Yoshiro Saito

Clinical Pharmacogenetics Implementation Consortium (CPIC) guidelines for human leukocyte antigen B (HLA-B) genotype and allopurinol dosing: 2015 update.

The Clinical Pharmacogenetics Implementation Consortium (CPIC) Guidelines for HLA‐B*58:01 Genotype and Allopurinol Dosing was originally published in February 2013. We reviewed the recent literature and concluded that none of the evidence would change the therapeutic recommendations in the original guideline; therefore, the original publication remains clinically current. However, we have updated the Supplemental Material and included additional resources for applying CPIC guidelines into the electronic health record. Up‐to‐date information can be found at PharmGKB (http://www.pharmgkb.org).

Ligand-induced internalization and phosphorylation-dependent degradation of growth hormone receptor in human IM-9 cells

The human growth hormone (hGH) induced a marked reduction in the number of human growth hormone receptors (hGHR) within 60 min, as assessed by immunoblotting of the crude membrane fraction from human IM-9 cells, without an increase in soluble forms of hGHR. The disappearance of hGH-induced hGHR was markedly inhibited by reagents that raise the internal pH of acidic organella and partially by protease inhibitors. These results suggest that hGH stimulation results in degradation of internalized hGHRs, where proteases in acidic compartments such as lysosomes may be involved. The relationship between the hGH concentration and the number of residual cell surface hGHRs 60 min after hGH stimulation yielded a curve with an inverted bell shape showing maximum internalization at 10 nM hGH. A similar relationship was shown in the hGHR degradation. The fact that the ligands in excess gave reduced internalization and degradation supports the idea that dimerization of hGHRs on the cell surface through the bivalent ligand hGH is required for their internalization and subsequent degradation. Following hGH stimulation, several hGHR-associated proteins including JAK2 were phosphorylated. These phosphorylations were inhibited by pretreatment with a protein kinase inhibitor, staurosporine. The hGHR internalization, however, was not markedly affected by the inhibitor. In contrast, the staurosporine inhibited the degradation of hGHR in a dose-dependent manner. These results suggest that staurosporine-sensitive phosphorylation is not required for the hGHR internalization, but the phosphorylation is involved in the degradation of hGHR.

Activation of protein kinase Cα enhances human growth hormone-binding protein release

Abstract The effect of phorbol ester on human growth hormone-binding protein (hGH-BP) release was investigated. The hGH-BP release from human IM-9 cells measured by immunoblotting was dose-dependently enhanced by a phorbol ester, phorbol 12, 13-dibutyrate (PDBu), and reached plateau at 100 nM. The increased hGH-BP release was shown after 10 min incubation with PDBu and reached a plateau at 60 min after stimulation. Similarly, a diacylglycerol analogue, 1-oleoyl-2-acetyl-sn-glycerol, enhanced hGH-BP release. The enhancement was not inhibited by cycloheximide pretreatment, suggesting that the enhanced hGH-BP release does not require de novo protein synthesis. The PDBu-enhanced hGH-BP release was strongly inhibited by extracellular EDTA, and was dose-dependently inhibited by protein kinase C (PKC)-specific inhibitor, Ro 31-8220. These results suggest that activation of PKC mediates the PDBu-enhanced hGH-BP release. Of the 11 known PKC isoforms in human cells, PKCα, δ, μ and ι were detected in IM-9 cells by immunoblotting. Of these isoforms, PKCα, δ and μ were present in the membrane fraction, which is a known activation marker of PKC. Furthermore, when several PKC-specific inhibitors (Go 6976, GF 109203X or bisindolylmaleimide III) with different specificities for each isoform were used, there was a good correlation between inhibition of the enhancement of hGH-BP release and inhibition of the phosphorylation of PKC isoforms, another activation marker of PKC, in PKCα but not in PKCδ and μ. These results suggest that activation of PKCα is involved in PDBu-enhanced hGH-BP release.

Genetic variations of orosomucoid genes associated with serum alpha‐1‐acid glycoprotein level and the pharmacokinetics of paclitaxel in Japanese cancer

Alpha-1-acid glycoprotein (AGP) encoded by orosomucoid genes (ORM1 and ORM2) is an acute-phase response protein and functions as a drug-binding protein that affects pharmacokinetics (PK)/pharmacodynamics of binding drugs. To explore the effects of genetic variations of ORMs and a role of AGP on paclitaxel (PTX) therapy, we analyzed the duplication and genetic variations/haplotypes of ORMs in 165 Japanese cancer patients and then investigated their associations with serum AGP levels and the PK parameters of PTX. No effects of ORM duplications on serum AGP levels at baseline or PK of PTX were observed, but close associations of ORM1 -559T > A with the increases of AGP levels and area under the curve (AUC) of PTX metabolites were detected. In addition, a significant correlation between the serum AGP level and the AUCs of PTX metabolites was observed, suggesting that AGP may function as a carrier of PTX from the blood into the liver via putative receptors. This study provided useful information on the possible clinical importance of ORM genetic polymorphisms and a novel role of AGP in PTX therapy.

CASEIN KINASE II-LIKE ECTOKINASE ACTIVITY ON RBL-2H3 CELLS

We studied the properties of the ectokinase activity on the outer cell surfaces of RBL-2H3 cells and examined the phosphorylation of exogenous substrates to clarify the substrate specificity of the ectokinases on RBL-2H3 cells. Among the several protein substrates tested, casein was the most strongly phosphorylated with [gamma-32P]ATP, and the net incorporation of 32P into casein was 0.65 pmol P/50 microg/10(6) cells. Casein kinase II peptide was also phosphorylated with [gamma-32P]ATP. The phosphorylation of casein and casein kinase II peptide was almost completely inhibited by the addition of 3 microg/ml of cell-impermeable K252b. Phosphorylation of casein and casein kinase II peptide was also observed by [gamma-32P]GTP. Western blot analysis using anti-casein kinase II antibody revealed a 44-kDa casein kinase band in the membrane fraction and Fc epsilonRI complexes. The immunofluorescence microscopic analysis using anti-casein kinase II antibody showed the existence of casein kinase II on the surface of the cells. This is the first report about the existence of ectokinase on mast cells.

Proteasome inhibitor enhances growth hormone-binding protein release.

We used murine Ba/F3 cells transfected with human growth hormone receptor (hGHR) cDNA to investigate the regulatory mechanisms of human growth hormone-binding protein (hGH-BP) release. The extracellular domain of hGHRs were cleaved and released as hGH-BPs (a soluble form of hGHR). The hGH-BP release was enhanced by phorbol 12,13-dibutyrate (PDBu), and suggested to be mediated by activation of PKC, the same as in human IM-9 cells. Thus, Ba/F3 cells have hGH-BP-releasing pathways similar to those of human cells. The proteasome inhibitors MG-132 and clasto-lactacystin beta-lactone also increased hGH-BP release from Ba/F3-hGHR cells, and MG-132 and PDBu synergistically increased hGH-BP release. The results obtained by using three PKC inhibitors Gö 6976, GF 109203X and Gö 6983 suggest that the enhancement of hGH-BP release by MG-132 and PDBu is mediated by different mechanisms probably involving different PKC isozymes.

Cross-Classification of Human Urinary Lipidome by Sex, Age, and Body Mass Index

Technological advancements in past decades have led to the development of integrative analytical approaches to lipidomics, such as liquid chromatography-mass spectrometry (LC/MS), and information about biogenic lipids is rapidly accumulating. Although several cohort-based studies have been conducted on the composition of urinary lipidome, the data on urinary lipids cross-classified by sex, age, and body mass index (BMI) are insufficient to screen for various abnormalities. To promote the development of urinary lipid metabolome-based diagnostic assay, we analyzed 60 urine samples from healthy white adults (young (c.a., 30 years) and old (c.a., 60 years) men/women) using LC/MS. Women had a higher urinary concentration of omega-3 12-lipoxygenase (LOX)-generated oxylipins with anti-inflammatory activity compared to men. In addition, young women showed increased abundance of poly-unsaturated fatty acids (PUFAs) and cytochrome P450 (P450)-produced oxylipins with anti-hypertensive activity compared with young men, whereas elderly women exhibited higher concentration of 5-LOX-generated anti-inflammatory oxylipins than elderly men. There were no significant differences in urinary oxylipin levels between young and old subjects or between subjects with low and high BMI. Our findings suggest that sex, but neither ages nor BMI could be a confounding factor for measuring the composition of urinary lipid metabolites in the healthy population. The information showed contribute to the development of reliable biomarker findings from urine.

Predictive genomic markers for severe adverse drug reactions

Severe adverse drug reactions are an important issue to be considered during proper drug usage in postmarketing period. Most severe adverse reactions are idiosyncratic and unrelated to their pharmacological actions via primary targets. Although these reactions were not predictable, recent developments in the field of genomics have revealed closely associated markers responsible for some severe adverse reactions, including Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN). This review demonstrates genomic biomarkers for SJS/TEN and drug-induced liver injury (DILI) that were found mainly in Japanese patients and reveal ethnic differences. We and other groups have found the following associations of SJS/TEN with susceptible drugs: 1) HLA-B*58:01 for allopurinol-related cases; 2) HLA-B*15:11 and HLA-A*31:01 for carbamazepine-related cases; 3) HLA-B*51:01 for phenobarbital-related cases; 4) HLA-A*02:07 for zonisamide-related cases; 5) CYP2C9*3 for phenytoin-related cases; and 6) HLA-A*02:06 for cold medicine-related cases. The allele frequencies of these related HLA types vary among Asian populations. In addition, direct (noncovalent) binding of carbamazepine or an allopurinol metabolite, oxypurinol, to the associated HLA-type proteins was suggested. Associated genomic biomarkers are also summarized for DILI in Japanese and Caucasian populations. The application of these genomic biomarkers to prevent the onset of a reaction has been utilized in a few countries. However, in Japan, the package inserts only contain precautions that cite the research findings. To overcome this limitation, the following points should be addressed: 1) factors responsible for the development of SJS/TEN should be identified in addition to the above-mentioned HLA alleles; and 2) an inexpensive genotyping strategy and assay methods should be developed to provide a pharmacoeconomical viewpoint. Further research on severe adverse reactions is warranted.

Development of a simple genotyping method for the HLA-A*31:01-tagging SNP in Japanese

AIM To construct a simple, low-cost typing method for the surrogate marker of HLA-A*31:01, a risk factor for carbamazepine (CBZ) related Stevens-Johnson syndrome/toxic epidermal necrolysis (SJS/TEN). MATERIALS & METHODS DNAs from Japanese SJS/TEN patients were used for genotyping and developing the assay. RESULTS HLA-A*31:01 was confirmed to be significantly associated with definite/probable cases of CBZ-related SJS/TEN (p = 0.0040). Three single nucleotide polymorphisms, rs1150738, rs3869066 and rs259945, were in absolute linkage disequilibrium with HLA-A*31:01 in 210 Japanese SJS/TEN patients. Robust genotyping of rs3869066 in ZNRD1-AS1 was developed using polymerase chain reaction-restriction fragment length polymorphism assays. CONCLUSION Single nucleotide polymorphism genotyping is less time consuming and cheaper than conventional HLA typing, and would be useful for identifying Japanese patients at risk of CBZ-related SJS/TEN.

Genetic polymorphisms of FCGR2A encoding Fcγ receptor IIa in a Japanese population and functional analysis of the L273P variant

Fcγ receptor IIa (FcγRIIa) plays an important role in antibody-dependent cellular cytotoxicity and inflammation. Changes in FcγRIIa expression levels or activity caused by genetic polymorphisms in FCGR2A, the gene encoding FcγRIIa, may lead to differences in disease progression as well as efficacy of antibody therapeutics between individuals. In this study, we sequenced the 5′-flanking region along with all exons and their flanking regions of FCGR2A from 111 Japanese subjects. Forty-eight genetic variations were found including 12 novel ones. Beside the well-known functional 497A > G (H166R) polymorphism, we detected 818T > C (L273P) at 0.005 frequency. Since the functional significance of this polymorphism has not been revealed, we next assessed the functions of the L273P substitution by expressing wild-type and the variant proteins in human Jurkat cells. The L273P variant protein showed similar cell surface expression and IgG-binding properties as the wild-type, but it exhibited a stronger signal transduction activity based on the approximately 2-fold enhancement of tyrosine phosphorylation of FcγRIIa itself. The current results suggest that L273P could have functional significance in the antibody-dependent clinical responses through FcγRIIa.