Expression of alcohol oxidase gene from Ochrobactrum sp. AIU 033 in recombinant Escherichia coli through the twin-arginine translocation pathway.

We cloned a set of genes encoding alcohol oxidase from Ochrobactrum sp. AIU 033 (OcAOD), which exhibits the appropriate substrate specificity for glyoxylic acid production from glycolic acid. The set of genes for OcAOD contained two open reading frames consisting of 555-bp (aodB) and 1572-bp (aodA) nucleotides, which encode the precursor for the ß-subunit and α-subunit of OcAOD, respectively. We expressed the cloned genes as an active product in Escherichia coli BL21(DE3). The recombinant OcAOD oxidized glycolic acid and primary alcohols with C2-C8 but not glyoxylic acid (as is the case for native OcAOD), whereas the Km and Vmax values for glycolic acid and the pH stability were higher than those of native OcAOD. A consensus sequence for the twin-arginine translocation (Tat) pathway was identified in the N-terminal region of the precursor for the ß-subunit, and the active form of OcAOD was localized in the periplasm of recombinant E. coli, which indicated that OcAOD would be transported from the cytoplasm to the periplasm by the hitchhiker mechanism through the Tat pathway. The OcAOD productivity of the recombinant E. coli was 24-fold higher than that of Ochrobactrum sp. AIU 033, and it was further enhanced by 1.2 times by the co-expression of additional tatABC from E. coli BL21(DE3). Our findings thus suggest a function of the ß-subunit of OcAOD in membrane translocation, and that the recombinant OcAOD has characteristics that are suitable for the enzymatic synthesis of glyoxylic acid as well as native OcAOD.

Highly Selective Tau-SPECT Imaging Probes for Detection of Neurofibrillary Tangles in Alzheimer's Disease.

Neurofibrillary tangles composed of aggregates of hyperphosphorylated tau proteins are one of the neuropathological hallmarks of Alzheimer's disease (AD) in addition to the deposition of ß-amyloid plaques. Since the deposition of tau aggregates is closely associated with the severity of AD, the in vivo detection of tau aggregates may be useful as a biomarker for the diagnosis and progression of AD. In this study, we designed and synthesized a new series of radioiodinated benzoimidazopyridine (BIP) derivatives, and evaluated their utility as single photon emission computed tomography (SPECT) imaging agents targeting tau aggregates in AD brains. Five radioiodinated BIP derivatives were successfully prepared in high radiochemical yields and purities. In in vitro autoradiographic studies using postmortem AD brains, all BIP derivatives displayed high accumulation of radioactivity in the brain sections with abundant neurofibrillary tangles, while no marked radioactivity accumulation was observed in the brain sections with only ß-amyloid aggregates, indicating that the BIP derivatives exhibited selective binding to tau aggregates. Biodistribution studies in normal mice showed high brain uptake at 2 min postinjection (3.5-4.7% ID/g) and rapid clearance at 60 min postinjection (0.04-0.23% ID/g), which is highly desirable for tau imaging agents. The results of the present study suggest that [123I]BIP derivatives may be useful SPECT agents for the in vivo imaging of tau aggregates in AD.

Imaging of Cerebral Amyloid Angiopathy with Bivalent (99m)Tc-Hydroxamamide Complexes.

Cerebral amyloid angiopathy (CAA), characterized by the deposition of amyloid aggregates in the walls of cerebral vasculature, is a major factor in intracerebral hemorrhage and vascular cognitive impairment and is also associated closely with Alzheimer's disease (AD). We previously reported (99m)Tc-hydroxamamide ((99m)Tc-Ham) complexes with a bivalent amyloid ligand showing high binding affinity for ß-amyloid peptide (Aß(1-42)) aggregates present frequently in the form in AD. In this article, we applied them to CAA-specific imaging probes, and evaluated their utility for CAA-specific imaging. In vitro inhibition assay using Aß(1-40) aggregates deposited mainly in CAA and a brain uptake study were performed for (99m)Tc-Ham complexes, and all (99m)Tc-Ham complexes with an amyloid ligand showed binding affinity for Aß(1-40) aggregates and very low brain uptake. In vitro autoradiography of human CAA brain sections and ex vivo autoradiography of Tg2576 mice were carried out for bivalent (99m)Tc-Ham complexes ([(99m)Tc]SB2A and [(99m)Tc]BT2B), and they displayed excellent labeling of Aß depositions in human CAA brain sections and high affinity and selectivity to CAA in transgenic mice. These results may offer new possibilities for the development of clinically useful CAA-specific imaging probes based on the (99m)Tc-Ham complex.

Structure-Activity Relationship Study of Heterocyclic Phenylethenyl and Pyridinylethenyl Derivatives as Tau-Imaging Agents That Selectively Detect Neurofibrillary Tangles in Alzheimer's Disease Brains.

In order to explore novel tau-imaging agents that can selectively detect neurofibrillary tangles in Alzheimer's disease (AD) brains, we designed and synthesized a series of heterocyclic phenylethenyl and (3-pyridinyl)ethenyl derivatives with or without a dimethyl amino group. In in vitro autoradiography using AD brain sections, all radioiodinated ligands with a dimethyl amino group bound to Aß deposits in the sections. In contrast, the ligands without a dimethyl amino group showed different patterns of radioactivity accumulation in the sections depending on the kind of heterocycle contained in their molecules. Particularly, a phenylethenyl benzimidazole derivative ([(125)I]64) showed marked radioactivity accumulation in the temporal lobe which corresponded with the distribution of tau deposits. [(125)I]64 also showed the most favorable pharmacokinetics in normal mouse brains (3.69 and 0.06% ID/g at 2 and 60 min postinjection, respectively) among all ligands in this study. Taken together, these results suggest that [(123)I]64 may be a new candidate tau-imaging agent.

[Iron, aluminum, and lanthanum-based drugs for treatment of hyperphosphatemia].

The control of the serum phosphorus (P) level in chronic kidney disease patients is important because elevated serum P levels are associated with progression of vascular calcification and increased mortality in these patients. In 2014, a novel phosphate binder, ferric citrate hydrate (Riona(®)), became available for the treatment of hyperphosphatemia in Japan, the first country to approve this medication. Ferric citrate hydrate, which relies upon the potent phosphate-binding capacity of ferric iron, inhibits P absorption by forming complexes between ferric iron and dietary phosphate in the gut. The active pharmaceutical ingredient in ferric citrate hydrate provides a larger specific surface area and higher water solubility; therefore, it is expected to have greater efficacy in terms of its phosphate-binding capacity. In clinical trials, ferric citrate hydrate significantly reduced the serum phosphate level and effectively maintained serum P concentrations throughout the duration of the trials. Moreover, in one clinical trial, ferric citrate hydrate significantly decreased levels of fibroblast growth factor-23 (FGF-23) in nondialysis patients. FGF-23 is an endocrine hormone that increases urinary phosphate excretion to maintain serum P at the proper level. A portion of the iron from ferric citrate hydrate is absorbed and transported throughout the body as transferrin-bound iron, where it is used for the synthesis of hemoglobin, enzymes, and others. Although safer and more effective phosphate binders are expected in the future, ferric citrate hydrate will become a new approach for the treatment of hyperphosphatemia.

A (68)Ga complex based on benzofuran scaffold for the detection of ß-amyloid plaques.

Since the imaging of ß-amyloid (Aß) plaques in the brain is believed to be a useful tool for the early diagnosis of Alzheimer's disease (AD), a number of imaging probes to detect Aß plaques have been developed. Because the radionuclide (68)Ga (t1/2=68 min) for PET imaging could become an attractive alternative to (11)C and (18)F, we designed and synthesized a benzofuran derivative conjugated with a (68)Ga complex ((68)Ga-DOTA-C3-BF) as a novel Aß imaging probe. In an in vitro binding assay, Ga-DOTA-C3-BF showed high affinity for Aß(1-42) aggregates (Ki=10.8 nM). The Ga-DOTA-C3-BF clearly stained Aß plaques in a section of Tg2576 mouse, reflecting the affinity for Aß(1-42) aggregates in vitro. In a biodistribution study in normal mice, (68)Ga-DOTA-C3-BF displayed low initial uptake (0.45% ID/g) in the brain at 2 min post-injection. While improvement of the brain uptake of (68)Ga complexes appears to be essential, these results suggest that novel PET imaging probes that include (68)Ga as the radionuclide for PET may be feasible.

Enhancement of binding affinity for amyloid aggregates by multivalent interactions of 99mTc-hydroxamamide complexes.

Deposition of amyloid aggregates has been regarded as an early stage of amyloidosis progression. An imaging probe that can image amyloid aggregates enables the early diagnosis of amyloidosis and contributes to the development of new medical therapies. High binding affinity for amyloid aggregates is essential to develop a useful molecular imaging probe. This article describes a new strategy to enhance the binding affinity of imaging agents targeting amyloid aggregates. We designed and synthesized novel (99m)Tc-hydroxamamide ((99m)Tc-Ham) complexes with a bivalent amyloid ligand and evaluated their binding affinity for amyloid aggregates by using ß-amyloid peptide (Aß(1-42)) aggregates as a model. In vitro inhibition assay indicated that bivalent (99m)Tc-Ham complexes had much higher binding affinity for amyloid aggregates than monovalent complexes. In vitro autoradiography using Tg2576 mice showed the specific binding of bivalent (99m)Tc-Ham complexes to Aß plaques in the mouse brain, as reflected in the results of the inhibition assay. The preliminary results suggest that a new molecular design based on bivalent (99m)Tc-Ham complexes may be reasonable to develop an imaging probe targeting amyloid aggregates.

Development of novel 123I-labeled pyridyl benzofuran derivatives for SPECT imaging of ß-amyloid plaques in Alzheimer's disease.

Imaging of ß-amyloid (Aß) plaques in the brain may facilitate the diagnosis of cerebral ß-amyloidosis, risk prediction of Alzheimer's disease (AD), and effectiveness of anti-amyloid therapies. The purpose of this study was to evaluate novel (123)I-labeled pyridyl benzofuran derivatives as SPECT probes for Aß imaging. The formation of a pyridyl benzofuran backbone was accomplished by Suzuki coupling. [(123)I/(125)I]-labeled pyridyl benzofuran derivatives were readily prepared by an iododestannylation reaction. In vitro Aß binding assays were carried out using Aß(1-42) aggregates and postmortem human brain sections. Biodistribution experiments were conducted in normal mice at 2, 10, 30, and 60 min postinjection. Aß labeling in vivo was evaluated by small-animal SPECT/CT in Tg2576 transgenic mice injected with [(123)I]8. Ex vivo autoradiography of the brain sections was performed after SPECT/CT. Iodinated pyridyl benzofuran derivatives showed excellent affinity for Aß(1-42) aggregates (2.4 to 10.3 nM) and intensely labeled Aß plaques in autoradiographs of postmortem AD brain sections. In biodistribution experiments using normal mice, all these derivatives displayed high initial uptake (4.03-5.49% ID/g at 10 min). [(125)I]8 displayed the quickest clearance from the brain (1.30% ID/g at 60 min). SPECT/CT with [(123)I]8 revealed higher uptake of radioactivity in the Tg2576 mouse brain than the wild-type mouse brain. Ex vivo autoradiography showed in vivo binding of [(123)I]8 to Aß plaques in the Tg2576 mouse brain. These combined results warrant further investigation of [(123)I]8 as a SPECT imaging agent for visualizing Aß plaques in the AD brain.

Molecular imaging of ß-amyloid plaques with near-infrared boron dipyrromethane (BODIPY)-based fluorescent probes.

The formation of ß-amyloid (Aß) plaques is a critical neurodegenerative change in Alzheimer disease (AD). We designed and synthesized novel boron dipyrromethane (BODIPY)-based Aß probes (BAPs) and evaluated their utility for near-infrared fluorescence imaging of Aß plaques in the brain. In binding experiments in vitro, BAPs showed high affinity for synthetic Aß aggregates (Kd  =  18-149 nM). Furthermore, BAPs clearly stained Aß plaques in sections of Tg2576 mice. In mouse brain tissue, BAPs showed sufficient uptake for optical imaging. In addition, ex vivo fluorescent staining of brain sections from Tg2576 mice after the injection of BAP-2 showed selective binding of Aß plaques with little nonspecific binding. BAPs may be useful as a near-infrared fluorescent probe for imaging Aß plaques.

Synthesis and biological evaluation of novel styryl benzimidazole derivatives as probes for imaging of neurofibrillary tangles in Alzheimer's disease.

This paper describes the synthesis and biological evaluation of styrylbenzimidazole (SBIM) derivatives as agents for imaging neurofibrillary tangles (NFT) in patients with Alzheimer's disease (AD). SBIM derivatives were prepared with 4-iodobenzene-1,2-diamine and substituted cinnamaldehydes. In binding experiments using recombinant tau and Aß(1-42) aggregates, SBIM-3 showed higher affinity for the tau aggregates than Aß(1-42) aggregates (ratio of K(d) values was 2.73). In in vitro autoradiography and fluorescent staining, [(125)I]SBIM-3 (or SBIM-3) bound NFT in sections of AD brain tissue. In biodistribution experiments using normal mice, all [(125)I]SBIM derivatives showed high initial uptake into (3.20-4.11%ID/g at 2 min after the injection) and rapid clearance from (0.12-0.33%ID/g at 60 min after the injection) the brain. In conclusion, appropriate structural modifications of SBIM derivatives could lead to more useful agents for the in vivo imaging of NFT in AD brains.

Structure-Activity Relationships and in Vivo Evaluation of Quinoxaline Derivatives for PET Imaging of ß-Amyloid Plaques.

This letter describes the synthesis, structure-activity relationships, and in vivo evaluation of a new series of 2-phenylquinoxaline (PQ) derivatives for imaging ß-amyloid (Aß) plaques in Alzheimer's disease (AD). In experiments in vitro, the affinity of the derivatives for Aß aggregates varied, with K i values of 0.895 to 1180 nM. In brain sections from AD patients, derivatives with a K i of less than 111 nM intensely labeled Aß plaques, while those with values over 242 nM showed no marked labeling. In biodistribution experiments using normal mice, the derivatives showed good uptake into (4.69-7.59 %ID/g at 2 or 10 min postinjection) and subsequent washout from (1.48-3.08 %ID/g at 60 min postinjection) the brain. In addition, [(18)F]PQ-6 labeled Aß plaques in vivo in APP transgenic mice, while it showed nonspecific binding in the white matter. Further structural optimization based on [(18)F]PQ-6 may lead to more useful PET probes for imaging Aß plaques.

4,8-Sphingadienine and 4-hydroxy-8-sphingenine activate ceramide production in the skin.

BACKGROUND: Ingestion of glucosylceramide improves transepidermal water loss (TEWL) from the skin, but the underlying mechanism by which a small amount of dietary glucosylceramide can vastly improve skin conditions remains unclear. In a previous report, glucosylceramides were shown to be digested to sphingoids, which were shown to be absorbed through the intestinal epithelium. Based on these observations, we hypothesized that sphingoids are the key molecules facilitating endogenous ceramide production. In this study, we assessed the effect of 4,8-sphingadienine (d18:2) and 4-hydroxy-8-sphingenine (t18:1), derived from konjac glucosylceramide, on stimulating ceramide production. METHODS: Konjac glucosylceramide acidolysis was performed using hydrochloric acid; the resulting d18:2 and t18:1 were fractionated by column chromatography. Real-time quantitative RT-PCR was performed to assess the effect of d18:2 and t18:1 on gene expression in normal human epidermal keratinocytes, while their effect on the nuclear receptor, peroxisome proliferator-activated receptor (PPAR)γ, was measured using a receptor-cofactor assay system. The effect of d18:2 and t18:1 on stimulating ceramide production was evaluated using HPTLC analysis in a 3-dimensional human skin model. RESULTS: We noted the upregulation of genes related to de novo ceramide synthesis as well as of those encoding the elongases of very long-chain fatty acids by d18:2 and t18:1, but not by glucosylceramide and 4-sphingenine. Both these sphingoids also facilitated the expression of PPARß/δ and PPARγ; moreover, they also demonstrated ligand activity for PPARγ. These results indicated that d18:2 and t18:1 promote the differentiation of keratinocytes. Analysis of the lipids within the 3-dimensional human skin model indicated that treatment with d18:2 and t18:1 not only upregulated gene expression but also increased ceramide production. CONCLUSIONS: The sphingoids d18:2 and t18:1 activated genes related to de novo ceramide synthesis and increased ceramide production, whereas glucosylceramide and 4-sphingenine could not. These results suggest that the effect of dietary glucosylceramides on the skin is mediated by d18:2 and t18:1.

Synthesis and biological evaluation of novel oxindole derivatives for imaging neurofibrillary tangles in Alzheimer's disease.

This letter describes the synthesis and biological evaluation of a novel series of radioiodinated oxindole (OI) derivatives for detecting neurofibrillary tangles (NFTs) in the brains of patients with Alzheimer's disease (AD). In binding experiments in vitro, 2-oxindole (2-OI) and 3-oxindole (3-OI) derivatives showed affinity for tau aggregates. The 3-OI derivative 14 showed the highest affinity of these derivatives. In biodistribution experiments using normal mice, the OI derivatives displayed good uptake (2.4-2.5%ID/g at 2 min) and clearance from the brain with time (0.6-1.4%ID/g at 30 min). In fluorescence staining experiments using AD brain sections, 14 clearly stained NFTs. 3-OI may serve as a new molecular scaffold for developing novel NFT imaging agents.

(18)F-labeled phenyldiazenyl benzothiazole for in vivo imaging of neurofibrillary tangles in Alzheimer's disease brains.

We synthesized and evaluated (E)-4-((6-(2-(2-(2-fluoroethoxy)ethoxy)ethoxy)benzo[d]thiazol-2-yl)diazenyl)-N,N-dimethylaniline (FPPDB) as a probe for the imaging of neurofibrillary tangles (NFTs) in patients with Alzheimer's disease (AD). In assays using thioflavin S (ThS) as a competitive ligand, FPPDB competed with ThS well and showed high affinity for both tau and Aß1-42 aggregates (K i = 13.0 and 20.0 nM, respectively). The results of saturation binding assays also verified that FPPDB bound to both tau and Aß1-42 aggregates with high affinity (K d = 44.8 nM and B max = 45.8 pmol/nmol protein for tau aggregates and K d = 45.4 nM and B max = 38.9 pmol/nmol protein for Aß1-42 aggregates). Furthermore, [(18)F]FPPDB substantially labeled NFTs and senile plaques in AD brain sections but not control brain sections. In biodistribution experiments using normal mice, [(18)F]FPPDB displayed higher uptake (4.28% ID/g at 2 min postinjection) into and washout (2.53% ID/g at 60 min postinjection) from the brain with time. On the basis of the chemical structure of FPPDB, further increases in selective binding to tau aggregates may lead to the development of more useful probes for the imaging of NFTs in AD brains.

Rhodanine and thiohydantoin derivatives for detecting tau pathology in Alzheimer's brains.

A novel series of rhodanin (RH) and thiohydantoin (TH) derivatives were designed and synthesized for detecting tau pathology in the brains of patients with Alzheimer's disease (AD). In experiments in vitro using tau and ß-amyloid (Aß) aggregates, the TH derivative, TH2, showed high specific binding to tau aggregates. In hippocampal sections obtained from AD patients, TH2 intensely stained neurofibrillary tangles. In experiments using normal mice, [(125)I]TH2 showed good uptake (1.54%ID/g, 2 min postinjection) into and a rapid washout (0.25%ID/g, 60 min postinjection) from the brain. [(123)I]TH2 should be further investigated as a potential imaging agent for detecting tau pathology.

The proteinase/proteinase-activated receptor-2/transient receptor potential vanilloid-1 cascade impacts pancreatic pain in mice.

AIMS: Proteinase-activated receptor-2 (PAR2) and transient receptor potential vanilloid-1 (TRPV1) are co-localized in the primary afferents, and the trans-activation of TRPV1 by PAR2 activation is involved in processing of somatic pain. Given evidence for contribution of PAR2 to pancreatic pain, the present study aimed at clarifying the involvement of TRPV1 in processing of pancreatic pain by the proteinase/PAR2 pathway in mice. MAIN METHODS: Acute pancreatitis was created by repeated administration of cerulein in conscious mice, and the referred allodynia/hyperalgesia was assessed using von Frey filaments. Injection of PAR2 agonists into the pancreatic duct was achieved in anesthetized mice, and expression of Fos in the spinal cord was determined by immunohistochemistry. KEY FINDINGS: The established referred allodynia/hyperalgesia following cerulein treatment was abolished by post-treatment with nafamostat mesilate, a proteinase inhibitor, and with capsazepine, a TRPV1 antagonist, in mice. Injection of trypsin, an endogenous PAR2 agonist, or SLIGRL-NH(2), a PAR2-activating peptide, into the pancreatic duct caused expression of Fos protein in the spinal superficial layers at T8-T10 levels in the mice. The spinal Fos expression caused by trypsin and by SLIGRL-NH(2) was partially blocked by capsazepine, the former effect abolished by nafamostat mesilate. SIGNIFICANCE: Our data thus suggest that the proteinase/PAR2/TRPV1 cascade might impact pancreatic pain, in addition to somatic pain, and play a role in the maintenance of pancreatitis-related pain in mice.

Development of dual functional SPECT/fluorescent probes for imaging cerebral beta-amyloid plaques.

The imaging of beta-amyloid (Abeta) aggregates in the brain may lead to the early detection of Alzheimer's disease (AD) and monitoring of the progression and effectiveness of treatment. The purpose of this study was to develop dual modality SPECT and fluorescent probes based on boron dipyrromethane (BODIPY) as a core structure. We designed and synthesized an (125)I-labeled derivative of BODIPY (BODIPY7). BODIPY7 had a K(i) value of 108nM for Abeta(1-42) aggregates and exhibited peaks of absorption/emission at 606/613nm. It detected Abeta plaques in sections of brain tissue from an animal model of AD and displayed low uptake in the brain and high uptake in the liver in normal mice. Although additional modifications of the BODIPY scaffold are necessary to improve brain uptake, these results should aid the development of dual functional SPECT/fluorescent probes for the imaging of Abeta plaques in the brain.

Identification of mutation sites on Delta12 desaturase genes from Mortierella alpina 1S-4 mutants.

The mutation sites on the Delta12 desaturase gene in Mortierella alpina Delta12 desaturase-defective mutants SR88, TM912, and Mut48 accumulating Mead acid were identified. Each mutation resulted in an amino acid replacement (H116Y and P166L) in the Delta12 desaturase gene from SR88 and Mut48, respectively.

Radioimmunoscintigraphy of pancreatic cancer in tumor-bearing athymic nude mice using (99m)technetium-labeled anti-KL-6/MUC1 antibody.

PURPOSE: KL-6 is an extracellular epitope of MUC1, a membrane-penetrating glycoprotein, and its overexpression has been reported in pancreatic cancer. The aim of this study was to examine whether radiolabeled anti-KL-6/MUC1 antibody could be used for molecular imaging of pancreatic cancer in vivo. MATERIALS AND METHODS: Anti-KL-6/MUC1 antibody was labeled with 99mTC by the stannous reduction method. Immunoreactivity of the 99mTc-labeled anti-KL-6/MUC1 antibody was evaluated by a whole-cell binding study. In vivo experiments were performed by injecting the 99mTc-labeled anti-KL-6/MUC1 antibody into athymic nude mice bearing the KP-1NL pancreatic cancer cell line. RESULTS: A whole-cell binding study showed that the radiolabeled antibody retained its immunoreactivity. On scintigrams, the density of the tumors remained unchanged during the 16-32 h after injection, whereas that of the kidneys decreased time-dependently. The radioactivity levels of the kidneys and tumors were measured densitometrically, and we found that the intensity in the tumors relative to that in the kidneys increased time-dependently. Radioactivity levels were the highest in the blood 32 h after injection, and those in the liver, kidney, lung, and tumor were also rather high. CONCLUSION: 99mTc-labeled anti-KL-6/MUC1 antibody appears to be a promising agent as a tumor-specific radiotracer for pancreatic cancer.

Involvement of human blood arylesterases and liver microsomal carboxylesterases in nafamostat hydrolysis.

Metabolism of nafamostat, a clinically used serine protease inhibitor, was investigated with human blood and liver enzyme sources. All the enzyme sources examined (whole blood, erythrocytes, plasma and liver microsomes) showed nafamostat hydrolytic activity. V(max) and K(m) values for the nafamostat hydrolysis in erythrocytes were 278 nmol/min/mL blood fraction and 628 microM; those in plasma were 160 nmol/min/mL blood fraction and 8890 microM, respectively. Human liver microsomes exhibited a V(max) value of 26.9 nmol/min/mg protein and a K(m) value of 1790 microM. Hydrolytic activity of the erythrocytes and plasma was inhibited by 5, 5'-dithiobis(2-nitrobenzoic acid), an arylesterase inhibitor, in a concentration-dependent manner. In contrast, little or no suppression of these activities was seen with phenylmethylsulfonyl fluoride (PMSF), diisopropyl fluorophosphate (DFP), bis(p-nitrophenyl)phosphate (BNPP), BW284C51 and ethopropazine. The liver microsomal activity was markedly inhibited by PMSF, DFP and BNPP, indicating that carboxylesterase was involved in the nafamostat hydrolysis. Human carboxylesterase 2 expressed in COS-1 cells was capable of hydrolyzing nafamostat at 10 and 100 microM, whereas recombinant carboxylesterase 1 showed significant activity only at a higher substrate concentration (100 microM). The nafamostat hydrolysis in 18 human liver microsomes correlated with aspirin hydrolytic activity specific for carboxylesterase 2 (r=0.815, p<0.01) but not with imidapril hydrolysis catalyzed by carboxylesterase 1 (r=0.156, p=0.54). These results suggest that human arylesterases and carboxylesterase 2 may be predominantly responsible for the metabolism of nafamostat in the blood and liver, respectively.