Synthesis and structure determination of a synthetic cannabinoid CUMYL-THPINACA metabolite with differentiation between the ortho-, meta-, and para-hydroxyl positions of the cumyl moiety.

Synthetic cannabinoids, a type of new psychoactive substances, are likely to be rapidly metabolized; thus, the detection of their metabolites, rather than the parent compound, is a common method used to prove drug consumption. Although the analysis of metabolites is generally performed by mass spectrometry, it is limited to structural estimation because of few commercially available standards. In particular, distinguishing between positional isomers is difficult. Synthetic cannabinoids with a cumyl moiety can be hydroxylated at the cumyl moiety during metabolism, but it remains unclear whether the hydroxylation occurs at the ortho, meta, or para position. This study determined the structures of a metabolite formed by mono-hydroxylation at the cumyl moiety of the synthetic cannabinoid CUMYL-THPINACA, used as a model compound. Chemical synthesis was performed to create possible metabolites with one hydroxyl group at the ortho, meta, or para positions of the cumyl moiety. Using the synthesized metabolites and liquid chromatography-quadrupole time-of-flight mass spectrometry, the metabolite detected in the microsomal reaction of CUMYL-THPINACA was identified as a compound mono-hydroxylated at the para position based on retention time and product ion spectra. Moreover, the rapid metabolism of CUMYL-THPINACA was demonstrated with an in vitro half-life of 4.9 min and the identified metabolite could be detected for a relatively long time in vitro. The synthesized metabolite may be utilized as a good reference standard for proof of CUMYL-THPINACA consumption. These findings have potential applications in the synthesis of metabolites of other synthetic cannabinoids bearing a cumyl moiety.

Rapid Determination of Preservatives in Cosmetics Using a Core-Shell Column.

Preservatives are frequently added to cosmetics to maintain product quality. Our laboratory quantifies 11 preservatives in cosmetics each year for regulatory purposes. In laboratories, many manufactures also analyze the preservatives in their products for quality control. To analyze many cosmetic samples, a rapid analysis method is required for efficiency. In this study, we developed a rapid method for the simultaneous determination of 11 regulated preservatives in cosmetics using a core-shell column by high-performance liquid chromatography. In this method, the 11 preservatives were separated within 17 min, which was approximately half the time reported in the previous study. The peak resolution for each preservative was >2.6, the correlation coefficients of the calibration curves were >0.9988, the percent recoveries were 92.0-111.9% and the relative standard deviations were <3.5% (n = 3). The relative standard deviations among 6 researchers were <4.7%. Thus, it is an effective rapid determination method for the analysis of preservatives in cosmetics.

Energy conversion efficiency comparison of different aqueous and semi-aqueous CO2 electroreduction systems.

An energy conversion efficiency index, that is independent of the anode reaction performance, is proposed for CO2 reduction in aqueous and semi-aqueous systems. The energy conversion efficiency of CO2 reduction under 107 typical conditions was calculated based on the derived formula. Notably, the resulting efficiency trends of the reduction products differed from their faradaic efficiency trends. When the products were CO, HCOOH, C2H4, and CH4, the electrocatalysts with the higher energy conversion efficiencies were Au, Pd, Cu, and Pt, respectively. Based on the discussion on the overall energy conversion efficiency of all products, Pt should be a specific energetically advantageous catalyst for CO2 reduction because the activation energy is negligibly small. Moreover, the energy conversion and faradaic efficiencies were discovered to not only depend on the electrocatalyst species, but also on the complexity of the reaction, including the number of reaction electrons. Our proposed method for evaluating the energy conversion efficiency of cathode reactions can potentially serve as a novel platform for comparing the CO2 reduction efficiencies of different electroreduction systems.

A Simple Flow Injection Analysis-Tandem Mass Spectrometry Method to Reduce False Positives of C5-Acylcarnitines Due to Pivaloylcarnitine Using Reference Ions.

Flow injection analysis−tandem mass spectrometry (FIA-TMS) has been applied in a first-tier test of newborn screening (NBS). Although isovalerylcarnitine (i-C5), which is a diagnostic indicator of isovaleric acidemia (IVA), is isobaric with pivaloylcarnitine (p-C5), 2-methylbutyrylcarnitine, and n-valerylcarnitine, these isomers cannot be distinguished by the FIA-TMS. There are many reports of false positives derived from p-C5 due to the use of pivalate-conjugated antibiotics. In this study, we developed a new FIA-TMS method to distinguish i-C5 and p-C5. We found that the intensity ratio of product ions for i-C5 and p-C5 was different in a certain range even under the same analytical conditions. The product ions with the most distinct differences in ionic intensity between the isomers and the collision energies that produce them were determined to be m/z 246.2 > 187.1 and −15 V, respectively. In addition to the quantification ion, a reference ion was defined, and the similarity of the i-C5 and p-C5 reference ion ratios (i-C5 score and p-C5 score, respectively) were used to estimate which isomer (i-C5 and p-C5) was responsible for elevated C5 acylcarnitine in dried blood spots (DBSs). As a result of analyses of 11 DBS samples derived from pivalate-conjugated antibiotics and four DBS samples from IVA patients using our method, it was found that our method was able to correctly determine the type of C5-acylcarnitine (i-C5 or p-C5) in the DBS samples. Implementation of this new FIA-TMS method into the current NBS protocol will allow for a reduction in false positives in IVA.

Diagnosis of Mucopolysaccharidoses and Mucolipidosis by Assaying Multiplex Enzymes and Glycosaminoglycans.

Mucopolysaccharidoses (MPS) and mucolipidosis (ML II/III) are a group of lysosomal storage disorders (LSDs) that occur due to a dysfunction of the lysosomal hydrolases responsible for the catabolism of glycosaminoglycans (GAGs). However, ML is caused by a deficiency of the enzyme uridine-diphosphate N-acetylglucosamine:lysosomal-enzyme-N-acetylglucosamine-1-phosphotransferase (GlcNAc-1-phosphotransferase, EC2.7.8.17), which tags lysosomal enzymes with a mannose 6-phosphate (M6P) marker for transport to the lysosome. A timely diagnosis of MPS and ML can lead to appropriate therapeutic options for patients. To improve the accuracy of diagnosis for MPS and ML in a high-risk population, we propose a combination method based on known biomarkers, enzyme activities, and specific GAGs. We measured five lysosomal enzymes (α-L-iduronidase (MPS I), iduronate-2-sulfatase (MPS II), α-N-acetylglucosaminidase (MPS IIIB), N-acetylglucosamine-6-sulfatase (MPS IVA), and N-acetylglucosamine-4-sulfatase (MPS VI)) and five GAGs (two kinds of heparan sulfate (HS), dermatan sulfate (DS), and two kinds of keratan sulfate (KS)) in dried blood samples (DBS) to diagnose suspected MPS patients by five-plex enzyme and simultaneous five GAGs assays. We used liquid chromatography-tandem mass spectrometry (LC-MS/MS) for both assays. These combined assays were tested for 43 patients with suspected MPS and 103 normal control subjects. We diagnosed two MPS I, thirteen MPS II, one MPS IIIB, three MPS IVA, two MPS VI, and six ML patients with this combined method, where enzymes, GAGs, and clinical manifestations were compatible. The remaining 16 patients were not diagnosed with MPS or ML. The five-plex enzyme assay successfully identified MPS patients from controls. Patients with MPS I, MPS II, and MPS IIIB had significantly elevated HS and DS levels in DBS. Compared to age-matched controls, patients with ML and MPS had significantly elevated mono-sulfated KS and di-sulfated KS levels. The results indicated that the combination method could distinguish these affected patients with MPS or ML from healthy controls. Overall, this study has shown that this combined method is effective and can be implemented in larger populations, including newborn screening.

Bevacizumab plus cisplatin/pemetrexed then bevacizumab alone for unresectable malignant pleural mesothelioma: A Japanese safety study.

AIMS: Malignant pleural mesothelioma (MPM) is an aggressive malignancy with poor prognosis and limited treatment options. Cisplatin plus pemetrexed is the only approved first-line treatment for patients with unresectable MPM. Recently, promising outcomes were observed with first-line bevacizumab combined with cisplatin/pemetrexed, leading to the recommendation of this regimen as a first-line treatment option for patients with MPM. Bevacizumab plus cisplatin/pemetrexed has been shown to be safe and effective in non-small cell lung cancer, however, there are no efficacy or safety data in Japanese patients with MPM treated with this regimen. We conducted a multicenter study to evaluate tolerability and safety for Japanese patients with chemotherapy-naïve, unresectable MPM. METHODS: Eligible patients (n = 7) received bevacizumab plus cisplatin/pemetrexed (up to six cycles), then single-agent bevacizumab until disease progression or onset of unacceptable adverse events (AEs), according to the 3+3 design analogy. RESULTS: One patient (14.3%) reported an AE (gastric ulcer) meeting tolerability criteria. All patients experienced gastrointestinal disorders, including nausea (grade 1/2 only, n = 6, 85.7%) and constipation (grade 1/2 only, n = 5, 71.4%). Five patients (71.4%) had grade 3 hypertension. Two patients discontinued treatment due to gastric ulcer (n = 1) and proteinuria (n = 1). At data cut-off, four patients had stable disease, two had partial response and one had non-complete response/non-progressive disease due to the absence of target lesions. CONCLUSIONS: Bevacizumab plus cisplatin/pemetrexed then bevacizumab was well tolerated in Japanese patients with MPM.

Validation of Liquid Chromatography-Tandem Mass Spectrometry-Based 5-Plex Assay for Mucopolysaccharidoses.

Mucopolysaccharidoses (MPSs) are rare lysosomal storage diseases caused by the accumulation of undegraded glycosaminoglycans in cells and tissues. The effectiveness of early intervention for MPS has been reported. Multiple-assay formats using tandem mass spectrometry have been developed. Here, we developed a method for simultaneous preparation and better measurement of the activities of five enzymes involved in MPSs, i.e., MPS I, MPS II, MPS IIIB, MPS IVA, and MPS VI, which were validated using 672 dried blood spot samples obtained from healthy newborns and 23 patients with MPS. The mean values of the enzyme activities and standard deviations in controls were as follows: α-iduronidase (IDUA), 4.19 ± 1.53 µM/h; iduronate-2-sulfatase (I2S), 8.39 ± 2.82 µM/h; N-acetyl-α-glucosaminidase (NAGLU), 1.96 ± 0.57 µM/h; N-acetylgalactosamine-6-sulfatase (GALNS), 0.50 ± 0.20 µM/h; and N-acetylgalactosamine-4-sulfatase (ARSB), 2.64 ± 1.01 µM/h. All patients displayed absent or low enzyme activity. In MPS I, IIIB, and VI, each patient group was clearly separated from controls, whereas there was some overlap between the control and patient groups in MPS II and IVA, suggesting the occurrence of pseudo-deficiencies. Thus, we established a multiplex assay for newborn screening using liquid chromatography tandem mass spectrometry, allowing simultaneous pretreatment and measurement of five enzymes relevant to MPSs.

Effect of an Enhanced Nose-to-Brain Delivery of Insulin on Mild and Progressive Memory Loss in the Senescence-Accelerated Mouse.

Insulin is now considered to be a new drug candidate for treating dementias, such as Alzheimer's disease, whose pathologies are linked to insulin resistance in the brain. Our recent work has clarified that a noncovalent strategy involving cell-penetrating peptides (CPPs) can increase the direct transport of insulin from the nasal cavity into the brain parenchyma. The present study aimed to determine whether the brain insulin level increased by intranasal coadministration of insulin with the CPP penetratin has potential for treating dementia. The pharmacological actions of insulin were investigated at different stages of memory impairment using a senescence-accelerated mouse-prone 8 (SAMP8) model. The results of spatial learning tests suggested that chronic intranasal administration of insulin with l-penetratin to SAMP8 slowed the progression of memory loss in the early stage of memory impairment. However, contrary to expectations, this strategy using penetratin was ineffective in recovering the severe cognitive dysfunction in the progressive stage, which involves brain accumulation of amyloid ß (Aß). Immunohistological examination of hippocampal regions of samples from SAMP8 in the progressive stage suggested that accelerated nose-to-brain insulin delivery had a partial neuroprotective function but unexpectedly increased Aß plaque deposition in the hippocampus. These findings suggest that the efficient nose-to-brain delivery of insulin combined with noncovalent CPP strategy has different effects on dementia during the mild and progressive stages of cognitive dysfunction.

A selective detection of lysophosphatidylcholine in dried blood spots for diagnosis of adrenoleukodystrophy by LC-MS/MS.

X-linked adrenoleukodystrophy (X-ALD) is a rare inherited metabolic disorder characterized by an impaired beta-oxidation of very long chain fatty acids in the peroxisomes. Recent studies have suggested that 1-hexacosanoyl-2-hydroxy-sn-glycero-3-phosphocholine (Lyso-PC 26:0) can be a sensitive biomarker for X-ALD. Although approximately 10-fold increase in the concentration of Lyso-PC 26:0 in DBSs from X-ALD-affected individuals were reported, whether the carriers might be distinguished from the healthy controls remained unclear. To address this question, we have validated previously developed LC-MS/MS-based analytical procedures using QC DBS. We found that the recovery of Lyso-PC 26:0 from the QC DBSs was 73.6 ± 0.3% when 2 µM of Lyso-PC 26:0 was spiked into the blood. Based on this result, the amounts of Lyso-PC 26:0 in the controls and ALD-affected individuals were 0.090 ± 0.004 (n = 11) and 1.078 ± 0.217 (n = 4) pmol/DBS, respectively. Interestingly, the concentration of Lyso-PC 26:0 in the carriers were 0.548 ± 0.095 pmol/DBS (n = 3), indicating that the carriers and the healthy controls can be distinguished. These results suggest that LC-MS/MS-based technique can be used for the detection of asymptomatic carriers and X-ALD-affected subjects in the newborn screening.

The levels of urinary glycosaminoglycans of patients with attenuated and severe type of mucopolysaccharidosis II determined by liquid chromatography-tandem mass spectrometry.

Glycosaminoglycans (GAGs) play important roles on the regulation of extracellular signaling, neuronal development, and cartilage maintenance. The extracellular concentration of total GAGs has been used as an established measure for the diagnosis of mucopolysaccharidoses (MPSs). Heparan sulfate (HS), Dermatan sulfate (DS) and chondroitin sulfate are known to be elevated in the GAGs under pathological conditions associated with MPS. Furthermore, the selective accumulation of disease-specific one of, or a combination of, them has also been used for the estimation of subtypes of MPS. A previously developed method [Auray-Blais C et al. Molecular Genetics and Metabolism 102 (2011) 49-56.] measures the concentration of GAGs using liquid chromatography with tandem mass spectrometry (LC-MS/MS) with higher precision. To ask whether the selective accumulation of HS and DS in the urine of MPS II patients discriminate the attenuated and severe type of MPS II, we examined the concentrations of HS and DS by this methodology. Compared to the healthy controls, we found a marked elevation of HS and DS in all of the MPS II-affected patients. Among patients who received ERT with confirmed elevation of antibody titer, the concentrations of HS in the urine of patients with attenuated type were lower than those with severe type of MPS II. In these patients, the concentrations of DS by LC-MS/MS and of total GAG by DMB failed to depend on the accumulation of antibody. These results suggest that the LC-MS/MS method employed in this study might discriminate the subtypes of MPS II in different clinical background.

Visualization and Quantitative Assessment of the Brain Distribution of Insulin through Nose-to-Brain Delivery Based on the Cell-Penetrating Peptide Noncovalent Strategy.

Our recent work suggested that intranasal coadministration with the cell-penetrating peptide (CPP) penetratin increased the brain distribution of the peptide drug insulin. The present study aimed to distinctly certify the ability of penetratin to facilitate the nose-to-brain delivery of insulin by quantitatively evaluating the distribution characteristics in brain using radioactive (64)Cu-NODAGA-insulin. Autoradiography and analysis using a gamma counter of brain areas demonstrated that the accumulation of radioactivity was greatest in the olfactory bulb, the anterior part of the brain closest to the administration site, at 15 min after intranasal administration of (64)Cu-NODAGA-insulin with l- or d-penetratin. The brain accumulation of (64)Cu-NODAGA-insulin with penetratin was confirmed by ELISA using unlabeled insulin in which intact insulin was delivered to the brain after intranasal coadministration with l- or d-penetratin. By contrast, quantification of cerebrospinal fluid (CSF) samples showed increased insulin concentration in only the anterior portion of the CSF at 15 min after intranasal coadministration with l-penetratin. This study gives the first concrete proof that penetratin can accelerate the direct transport of insulin from the nasal cavity to the brain parenchyma. Further optimization of intranasal administration with CPP may increase the efficacy of delivery of biopharmaceuticals to the brain while reducing the risk of systemic drug exposure.

Multi-locus genotyping of bottom fermenting yeasts by single nucleotide polymorphisms indicative of brewing characteristics.

Yeast plays a capital role in brewing fermentation and has a direct impact on flavor and aroma. For the evaluation of competent brewing strains during quality control or development of novel strains it is standard practice to perform fermentation tests, which are costly and time-consuming. Here, we have categorized DNA markers which enable to distinguish and to screen brewing strains more efficiently than ever before. Sequence analysis at 289 loci in the genomes of six bottom fermenting Saccharomyces pastorianus strains revealed that 30 loci contained single nucleotide polymorphisms (SNPs). By determining the nucleotide sequences at the SNP-loci in 26 other S. pastorianus strains and 20 strains of the top fermenting yeast Saccharomyces cerevisiae, almost all these strains could be discriminated solely on the basis of the SNPs. By comparing the fermentative phenotypes of these strains we found that some DNA markers showed a strong association with brewing characteristics, such as the production of ethyl acetate and hydrogen sulphide (H2S). Therefore, the DNA markers we identified will facilitate quality control and the efficient development of brewing yeast strains.

Acute renal failure after exercise in a Japanese sumo wrestler with renal hypouricemia.

Familial renal hypouricemia is a hereditary disease characterized by extraordinary high renal uric acid clearance and is associated with acute renal failure (ARF). An 18-year-old sumo wrestler developed ARF after anaerobic exercise. Several hours after the exercise, he had a pain in the loins with oliguria, headache, and nausea. On admission, his serum uric acid was decreased despite the elevation of serum creatinine (9.5 mg/dL). The level of creatine kinase was normal and there was no myoglobinuria or urolithiasis. Magnetic resonance imaging showed no significant abnormality. Renal function improved completely within 2 weeks of hydration treatment. After remission, hypouricemia became obvious (1.0 mg/dL) from the initial level of uric acid (6.1 mg/dL) and fractional excretion of uric acid was 49%. Polymerase chain reaction of a urate anion exchanger known to regulate blood urate level (SLC22A12 gene: URAT1) demonstrated that homozygous mutations in exon 4 (W258X). Both parents showed heterozygous mutation of the URAT1 gene, but both siblings showed no mutation. Thus, we describe a Japanese sumo wrestler of familial renal hypouricemia complicated with anaerobic exercise-induced ARF, with definite demonstration of genetic abnormality in the responsible gene, URAT1.

Glomerulonephritis induced by methicillin-resistant Staphylococcus aureus infection that progressed during puerperal period.

A 28-year-old Japanese woman developed fever, leg edema, purpura, and abdominal pain during the puerperal period, showing nephrotic syndrome with microscopic hematuria. At first she was thought to have Henoch-Shönlein purpura nephritis and was given steroids at another hospital. Because anasarca and massive urinary protein excretion developed, she was referred to our hospital. Renal biopsy specimens showed endocapillary proliferative glomerulonephritis with massive IgA and C3d deposition along the capillary loops and in the mesangium. A bacteriological study detected methicillin-resistant Staphylococcus aureus (MRSA) in cultures of vaginal secretions, urine, stool, and pharyngeal mucus samples. Based on the diagnosis of MRSA nephritis, anti-MRSA therapy with antibiotics was started, and MRSA became negative for each culture, and urinary protein decreased. Two months after the first renal biopsy, a second renal biopsy was performed, which revealed feeble endocapillary proliferation with mild IgA and C3d deposition in the mesangium. This case showed that the delivery procedure can cause MRSA nephritis after MRSA infection, and that steroid therapy should be avoided in the early phase of this type of nephritis.

Roles of coagulation pathway and factor Xa in rat mesangioproliferative glomerulonephritis.

Tissue factor initiates the extrinsic coagulation pathway by activating coagulation factor X to factor Xa, and factor V is a cofactor for the prothrombin activation by factor Xa. As factor Xa is known to promote the proliferation of mesangial cells in culture, the roles of the coagulation pathway and factor Xa were studied in an animal model of mesangioproliferative glomerulonephritis (MsPGN). MsPGN was induced in Wistar rats by an intravenous injection of anti-Thy 1.1 monoclonal antibody, OX-7. To clarify the role of factor Xa in MsPGN, a specific factor Xa inhibitor, DX-9065a, was injected intravenously at 2.5 or 10 mg/kg at the same time as OX-7, and kidney involvement was assessed by immunohistological analyses. We also examined p44/42 mitogen-activated protein (MAP) kinase activation. Time-course study revealed that expressions of tissue factor, factor V, and protease-activated receptor 2 (PAR2) were peaked on day 3, followed by factor X accumulation and mesangial proliferation. DX-9065a treatment significantly ameliorated proteinuria in a dose-dependent manner on day 8. Histological analyses showed a significant reduction in the size of glomeruli, the total number of glomerular cells, and crescent formation by DX-9065a treatment. Macrophage infiltration, which was rapidly observed on day 1 in disease control rats was not inhibited on days 1-3 by DX-9065a treatment, however it was suppressed on days 5-8. The deposition of fibrin, the number of PCNA-positive cells, and phosphorylation of p44/42 MAP kinase were markedly increased in the disease control group, whereas they were significantly reduced in the treatment group. Tissue factor and factor V induction may accelerate MsPGN through the activation and accumulation of factor X via proinflammatory and procoagulant mechanisms, and the inhibition of factor Xa would be a promising method to regulate the disease process.

The Na+-excreting efficacy of indapamide in combination with furosemide in massive edema.

BACKGROUND: Massive systemic edema is often observed in patients with severe nephrotic syndrome, including diabetic nephropathy. Although furosemide, a loop diuretic, is often administered to these patients, some patients do not respond to this treatment, still showing massive edema. METHODS: The efficacy of indapamide which has a thiazide-like effect on distal convoluted tubules in combination with furosemide, was evaluated in eight patients with massive edema, in regard to both Na+ excretion and diuresis. Indapamide 2 mg was administered once a day, in the morning, to patients in whom it was considered that furosemide treatment of 40-120 mg a day for 1 week was ineffective. RESULTS: Urinary Na+ excretion was markedly increased, from 83.7 +/- 82.2 mEq/day to 140.7 +/- 33.8 mEq/day after 1 week of the combination therapy compared with furosemide alone (P < 0.01); urine volume was also increased, from 1070 +/- 230 ml to 1359 +/- 296 ml after 1 week of the combination therapy (P < 0.05). In this context, body weight was significantly decreased, from 57.2 +/- 12.3 kg to 53.4 +/- 12.8 kg, after the combination therapy (P = 0.01). Indapamide in combination with furosemide was well tolerated, and no significant changes in serum levels of creatinine and potassium were observed. CONCLUSIONS: This combination therapy appears to be effective in patients with massive edema, as it increased diuresis, and achieved potent Na+ excretion.

Role of coagulation factor Xa and protease-activated receptor 2 in human mesangial cell proliferation.

BACKGROUND: Fibrin deposition and mesangial cell proliferation are frequently observed in the active type of mesangioproliferative glomerulonephritis. Coagulation factors, such as factor V and factor Xa are colocalized with fibrin in the mesangial areas in active type of IgA nephropathy with mesangial cell proliferation. In this study, therefore, we studied the role of factor Xa and its receptor, protease-activated receptor 2 (PAR2) in mesangial cell proliferation and fibrin deposition, and examined ant-proliferative effects of a specific factor Xa inhibitor, DX-9065a, in cultured human mesangial cells. METHODS: To examine the effect of DX-9065a on the factor Xa-induced proliferation of cultured human mesangial cells, we measured thymidine incorporation and cell numbers. We also examined the effect of DX-9065a on extracellular regulated kinase (ERK) activation and fibrin production induced by factor Xa in human mesangial cells. RESULTS: Factor Xa increased [(3)H]-thymidine incorporation and cell numbers in a dose-dependent manner in mesangial cells, which was inhibited by DX-9065a. DX-9065a also suppressed factor Xa-triggered fibrin deposition on mesangial cell surface. Factor Xa induced the activation of ERK in mesangial cells and this activation was also completely inhibited by DX-9065a, but not inhibited by PAR1 antagonist. Factor Xa-induced cell proliferation and ERK activation were inhibited by PD98059. CONCLUSION: There results suggest that factor Xa can induce mesangial cell proliferation through the activation of ERK via PAR2 in mesangial cells and that PAR2 may play a crucial role in the cell proliferation induced by factor Xa. Our results implicate that DX-9065a may be a promising agent to regulate proliferation of mesangial cellss and inhibit the coagulation process in mesangium.