expression in the Chinese population and have developed a database that correlates POR expression with the expression and activity of 10 P450s important in drug metabolism.