Comparison of Different Scoring Systems Based on Both Donor and Recipient Characteristics for Predicting Outcome after Living Donor Liver Transplantation

Background and Objectives

In order to provide a good match between donor and recipient in liver transplantation, four scoring systems [the product of donor age and Model for End-stage Liver Disease score (D-MELD), the score to predict survival outcomes following liver transplantation (SOFT), the balance of risk score (BAR), and the transplant risk index (TRI)] based on both donor and recipient parameters were designed. This study was conducted to evaluate the performance of the four scores in living donor liver transplantation (LDLT) and compare them with the MELD score.

Patients and Methods

The clinical data of 249 adult patients undergoing LDLT in our center were retrospectively evaluated. The area under the receiver operating characteristic curves (AUCs) of each score were calculated and compared at 1-, 3-, 6-month and 1-year after LDLT.

Results

The BAR at 1-, 3-, 6-month and 1-year after LDLT and the D-MELD and TRI at 1-, 3- and 6-month after LDLT showed acceptable performances in the prediction of survival (AUC>0.6), while the SOFT showed poor discrimination at 6-month after LDLT (AUC = 0.569). In addition, the D-MELD and BAR displayed positive correlations with the length of ICU stay (D-MELD, p = 0.025; BAR, p = 0.022). The SOFT was correlated with the time of mechanical ventilation (p = 0.022).

Conclusion

The D-MELD, BAR and TRI provided acceptable performance in predicting survival after LDLT. However, even though these scoring systems were based on both donor and recipient parameters, only the BAR provided better performance than the MELD in predicting 1-year survival after LDLT.     

Q-FISH Measurement of Hepatocyte Telomere Lengths in Donor Liver and Graft after Pediatric Living-Donor Liver Transplantation: Donor Age Affects Telomere Length Sustainability

Along with the increasing need for living-donor liver transplantation (LDLT), the issue of organ shortage has become a serious problem. Therefore, the use of organs from elderly donors has been increasing. While the short-term results of LDLT have greatly improved, problems affecting the long-term outcome of transplant patients remain unsolved. Furthermore, since contradictory data have been reported with regard to the relationship between donor age and LT/LDLT outcome, the question of whether the use of elderly donors influences the long-term outcome of a graft after LT/LDLT remains unsettled. To address whether hepatocyte telomere length reflects the outcome of LDLT, we analyzed the telomere lengths of hepatocytes in informative biopsy samples from 12 paired donors and recipients (grafts) of pediatric LDLT more than 5 years after adult-to-child LDLT because of primary biliary atresia, using quantitative fluorescence in situ hybridization (Q-FISH). The telomere lengths in the paired samples showed a robust relationship between the donor and grafted hepatocytes (r = 0.765, p = 0.0038), demonstrating the feasibility of our Q-FISH method for cell-specific evaluation. While 8 pairs showed no significant difference between the telomere lengths for the donor and the recipient, the other 4 pairs showed significantly shorter telomeres in the recipient than in the donor. Multiple regression analysis revealed that the donors in the latter group were older than those in the former (p = 0.001). Despite the small number of subjects, this pilot study indicates that donor age is a crucial factor affecting telomere length sustainability in hepatocytes after pediatric LDLT, and that the telomeres in grafted livers may be elongated somewhat longer when the grafts are immunologically well controlled.     

Pyrosequencing to Identify Homogeneous Phenomenon When Using Recipients/Donors with Different CYP3A5*3 Genotypes in Living Donor Liver Transplantation

This study used pyrosequencing to determine the proportional distribution of CYP3A5*3 genotypes to further confirm the homogeneous phenomenon that is observed when recipients and donors in living donor liver transplantation (LDLT) have a different single nucleotide polymorphism (SNP) genotype. We enrolled 42 recipient/living donor pairs and the SNPs of CYP3A5*3 were identified by polymerase chain reaction-restriction fragment length polymorphism. We performed 120 liver graft biopsies as part of clinical investigations after LDLT. Pyrosequencing of the CYP3A5*3 SNPs revealed that among the 16 recipients with the G/G genotype, 94.68% had the G and 5.32% the A allele. Among the 14 recipients with the A/G genotype, 78.08% had the G and 21.92% the A allele, and among the 12 recipients with the A/A genotype, 18.45% had the G and 81.55% the A allele. Among the 12 donors with the G/G genotype, 93.85% had the G and 6.14% the A allele. Among the 26 donors with the A/G genotype, 75.73% had the G and 24.27% the A allele, and among the 4 donors with the A/A genotype, 11.09% had the G and 88.91% the A allele. There were a total of 120 liver graft biopsy samples; among the 37 recipients with the G/G genotype, 89.74% had the G and 10.26% the A allele, among the 70 recipients with the A/G genotype, 71.57% had the G and 28.43% the A allele, and among the 13 recipients with the A/A genotype, 48.25% had the G and 51.75% the A allele. The proportional distribution of G and A alleles of the CYP3A5*3 SNP between recipients/donors and liver grafts after LDLT was significantly different (p<0.001). Pyrosequencing was useful in identifying detailed proportional changes of the CYP3A5*3 SNP allele distribution, and to confirm the homogeneous phenomenon when recipients and donors in LDLT have a different genotype.     

Epitope Analysis of the Collagen Type V-Specific T Cell Response in Lung Transplantation Reveals an HLA-DRB1*15 Bias in Both Recipient and Donor

Background

IL-17-dependent cellular immune responses to the α1 chain of collagen type V are associated with development of bronchiolitis obliterans syndrome after lung transplantation, and with idiopathic pulmonary fibrosis and coronary artery disease, primary indications for lung or heart transplantation, respectively.

Methodology/Principal Findings

We found that 30% of the patients awaiting lung transplantation exhibited a strong cell-mediated immune response to col(V). Of these, 53% expressed HLA-DR15, compared to a 28% HLA-DR15 frequency in col(V) low-responders (p=0.02). After transplantation, patients with HLA-DR1 and -DR17, not -DR15, developed anti-col(V) responses most frequently (p=0.04 and 0.01 vs. controls, respectively). However, recipients of a lung from an HLA-DR15⁺ donor were at significantly elevated risk of developing anti-col(V) responses (p=0.02) and BOS (p=0.03). To determine the molecular basis of this unusual pattern of DR allele bias, a peptide library comprising the collagenous region of the α1(V) protein was screened for binding to HLA-DR0101, -DR1501, -DR0301 (DR17) or to HLA-DQ2 (DQA1*0501: DQB1*0201; in linkage disequilibrium with -DR17) and -DQ6 (DQA1*0102: DQB1*0602; linked to -DR15). Eight 15-mer peptides, six DR-binding and two DQ-binding, were identified. HLA-DR15 binding to two peptides yielded the highest binding scores: 650 (where 100 = positive control) for p799 (GIRGLKGTKGEKGED), and 193 for p1439 (LRGIPGPVGEQGLPG). These peptides, which also bound weakly to HLA-DR1, elicited responses in both HLA-DR1⁺ and -DR15⁺ col(V) reactive hosts, whereas binding and immunoreactivity of p1049 (KDGPPGLRGFPGDRG) was DR15-specific. Remarkably, a col(V)-reactive HLA-DR1⁺DR15^neg lung transplant patient, whose donor was HLA-DR15⁺, responded not only to p799 and p1439, but also to p1049.

Conclusions/Significance

HLA-DR15 and IPF disease were independently associated with pre-transplant col(V) autoimmunity. The increased risk of de novo immunity to col(V) and BOS, associated with receiving a lung transplant from an HLA-DR15⁺ donor, may result from presentation by donor-derived HLA- DR15, of novel self-peptides to recipient T cells.     

Influence of CYP3A5, ABCB1 and NR1I2 polymorphisms on prednisolone pharmacokinetics in renal transplant recipients

The objective of this study was to evaluate whether genetic polymorphisms of CYP3A5 (A6986G, CYP3A5*3), ABCB1 (C1236T, G2677T/A, C3435T) and NR1I2 (A7635G) significantly impact the pharmacokinetics of prednisolone in renal transplant recipients. Ninety-five recipients were given repeated doses of triple therapy immunosuppression consisting of prednisolone, tacrolimus and mycophenolate mofetil. Twenty-eight days after renal transplantation, plasma prednisolone concentrations were measured by high-performance liquid chromatography. Comparisons of the CYP3A5 and ABCB1 genotypes revealed no significant differences in the prednisolone pharmacokinetics. The mean prednisolone C(max) for recipients (n=14) having both the ABCB1 3435CC genotype and the CYP3A5*3/*3 genotype was significantly higher than those (n=11) having both ABCB1 3435TT and CYP3A5*3/*3 genotypes (180ng/mL versus 129ng/mL, P=0.0392). The plasma concentrations of prednisolone in recipients having both ABCB1 3435CC and CYP3A5*3/*3 genotypes tended to be higher than those having both ABCB1 3435TT and CYP3A5*3/*3 genotypes. The mean AUC(0-24) and C(max) values for prednisolone in recipients having the NR1I2 7635G allele (AG: n=45, GG: n=32) were significantly lower than in patients having the 7635AA allele (n=18) (7635GG versus 7635AA, P=0.0308 for AUC(0-24), P=0.0382 for C(max) of prednisolone). In conclusion, NR1I2 (A7635G) rather than CYP3A5 or ABCB1 allelic variants affected patient variability of plasma prednisolone concentration. Recipients carrying the NR1I2 7635G allele seemed to possess higher metabolic activity for prednisolone in the intestine, greatly reducing its maximal plasma concentration.     

Distribution of ABCB1, CYP3A5, CYP2C19, and P2RY12 gene polymorphisms in a Mexican Mestizos population

The aim of the present study was to establish the gene frequency of six polymorphisms of the ABCB1, CYP3A5, CYP2C19, and P2RY12 genes in a population resident of Mexico City. The proteins encoded by these genes have been associated with the absorption, and biotransformation of clopidogrel. The ABCB1 T3435C, CYP3A5 V3* A6986G, P2RY12 G52T, P2RY12 C34T, CYP2C19 V2* and V3* (positions G681A and G636A, respectively), polymorphisms were analyzed by 5′ exonuclease TaqMan genotyping assays in a group of 269 healthy unrelated Mexican Mestizo individuals. The CYP2C19 V3* G636A polymorphism was not detected in the Mexican Mestizos population. However, the studied population presented significant differences (P < 0.05) in the distribution of the T3435C, A6986G, G681A, G52T and C34T polymorphisms when compared to reported frequencies of Amerindian of South America, Caucasian, Asian, and African populations. In summary, the distribution of the ABCB1, CYP3A5, CYP2C19, and P2RY12 gene polymorphisms distinguishes to the Mexican Mestizos population from other ethnic groups.     

Characterization of CYP1A2, CYP2C19, CYP3A4 and CYP3A5 polymorphisms in South Brazilians

Potential causes of variability in drug response include intrinsic factors such as ethnicity and genetic differences in the expression of enzymes that metabolize drugs, such as those from Cytochrome P450 (CYPs) superfamily. Pharmacogenetic studies search for genetic differences between populations since relevant alleles occur with varying frequencies among different ethnic populations. The Brazilian population is one of the most heterogeneous in the world, resulting from multiethnic admixture of Amerindians, Europeans, and Africans across centuries. Since the knowledge of CYP allele frequency distributions is relevant to pharmacogenetic strategies and these data are scarce in the Brazilian population, this study aimed to describe genotype and allele distributions of 15 single nucleotide polymorphisms (SNPs) at CYP 1A2, 2C19, 3A4, and 3A5 genes in African and European descents from South Brazil. A sample of 179 healthy individuals of European and African ancestry was genotyped by the MassARRAY SNP genotyping system. CYP3A5*3, CYP1A2*1F, CYP3A4*1B, and CYP2C19*2 were the most frequent alleles found in our sample. Significant differences in genotype and allelic distribution between African and European descents were observed for CYP3A4 and CYP3A5 genes. CYP3A4*1B was observed in higher frequency in African descents (0.379) than in European descents (0.098), and European descents showed higher frequency of CYP3A5*3 (0.810) than African descents (0.523). Our results indicate that only a few polymorphisms would have impact in pharmacogenetic testing in South Brazilians. Further studies with larger sample sizes are required also among other Brazilian regions.     

Impact of the CYP3A5, CYP3A4, COMT,IL-10 and POR Genetic Polymorphisms on Tacrolimus Metabolism in Chinese Renal Transplant Recipients

Tacrolimus is a widely used immunosuppressive drug for preventing the rejection of solid organ transplants. The efficacy of tacrolimus shows considerable variability, which might be related to genetic variation among recipients. We conducted a retrospective study of 240 Chinese renal transplant recipients receiving tacrolimus as immunosuppressive drug. The retrospective data of all patients were collected for 40 days after transplantation. Seventeen SNPs of CYP3A5, CYP3A4, COMT, IL-10 and POR were identified by the SNaPshot assay. Tacrolimus blood concentrations were obtained on days 1–3, days 6–8 and days 12–14 after transplantation, as well as during the period of the predefined therapeutic concentration range. Kruskal–Wallis test was used to examine the effect of genetic variation on the tacrolimus concentration/dose ratio (C ₀/D) at different time points. Chi-square test was used to compare the proportions of patients who achieved the target C ₀ range in the different genotypic groups at weeks 1, 2, 3 and 4 after transplantation. After correction for multiple testing, there was a significant association of C ₀/D with CYP3A5*3, CYP3A4*1G and CYP3A4 rs4646437 T>C at different time points after transplantation. The proportion of patients in the IL-10 rs1800871-TT group who achieved the target C ₀ range was greater (p = 0.004) compared to the IL-10 rs1800871-CT and IL-10 rs1800871-CC groups at week 3 after transplantation. CYP3A5*3, CYP3A4 *1G, CYP3A4 rs4646437 T>C and IL-10 rs1800871 C>T might be potential polymorphisms affecting the interindividual variability in tacrolimus metabolism among Chinese renal transplant recipients.     

Olomoucine II,but Not Purvalanol A,Is Transported by Breast Cancer Resistance Protein (ABCG2) and P-Glycoprotein (ABCB1)

Purine cyclin-dependent kinase inhibitors have been recognized as promising candidates for the treatment of various cancers; nevertheless, data regarding interaction of these substances with drug efflux transporters is still lacking. Recently, we have demonstrated inhibition of breast cancer resistance protein (ABCG2) by olomoucine II and purvalanol A and shown that these compounds are able to synergistically potentiate the antiproliferative effect of mitoxantrone, an ABCG2 substrate. In this follow up study, we investigated whether olomoucine II and purvalanol A are transported by ABCG2 and ABCB1 (P-glycoprotein). Using monolayers of MDCKII cells stably expressing human ABCB1 or ABCG2, we demonstrated that olomoucine II, but not purvalanol A, is a dual substrate of both ABCG2 and ABCB1. We, therefore, assume that pharmacokinetics of olomoucine II will be affected by both ABCB1 and ABCG2 transport proteins, which might potentially result in limited accumulation of the compound in tumor tissues or lead to drug-drug interactions. Pharmacokinetic behavior of purvalanol A, on the other hand, does not seem to be affected by either ABCG2 or ABCB1, theoretically favoring this drug in the potential treatment of efflux transporter-based multidrug resistant tumors. In addition, we observed intensive sulfatation of olomoucine II in MDCKII cell lines with subsequent active efflux of the metabolite out of the cells. Therefore, care should be taken when performing pharmacokinetic studies in MDCKII cells, especially if radiolabeled substrates are used; the generated sulfated conjugate may largely contaminate pharmacokinetic analysis and result in misleading interpretation. With regard to chemical structures of olomoucine II and purvalanol A, our data emphasize that even drugs with remarkable structure similarity may show different pharmacokinetic behavior such as interactions with ABC transporters or biotransformation enzymes.     

紫杉醇对食蟹猴和人CYP1A2、CYP3A4及CYP2A6酶代谢的影响

目的探讨紫杉醇对食蟹猴和人肝微粒体CYP1A2、CYP2A6和CYP3A4酶活性的影响。方法采用食蟹猴和人肝脏微粒体,分别以非那西汀、睾丸酮和香豆素分别作为CYP1A2、CYP2A6、CYP3A4的底物,建立CYP1A2、CYP2A6和CYP3A4体外代谢体系。采用不同浓度的紫杉醇分别与上述3种底物共同孵育于肝微粒体代谢体系中。用HPLC法分别测定各底物的代谢产物扑热息痛、6β-羟基睾丸酮、7-羟基香豆素的产生量,计算IC50值,以评估紫杉醇对CYP1A2、CYP2A6和CYP3A4代谢的影响。结果紫杉醇对食蟹猴肝微粒体3种酶的IC50值分别为570±5.9μmol/L、140±2.9μmol/L和无影响;紫杉醇对人肝微粒体3种酶的IC50值分别为193±6.6μmol/L、253±3.6μmol/L和24±1.6μmol/L。结论紫杉醇对食蟹猴肝微粒体CYP1A2和CYP3A4活性具有一定的抑制作用,但对CYP2A6酶的活性几乎没有影响。紫杉醇对人肝微粒体CYP1A2和CYP3A4活性的抑制作用较弱,但对CYP2A6酶的活性抑制作用较强,提示临床上紫杉醇与作为上述酶底物的药物联合用药时应慎重,以避免因中西药物相互作用所导致的不良反应发生。     

Polymorphisms in CYP1B1, CYP3A5, GSTT1, and SULT1A1 Are Associated with Early Age Acute Leukemia

Based on observational studies, early age leukemia (EAL) was associated with maternal hormone exposure during pregnancy. We studied the association between genetic polymorphisms of estrogen metabolism and EAL. Using data from the Brazilian Collaborative Study Group of Infant Acute Leukemia (2000–2012), 350 cases and 404 age-matched controls and 134 mothers of cases and controls were genotyped to explore polymorphisms in genes of the estrogen metabolism pathway: CYP1B1 (c.1294C>G, rs1056836), CYP3A4 (c.-392A>G, rs2740574), CYP3A5 (c.219-237G>A, rs776746), GSTM1/GSTT1 deletions, and SULT1A1 (c.638G>A, rs9282861; and c.667A>G, rs1801030). Logistic regression was used to calculate the odds ratios (OR) with 95% confidence intervals (CIs), and unconditional logistic regression was used to estimate adjusted odds ratios (aORs) by ethnicity. Because of multiple testing, p values < 0.01 were significant after Bonferroni correction. SULT1A1 (c.638G>A) was associated to infant acute lymphoblastic leukemia and acute myeloid leukemia (AML) risk in males (additive model: aOR = 0.52; 95% CI: 0.29–0.95, p = 0.03; dominant model: aOR = 2.18; 95% CI: 1.17–4.05, p = 0.01, respectively). CYP1B1 polymorphism was associated with a decreased risk of AML either for non-white or female children (additive model: OR = 0.24; 95% CI: 0.08–0.76, p < 0.01; additive model: aOR = 0.27; 95% CI: 0.08–0.89, p = 0.03, respectively). Since polymorphisms of Cytochrome P450 genes presented gender-specific risk associations, we also investigated their expression. CYP1B1 was not expressed in 57.1% of EAL cases, and its expression varied by genotype, gender, and leukemia subtype. Maternal-fetal GSTT1 null genotype was associated with risk of EAL. This study shows that polymorphisms in genes of estrogen metabolism confer genetic susceptibility to EAL, mainly in males, and maternal susceptibility genes modify the risk for developing EAL in newborns.     

Allele and genotype frequencies of the polymorphic cytochrome P450 genes (CYP1A1, CYP3A4, CYP3A5, CYP2C9 and CYP2C19) in the Jordanian population

Drug metabolizing enzymes participate in the neutralizing of xenobiotics and biotransformation of drugs. Human cytochrome P450, particularly CYP1A1, CYP2C9, CYP2C19, CYP3A4 and CYP3A5, play an important role in drug metabolism. The genes encoding the CYP enzymes are polymorphic, and extensive data have shown that certain alleles confer reduced enzymatic function. The goal of this study was to determine the frequencies of important allelic variants of CYP1A1, CYP2C9, CYP2C19, CYP3A4 and CYP3A5 in the Jordanian population and compare them with the frequency in other ethnic groups. Genotyping of CYP1A1(m1 and m2), CYP2C9 (*2 and *3), CYP2C19 (*2 and *3), CYP3A4*5, CYP3A5 (*3 and *6), was carried out on Jordanian subjects. Different variants allele were determined using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). CYP1A1 allele frequencies in 290 subjects were 0.764 for CYP1A1*1, 0.165 for CYP1A1*2A and 0.071 for CYP1A1*2C. CYP2C9 allele frequencies in 263 subjects were 0.797 for CYP2C9*1, 0.135 for CYP2C9*2 and 0.068 for CYP2C9*3. For CYP2C19, the frequencies of the wild type (CYP2C19*1) and the nonfunctional (*2 and *3) alleles were 0.877, 0.123 and 0, respectively. Five subjects (3.16?%) were homozygous for *2/*2. Regarding CYP3A4*1B, only 12 subjects out of 173 subjects (6.9?%) were heterozygote with none were mutant for this polymorphism. With respect to CYP3A5, 229 were analyzed, frequencies of CYP3A5*1,*3 and *6 were 0.071, 0.925 and 0.0022, respectively. Comparing our data with that obtained in several Caucasian, African-American and Asian populations, Jordanians are most similar to Caucasians with regard to allelic frequencies of the tested variants of CYP1A1, CYP2C9, CYP2C19, CYP3A4 and CYP3A5.     

Efficient deamination of 5-methylcytosines in DNA by human APOBEC3A, but not by AID or APOBEC3G

The AID/APOBEC family of enzymes in higher vertebrates converts cytosines in DNA or RNA to uracil. They play a role in antibody maturation and innate immunity against viruses, and have also been implicated in the demethylation of DNA during early embryogenesis. This is based in part on reported ability of activation-induced deaminase (AID) to deaminate 5-methylcytosines (5mC) to thymine. We have reexamined this possibility for AID and two members of human APOBEC3 family using a novel genetic system in Escherichia coli. Our results show that while all three genes show strong ability to convert C to U, only APOBEC3A is an efficient deaminator of 5mC. To confirm this, APOBEC3A was purified partially and used in an in vitro deamination assay. We found that APOBEC3A can deaminate 5mC efficiently and this activity is comparable to its C to U deamination activity. When the DNA-binding segment of AID was replaced with the corresponding segment from APOBEC3A, the resulting hybrid had much higher ability to convert 5mC to T in the genetic assay. These and other results suggest that the human AID deaminates 5mC’s only weakly because the 5-methyl group fits poorly in its DNA-binding pocket.     

Influence of CYP3A4, CYP3A5 and MDR-1 polymorphisms on tacrolimus pharmacokinetics and early renal dysfunction in liver transplant recipients

Objectives

Tacrolimus is a widely used immunosuppressive drug in organ transplantation. The oral bioavailability of tacrolimus varies greatly between individuals and depends largely on the activity of both the cytochrome P450 3A (CYP3A) subfamily and P-glycoprotein (P-gp). The possible influence of single nucleotide polymorphisms (SNPs) of CYP3A subfamily and P-gp (MDR-1) in liver transplant recipients has recently been indicated as one of the most important variables affecting the pharmacokinetics of tacrolimus and the renal injury induced by tacrolimus.

Methods

A total of 216 liver transplant recipients were enrolled in this study. The recipients' mean follow-up time was 52 mo (range from 16 to 96 mo). All liver transplant recipients were all in a stable stage with normal serum creatinine (SCr). All liver transplant recipients treated with tacrolimus were genotyped for CYP3A5 (6986A>G), CYP3A4 intron 6 (CYP3A4*22), MDR-1 exon 26 (3435C>T) and exon 12 (1236 C>T) SNPs by HRM analysis (high-resolution melting curve analysis). Recipients were defined as the early renal injury by the elevation of different microproteins in the urine including microalbumin (MA), urine immunoglobulin G (IGU), urine transferrin (TRU) and α1-microglobulin (A1M).

Results

The daily dose of tacrolimus was higher for recipients with CYP3A5*1/*1 (AA) genotype than those with CYP3A5*3/*3 (GG) genotype [3.0 (2.0–4.0) versus 2.0 (1.5–2.5) mg/d, P < 0.05]. The concentration/dose ratio of recipients with CYP3A5*1 homozygotes was lowest compared to recipients with CYP3A5*3/*3 and CYP3A5*1/*3 genotypes. Furthermore, the recipients carrying CYP3A5*3 allele were associated with increased risk of early renal glomerular injury compared to the recipients carrying CYP3A5*1 allele (P = 0.01). MDR-1 polymorphisms were not related with tacrolimus pharmacokinetics and early renal injury.

Conclusion

CYP3A5 6986A>G genetic polymorphism affected daily dose requirements, concentration and nephrotoxicity of tacrolimus. Screening for this single nucleotide polymorphism before the transplantation might be helpful for the selection of adequate initial daily dose and to achieve the desired immunosuppression outcome.     

Impact of ABCB1 and CYP2B6 Genetic Polymorphisms on Methadone Metabolism,Dose and Treatment Response in Patients with Opioid Addiction: A Systematic Review and Meta-Analysis

Background

Genetic variability may influence methadone metabolism, dose requirements, and risk of relapse.

Objectives

To determine whether the CYP2B6*6 or ABCB1 (rs1045642) polymorphisms are associated with variation in methadone response (plasma concentration, dose, or response to treatment).

Methods

Two independent reviewers searched Medline, EMBASE, CINAHL, PsycINFO, and Web of Science databases. We included studies that reported methadone plasma concentration, methadone response, or methadone dose in relation to the CYP2B6*6 or ABCB1 polymorphisms.

Results

We screened 182 articles and extracted 7 articles for inclusion in the meta-analysis. Considerable agreement was observed between the two independent raters on the title (kappa, 0.82), abstract (kappa, 0.43), and full text screening (kappa, 0.43). Trough (R) methadone plasma concentration was significantly higher in CYP2B6*6 homozygous carriers when compared to non-carriers (standardized mean difference [SMD] = 0.53, 95% confidence interval [CI], 0.05–1.00, p = 0.03) with minimal heterogeneity (I² = 0%). Similarly, trough (S) methadone plasma concentration was higher in homozygous carriers of the *6 haplotype when compared to non-carriers, (SMD = 1.44, 95% CI 0.27–2.61, p = 0.02) however significant heterogeneity was observed (I² = 69%). Carriers of the CYP2B6*6 haplotype were not found to be significantly different from non-carriers with respect to dose or response to treatment. We found no significant association between the ABCB1 polymorphism and the trough (R), (S) plasma concentrations, methadone dose, or methadone response.

Conclusion

Although the number of studies included and sample size were modest, this is the first meta analysis to show participants homozygous for the CYP2B6*6 genotype have higher trough (R) and (S) methadone plasma concentrations, suggesting that methadone metabolism is significantly slower in *6 homozygous carriers.     

Induction of cytochrome P450 (CYP)1A1, CYP1A2, and CYP3A4 but not of CYP2C9, CYP2C19, multidrug resistance (MDR-1) and multidrug resistance associated protein (MRP-1) by prototypical inducers in human hepatocytes

Human hepatocytes cultured serum-free for up to 6 weeks were used to study expression and induction of enzymes and membrane transport proteins involved in drug metabolism. Phase I drug metabolizing enzymes cytochrome P450 (CYP)1A1, CYP1A2, CYP2C9, CYP2C19, CYP2E1, and CYP3A4 were detected by Western blot analyses and, when appropriate, by enzymatic assays for ethoxyresorufin-O-deethylase(EROD)-activity and testosterone-6beta-hydroxylase(T6H)-activity. Expression of the membrane transporter multi-drug resistance protein (P-glycoprotein, MDR-1), multidrug resistance-associated protein (MRP-1), and lung-resistance protein (LRP) was maintained during the culture as detected by RT-PCR and Western blot analyses. Model inducers like rifampicin, phenobarbital, or 3-methylcholanthrene and beta-naphtoflavone were able to induce CYP1A or CYP3A4 as well as EROD or T6H activities for up to 30 days. CYP2C9, CYP2C19 and CYP2E1 expression was maintained but not inducible for 48 days. Also, rifampicin and phenobarbital were unable to increase MDR-1 and MRP-1 protein levels significantly.     

Granuloma Formation and Host Defense in Chronic Mycobacterium tuberculosis Infection Requires PYCARD/ASC but Not NLRP3 or Caspase-1

The NLR gene family mediates host immunity to various acute pathogenic stimuli, but its role in chronic infection is not known. This paper addressed the role of NLRP3 (NALP3), its adaptor protein PYCARD (ASC), and caspase-1 during infection with Mycobacterium tuberculosis (Mtb). Mtb infection of macrophages in culture induced IL-1β secretion, and this requires the inflammasome components PYCARD, caspase-1, and NLRP3. However, in vivo Mtb aerosol infection of Nlrp3^−/−, Casp-1^−/−, and WT mice showed no differences in pulmonary IL-1β production, bacterial burden, or long-term survival. In contrast, a significant role was observed for Pycard in host protection during chronic Mtb infection, as shown by an abrupt decrease in survival of Pycard^−/− mice. Decreased survival of Pycard^−/− animals was associated with defective granuloma formation. These data demonstrate that PYCARD exerts a novel inflammasome-independent role during chronic Mtb infection by containing the bacteria in granulomas.     

CYP 3A proteins are expressed in human neutrophils and lymphocytes but are not induced by rifampicin

Cytochrome P-450 3A (CYP 3A) enzymes, the prominent subfamily in the cytochrome system, are expressed in various extrahepatic tissues. Until now, their expression has been demonstrated in human polymorphic neutrophils (PMNs) but not in lymphocytes using immunohistochemistry and immunoblot analysis. Moreover, their potential modulation has not been determined yet. To study such an expression in different peripheral blood cell populations, rifampicin (600 mg/day during 6 days) was given to 8 healthy subjects. PMNs and lymphocytes were isolated by centrifugation of whole white blood cell fractions using Ficoll gradients before drug administration, immediately after, and 3 days after drug withdrawal. PMN and lymphocyte smears and homogenates were subjected to immunostaining and immunoblotting, respectively, with a mouse monoclonal antibody recognizing all CYP 3A proteins. These proteins were quantified by densitometric analysis. Before and after rifampicin administration, a positive cytoplasmic staining was observed in all PMNs and in about 50% of lymphocytes. CYP 3A expression in lymphocytes was further confirmed by positive immunoblots for lymphocyte homogenates. Neither in PMNs nor in lymphocytes, induction of CYP 3A protein expression was observed after rifampicin treatment despite overall induction of CYP 3A activity assessed by the urinary excretion of 6beta-hydroxycortisol. These results demonstrate that CYP 3A proteins are constitutively expressed not only in PMNs but also in lymphocytes. However, in both cell lineages CYP 3A protein expression was not induced by rifampicin.     

Inhibition of CYP450 1A and 3A by berberine in crucian carp Carassius auratus gibelio

Berberine has long been considered as an antibiotic candidate in aquaculture. However, studies regarding its effects on drug-metabolizing enzymes in fish are still limited. In the present study, the effects of berberine on cytochrome P4501A (CYP1A) and CYP3A in crucian carp were investigated. Injection of different concentrations of berberine (0, 5, 25, 50, and 100 mg/kg) inhibited the CYP1A mRNA expression, thereby inhibiting further the catalytic activity of CYP1A-related ethoxyresorufin-O-deethylase (EROD). Furthermore, both CYP1A expression and EROD activity were further inhibited with increasing berberine concentrations. In addition, the CYP3A expressions at both the mRNA and the protein levels were downregulated by higher berberine concentrations. The catalytic activity of CYP3A-related erythromycin N-demethylase (ERND) was also inhibited by berberine at a dose of no less than 25 mg/kg. Moreover, at the berberine concentration exceeding 25 mg/kg, the inhibition of CYP3A expression and ERND activity increased with increasing berberine concentrations. In vitro experiments were also performed. When berberine was pre-incubated with the crucian carp liver microsomes, it competitively inhibited the corresponding EROD activity with the IC₅₀ of 11.7 μM. However, the ERND activity was slightly inhibited by berberine with the IC₅₀ of 206.4 μM. These results suggest that, in crucian carp, berberine may be a potent inhibitor to CYP1A, whereas the CYP3A inhibition needs a higher concentration of berberine.