Functional characterization of seven single-nucleotide polymorphisms of the steroid sulfatase gene found in a Japanese population.

Steroid sulfatase (STS) is an enzyme that hydrolyzes steroid sulfates such as dehydroepiandrosterone sulfate (DHEA-S) and estrone sulfate. STS has a key role in the synthesis of steroid hormones in placenta and breast cancer cells. Recently, we have identified six novel single-nucleotide polymorphisms (SNPs) and one nonsynonymous SNP (V476M) in the STS gene in a Japanese population. To clarify the effects of SNPs in the 5'-flanking region or 5' untranslated region on transcriptional activity, a reporter gene assay was conducted. In addition, DHEA-S desulfatase activity of a variant (Met at codon 476)-type enzyme was compared with that of the wild (Wd)-type enzyme in COS-1 cells. The transcriptional activities were significantly decreased (155A) and increased (-2837A and -1588C) in MCF-7 cells. On the other hand, no significant difference was found in expression levels of STS protein or specific activities of DHEA-S desulfation between Wd and the variant enzymes. This is the first report on the effects of various SNPs in the STS gene detected in Japanese healthy subjects.

Carbamoyl-PROXYL-enhanced MRI detects very small disruptions in brain vascular permeability induced by dietary cholesterol.

BACKGROUND: Gd-DTPA-enhanced magnetic resonance imaging (MRI) is a conventional method for non-invasive investigation of blood-brain-barrier (BBB) permeability in animal models. It allows the visualization of serious injury to the BBB. We developed a novel approach for detecting very small disruptions in BBB permeability induced by dietary cholesterol by using carbamoyl-PROXYL (CMP) as an MRI contrast probe. METHODS: Mice were separated into two groups: normal diet (ND-mice) and high cholesterol diet (CD-mice). MRI-signal dynamics, plasma cholesterol, matrix metalloproteinase (MMP-9, MMP-2), and the white blood cell profile were analyzed. For the MRI analysis, two regions-of-interest (ROI) were selected: brain (ROI-1) and surrounding area (ROI-2). RESULTS: In the ROI-2 of ND-mice, CMP- or Gd-enhanced MRI-signal followed typical kinetics with a half-life of signal decay (τ(1/2)) approximately 8 or approximately 15 min, respectively. In CD-mice, the MRI-signal increased continuously without decay. In the ROI-1 of ND- and CD-mice, MRI-signal enhancement was not detected by Gd-DTPA. In the ROI-1 of ND-mice, CMP-induced MRI-signal enhancement was negligible, while in CD-mice, it was significant (τ(1/2)>15 min). Hypercholesterolemia increased the plasma levels of MMP-9 and neutrophils. CONCLUSIONS: Hypercholesterolemia increases vascular permeability, which is mediated by MMP-9 and neutrophils. GENERAL SIGNIFICANCE: Even very small disruptions in brain vascular permeability could be detected by CMP-enhanced MRI but not by Gd-DTPA-enhanced MRI.

Correlation between antizyme 1 and differentiation of vascular smooth muscle cells cultured in honeycomb-like type-I collagen matrix.

Vascular smooth muscle cells (SMC) are able to proliferate when cultured on plates, but become differentiated when maintained in three-dimensional type I collagen matrices (honeycombs). SMC grown in honeycombs contained a low level of polyamines due to the presence of antizyme 1 (AZ1), a negative regulator of ornithine decarboxylase (ODC) and of polyamine uptake. To clarify the role of AZ1 in differentiation of SMC in honeycombs, an ODC gene was stably transfected into SMC (ODC-SMC). Although proliferation of ODC-SMC on plates was accelerated together with an increase in phosphorylated focal adhesion kinase (FAK) and a decrease in α-actin and myosin, maker proteins of differentiation, growth of ODC-SMC ceased in honeycombs similarly to normal SMC with a low level of phosphorylated FAK and a high level of α-actin and myosin. AZ1 expression in ODC-SMC on plates was low, but that in honeycombs was high. Antizyme in ODC-SMC in honeycombs not only decreased the level of ODC but also inhibited polyamine uptake activity. These results taken together suggest that low levels of polyamines caused by AZ1 in SMC in honeycombs inhibit phosphorylation of FAK and enhance expression of α-actin and myosin, resulting in differentiation through inhibition of focal adhesions.

Degradation of filamin induces contraction of vascular smooth muscle cells in type-I collagen matrix honeycombs.

BACKGROUND: Dedifferentiated rabbit vascular smooth muscle cells (SMCs) exhibit similar features to differentiated SMCs when cultured in three-dimensional matrices of type-I collagen called "honeycombs," but the mechanism is unknown. The role of filamin, an actin-binding protein that links actin filaments in SMCs, was investigated. METHODS: Filamin and other related proteins were detected by western blot analysis and immunofluorescence staining. Honeycomb size was measured to confirm the contraction of SMCs. RESULTS: Full-length filamin was expressed in subconfluent SMCs cultured on plates; however, degradation of filamin, which might be regulated by calpain, was observed in confluent SMCs cultured on plates and in honeycombs. While filamin was co-localized with ß-actin in subconfluent SMCs grown on plates, filamin was detected in the cytoplasm in SMCs cultured in honeycombs, and degraded filamin was mainly detected in the cytoplasmic fraction of these cells. In addition, ß-actin expression was low in the cytoskeletal fraction of SMCs cultured in honeycombs compared with cells cultured on plates, and the size of the honeycombs used for culturing SMCs was significantly reduced. CONCLUSION: These data suggest that degradation of filamin in SMCs cultured in honeycombs induces structural weakness of ß-non-muscle actin filaments, thereby permitting SMCs in honeycombs to achieve contractility.

Q172H replacement overcomes effects on the metabolism of cyclophosphamide and efavirenz caused by CYP2B6 variant with Arg262.

There are a number of reports indicating that CYP2B6*6 (c.516G>T and c.785A>G) is responsible for decreased clearance of efavirenz (EFV), although increased disposition of cyclophosphamide (CPA) in individuals with this polymorphism was observed. Thus, we hypothesized that the effects of the two single nucleotide polymorphisms (SNPs) of CYP2B6*6 on the metabolism of drugs might be considerably different between these two agents. To clarify this possibility, we expressed two major variants of this enzyme, CYP2B6.6 (Q172H and K262R) and CYP2B6.4 (K262R), and investigated metabolic activities of these variants toward EFV and CPA. Kinetic analyses clearly indicated that CYP2B6.4 possessed enhanced metabolic activity toward EFV compared with that of the wild-type enzyme (CYP2B6.1), whereas CPA was metabolized less efficiently by CYP2B6.4 than by CYP2B6.1. On the other hand, CYP2B6.6 showed a completely opposite character, suggesting that Q172H gives inverse effects on metabolic activities of CYP2B6 affected by K262R. Although it is recognized that effects of amino acid change in cytochrome P450 on the metabolic activity depend on substrates, this study revealed SNPs giving an opposite effect on the metabolism of two clinically important drugs currently used. Furthermore, this study provides the first evidence that Q172H can reverse the direction of the effect caused by K262R in CYP2B6 on the metabolism of certain drugs.

Nitroxide derivatives for imaging of hypercholesterolemia-induced kidney dysfunction and assessing the effectiveness of antilipidemic drugs.

The present study was designed to clarify the possibility for application of nitroxide derivatives in magnetic resonance imaging (MRI) of hypercholesterolemia-mediated renal dysfunction in mice, as well as to assess the effectiveness of antilipidemic drugs (cholestyramine and ezetimibe). The mice were separated in four groups: (i) on a normal diet (ND) without medication (control); (ii) on a high cholesterol diet (CD) without medication; (iii) CD mice receiving cholestyramine; and (iv) CD mice receiving ezetimibe. In CD mice without medication, a hypercholesterolemia was developed, detected by the increasing of total plasma cholesterol and non-HDL cholesterol, and decreasing of HDL cholesterol. The hypercholesterolemia compromised renal function: blood urea nitrogen, creatine and uric acid increased significantly, accompanied with development of glomerulosclerosis, enhancement of the amount of neutrophils and overexpression of metalloproteinase-9. The mice were subjected to anesthesia and MR imaging was performed on 7 T magnet (T1-weighted incoherent gradient-echo sequence; fast low-angle shot). The region-of-interest was selected within the kidney. The images were obtained before and after injection of contrast probe [carbamoyl-PROXYL (CMP) or Gd-DTPA]. In the kidney of ND mice, the MRI signal intensity increased after injection of CMP, reached a maximum (very well-defined renal filtration peak) and decreased to the baseline level within 14 min. In kidney of CD mice, the CMP-mediated enhancement of MRI signal was not detected. Antilipidemic drugs patially abolished the effect of hypercholesterolemia on CMP-enhanced MRI in the kidney. The kinetic curves of Gd-enhanced MRI signal had also different profiles in the kidney of ND and CD mice. They were similar to the profiles of the kinetic curves, obtained from MR urography of healthy human and human with renal pathology, respectively. The present study suggests that CMP is a suitable MRI contrast probe for visualization of hypercholesterolemia-induced renal dysfunction in intact animals and the assessment of the efficacy of antilipidemic drugs. The probe was applied at a concentration that was 3 times lower than the LD50 for intravenous administration in mice. Since the probe is excreted by the kidney, it could be considered harmless for mammalians in the selected dose and appropriate candidate for translational research.

Growth inhibition and differentiation of cultured smooth muscle cells depend on cellular crossbridges across the tubular lumen of type I collagen matrix honeycombs.

Although rabbit vascular smooth muscle cells (SMCs) showed a differentiated phenotype in three-dimensional type I collagen matrices (honeycombs, diameter of pores=200-500 microm), mouse vascular SMCs proliferated in honeycombs having the same pore size. Here we investigated the relationship between pore sizes of honeycombs and differentiation of SMCs using various pore sizes of honeycombs. Rabbit SMCs (length: 200+/-32 microm) and mouse SMCs (49+/-10 microm) formed crossbridges in honeycombs with 200-300 microm and less than 200 microm of pores, respectively. Both SMCs spread on the inner wall but did not form crossbridges in honeycombs with larger pores. [(3)H]Thymidine incorporation and cell number of both SMCs were decreased when the crossbridges were formed in honeycombs. Because proliferation inhibition and crossbridge formation were observed in the culture of rabbit and mouse SMCs using 200-300 microm and less than 200 microm pore sized honeycombs, respectively, these data suggested that forming crossbridges was important for the inhibition of proliferation of SMCs. Rabbit SMCs differentiation was accompanied by the expression of caldesmon heavy chain when cultured in honeycombs having less than 300 microm pores. Proliferation of mouse SMCs stopped in honeycombs having less than 200 microm pores, but caldesmon heavy chain was not detected despite the expression of its mRNA. Proliferation of SMCs stopped on plates when cells reached confluent state, however, caldesmon heavy chain was not expressed. These data suggested that an appropriate structure and suitable honeycomb pore size are important for the differentiation of SMCs.

Theoretical calculation of triazolam hydroxylation and endogenous steroid inhibition in the active site of CYP3A4.

CYP3A4 has unusual kinetic characteristics because it has a large active site. CYP3A4 produced more 4-hydroxytriazolam than alpha-hydroxytriazolam at concentrations of more than 60 muM triazolam, and different steroids had different inhibitory effects on the system. To clarify these interesting observations, the interactions between substrate and substrate/steroid were investigated by theoretical calculations. When two triazolam molecules were docked into the active site, the distance between the O-atom and the 4-hydroxylated site was less than the distance to the alpha-hydroxylated site because of interaction between the two triazolam molecules. Estradiol inhibited both alpha- and 4-hydroxytriazolam formation by 50%. Dehydroepiandrosterone (DHEA) inhibited alpha-hydroxylation more than 4-hydroxytriazolam formation, whereas aldosterone had no effect. When one triazolam molecule and one steroid molecule were simultaneously docked, estradiol increased the distance between the O-atom and the two hydroxylated sites, DHEA only increased the distance between the O-atom and alpha-hydroxylated site, and aldosterone did not change the distances. The relevant angles of Fe-O-C in the hydroxylated site of triazolam also widened, together with increased distance. These findings indicate that formation of a substrate and substrate/effector complex in the active site may be a factor for determining the enzyme kinetic parameters of CYP3A4.

Probable interaction between warfarin and antitumor agents used in R-ESHAP chemotherapy.

BACKGROUND: The pharmacologic effects of warfarin might be altered by various factors, including drug-drug interaction. CASE SUMMARY: A 49-year-old Japanese man (height, 174 cm; weight, 68 kg) presented with a 20-month history of malignant lymphoma (diffuse large B cell lymphoma, clinical stage IV). He was treated with a combination of rituximab chemotherapy and etoposide, cisplatin, high-dose cytarabine, and methyl-prednisolone (R-ESHAP). He had been receiving warfarin for the secondary prevention of pulmonary embolism with deep venous thrombosis. When R-ESHAP was started, international normalized ratio (INR) increased from 1 to 5. This phenomenon was observed again in the second R-ESHAP. The INR was increased from 2.44 to 4.71 during chemotherapy but was returned to within the normal range (1.05; normal range: 0.81-1.009) 5 days after chemotherapy was completed. CONCLUSION: In this patient, R-ESHAP chemotherapy might have affected warfarin anticoagulation sensitivity; thus, careful monitoring of INR is essential, particularly in patients receiving warfarin who undergo R-ESHAP chemotherapy.

An epoxidation mechanism of carbamazepine by CYP3A4.

Human CYP3A4 catalyzes the 10,11-epoxidation of carbamazepine (CBZ). However, the epoxide is less stable in terms of potential energy than hydroxides of the six-membered aromatic ring. To clarify the reason why CYP3A4 produces such an energetically unfavorable compound, the mechanism of epoxidation of CBZ by CYP3A4 was investigated by theoretical calculations. The reaction consisted of two elementary processes in which two C-O bonds were generated stepwise. The rate-determining step was the first one and the activation energy was 21.3kcal/mol at the DFT (B3LYP/6-31G( * *)) level. The activation energy level of the first step of the 10,11-epoxidation was lower than that of the hydroxylation of the aromatic ring. For this reason, 10,11-epoxidation is more probable than hydroxylation of the aromatic ring, and only 10,11-epoxide is formed.

[Effect of pitavastatin on macrophage cholesterol metabolism].

OBJECTIVES: Pitavastatin is the first totally synthetic HMG-Co A reductase inhibitor in Japan that significantly reduces LDL cholesterol while raising HDL cholesterol. Clinical trial showed that pitavastatin has potent effects for LDL cholesterol lowering and is expected effectively to prevent atherosclerosis. To clarify the mechanism of reduction of atherosclerosis by pitavastatin, we examined the effect of pitavastatin on foam cell formation of RAW264.7 macrophages. METHODS & RESULTS: Macrophages were cultured with pitavastatin for 24 h and exposed to oxidized LDL with pitavastatin for 3 days. Pitavastatin decreased the cellular cholesteryl ester content in a dose-dependent manner, and this effect was not via inhibition of HMG-CoA reductase because the 3-30 nM pitavastatin did not inhibit [14C]cholesterol synthesis from [14C]acetic acid and the effect was not influenced by addition of mevalonic acid. Pitavastatin increased neutral cholesterol esterase (NCEase) activity and did not affect ACAT activity, and decreased the expression of CD36 and ABCA1 mRNA. The mechanism of the increase of NCEase activity was that pitavastatin directly modified the substrate state, which was cholesterol oleate emulsified with lecithin. CONCLUSION: Clinical blood concentrations of pitavastatin prevent foam cell formation of RAW macrophages by oxidized LDL, and this was not via inhibition of HMG-CoA reductase, and modify substrate condition.

[Appropriate use of anti-methicillin-resistant Staphylococcus aureus agents in a retrospective study].

The track records of the use of anti-methicillin-resistant Staphylococcus aureus agents (anti-MRSA agents) in a 5-year period (2001.4-2006.3) were collected, and cases in which anti-MRSA agents were used for >4 days were selected. In each case, the results of laboratory data and bacterial examination before and after administering the anti-MRSA agents were investigated retrospectively. In addition, it was also investigated in each case whether therapeutic drug monitoring (TDM) was carried out. It was observed that the number of patients treated with anti-MRSA agents and the total dose of anti-MRSA agents used tended to increase over time, except for arbekacin sulfate. It was, however, shown that treatment with anti-MRSA agents resulted in significant decreases in body temperature, C-reactive protein, and white blood cell counts. Bacterial examination was conducted in 75.6% of the patients treated with anti-MRSA agents, with MRSA being detected in 72.4% of the cases examined. On the other hand, TDM was also conducted in 60% of the cases, but this was at a lower percentage than that of the other examinations. Quantitative bacterial examination after treatment with anti-MRSA agents indicates that TDM can be considered important for the appropriate use of anti-MRSA agents.

Identification and partial characterization of a novel CYP2C9 splicing variant encoding a protein lacking eight amino acid residues.

CYP2C9 is known as an enzyme responsible for the metabolism of various clinically important drugs. Recently, we cloned a cDNA corresponding to a CYP2C9 splicing variant (SV), which seemed to have an open reading frame of a protein with 482 amino acid residues. To investigate whether or not the SV can be translated as a functionally active protein, we expressed the CYP2C9SV in insect cells, and spectrophotometric and enzymatic properties were characterized. The CYP2C9SV protein showed a typical reduced CO-difference spectrum, indicating that the translated protein binds a heme moiety. However, CYP2C9SV did not metabolize tolbutamide or diclofenac at all, suggesting that the SV protein appeared to lack the ability to catalyze reactions mediated by CYP2C9. Although the CYP2C9SV mRNA was detected in all human liver samples examined in this study by real-time PCR, the level was generally low, ranging between 0.7 and 9.6% of the normal CYP2C9 mRNA. These results suggest that the CYP2C9SV protein is unlikely to contribute to CYP2C9 activities, although it appears to be expressed in most individuals.

Current opinion: safety evaluation of drug metabolites in development of pharmaceuticals.

Safety assessment of drug metabolites in the development of pharmaceuticals was discussed in January 2007 at the kick-off meeting of a "Drug Evaluation Forum", with reference to the views of clinicians and other academic representatives. Safety evaluation of metabolites cannot readily be based on a single theoretical framework, and basically a case-by-case approach is called for. These evaluations should be performed precisely and an accurate profile secured taking into account adverse reactions that are unpredictable from the parent compound administered in clinical studies and any signs or symptoms that may be associated with the metabolites. In addition, elimination of scientifically meaningless metabolite safety assessment studies is essential for prompt supply of high-quality drugs to the medical frontline. Preparation of an outline concept paper would be useful for achievement of shared understanding of issues of this type. Collective viewpoints obtained in this fashion are also relevant to the discussion on the need for guidance, and given a degree of flexibility may also be helpful for drug development and, in turn, society at large.

Relationship between Kaposi's varicelliform eruption in Japanese patients with atopic dermatitis treated with tacrolimus ointment and genetic polymorphisms in the IL-18 gene promoter region.

Kaposi's varicelliform eruption is the most important problem in treating patients with atopic dermatitis (AD) with tacrolimus ointment. It has been considered that Kaposi's varicelliform eruption occurs due to decreased levels of interleukin (IL)-18. The aim of this study was to examine the relationship between Kaposi's varicelliform eruption and genetic polymorphisms in the IL-18 gene. IL-18 gene promoter polymorphisms were analyzed in 21 AD patients treated with tacrolimus ointment and in 100 healthy volunteers. Six AD patients with Kaposi's varicelliform eruption during the treatment with tacrolimus ointment showed significantly higher frequency in G-to-C mutations at the IL-18 gene promoter region -137 compared with 15 AD patients without Kaposi's varicelliform eruption. The 15 AD patients without Kaposi's varicelliform eruption as well as 100 healthy volunteers did not have mutations of G-to-C at the IL-18 gene promoter region -137. These results suggest that the onset of Kaposi's varicelliform eruption following the treatment with tacrolimus ointment is associated with the mutation of G-to-C in the IL-18 gene promoter region -137, and that caution is required when using tacrolimus ointment for treating AD patients with this mutation.

Intimal smooth muscle cells up-regulate beta-very low density lipoprotein-mediated cholesterol accumulation by enhancing beta-very low density lipoprotein uptake and decreasing cholesterol efflux.

To clarify the mechanism of smooth muscle cell (SMC)-derived foam cell formation, we investigated beta-very low density lipoprotein (beta-VLDL) cholesterol metabolism in vascular medial SMCs (M-SMCs) from normal rabbits compared with intimal SMCs (I-SMCs) from normal rabbits fed a high-cholesterol diet and LDL receptor-deficient rabbits. For both types of I-SMCs, uptake of [3H]cholesteryl oleate labeled beta-VLDL increased 1.6 times and release of [3H]cholesterol decreased 40% compared with M-SMCs. M-SMCs took up part of the beta-VLDL through the LDL receptor but I-SMCs did not. mRNAs for the VLDL receptor and the LDL receptor relative with 11 ligand binding repeats were expressed at similar levels in all SMCs. M-SMCs expressed more LDL receptor-related protein than I-SMCs. Ligand blotting analysis revealed greater 125I-beta-VLDL binding to a 700-kDa protein in I-SMCs compared with M-SMCs. I-SMCs had higher activities of acid cholesterol esterase and acyl-CoA:cholesterol acyltransferase, and lower activity of neutral cholesterol esterase than M-SMCs in both the absence and the presence of beta-VLDL. These results indicate that I-SMCs accumulate more cholesteryl ester than M-SMCs by taking up more beta-VLDL and by effluxing less cholesterol.

Molecular cloning of monkey CYP2C43 cDNA and expression in yeast.

A cDNA clone designated as CYP2C43 was isolated from the rhesus monkey liver cDNA library. The first 16 amino acid residues at the N-terminal region of this cDNA product were identical with those of P450 CMLd which have been purified and characterized as S-mephenytoin 4'-hydroxylase in monkey liver. The respective nucleotide and deduced amino acid sequences of CYP2C43 were 83% and 77%, identical to those of monkey CYP2C20. Antibody against CYP2C9 detected a protein in the microsomes of yeast transformed CYP2C43 expression plasmid. The specific content of recombinant CYP2C43 was 78.0 pmol/mg protein and the yield was 4.23 nmol/l of the culture. CYP2C43 was able to metabolize S-mephenytoin stereo-selectively. The activity for S-mephenytoin in the microsomes reconstituted with or without cytochrome b(5) was found to be 96.2 or 23.7 pmol/min/nmol P450, respectively. CYP2C43, however, did not show any oxidative activity for tolbutamide. These results indicate that CYP2C43 is the second identified member of the monkey CYP2C subfamily and a cDNA clone encoding P450 CMLd in monkey.

Identification of inhibitory component in cinnamon--O-methoxycinnamaldehyde inhibits CYP1A2 and CYP2E1-.

The Cinnamomi Cortex and Ephedra Herba were found to more strongly inhibit aminopyrine N-demethylation in rat liver microsomes compared to other constituents included in Sho-seiryu-to. The component inhibiting drug oxidations catalyzed by CYP1A2 and CYP2E1 was isolated from Cinnamomi Cortex, and was identified as o-methoxycinnamaldehyde (OMCA). When phenacetin and 4-nitrophenol were used as probe substrates for CYP1A2 and CYP2E1, respectively, the OMCA was shown to be a competitive inhibitor against CYP1A2 while it was a mixed type inhibitor against CYP2E1. The inhibitory effect of OMCA on 4-nitrophenol 2-hydroxylation (K(i)=6.3 microM) was somewhat potent compared to that observed on phenacetin O-deethylation (K(i)=13.7 microM) in rat liver microsomes.

Effects of continuous ingestion of green tea or grape seed extracts on the pharmacokinetics of midazolam.

Limited systematic data on herb-drug interaction are available, despite many opportunities to concomitant use of herb with prescribed drugs. We investigated the effects of 15 herbal extracts in dietary supplements on CYP2C9, CYP2D6 and CYP3A4 activities in human liver microsomes. Strong inhibition of these CYP activities was found by the addition of green tea extracts (GTE) or grape seed extracts (GSE) in vitro. To examine the effects of these extracts on CYP3A activities in vivo, the pharmacokinetics of midazolam (MDZ) was analyzed in rats. Although single treatments with these extracts had negligible effects, 1 week of treatment with them resulted in a significant increase in the ke of intravenously administered MDZ, indicating the induction of CYP3A in the liver. In contrast, 1 week of treatment with GTE, but not GSE, caused a significant increase in the C(max) and AUC(0-infinity) of orally administered MDZ without change in the t(1/2), suggesting a reduction in CYP3A activity in the small intestines. These studies indicate that subchronic ingestion of GTE or GSE may alter the pharmacokinetics of MDZ, and the effects of GTE on CYP3A activity appear opposite between liver and small intestine, which could not be predicted from in vitro experiments.