Hemistepsin A inhibits T0901317-induced lipogenesis in the liver.

Hemistepsin A (HsA) is a guaianolide sesquiterpene lactone that inhibits hepatitis and liver fibrosis. We evaluated the effects of HsA on liver X receptor (LXR)-mediated hepatic lipogenesis in vitro and in vivo. Up to 10 µM, HsA did not affect the viability of HepG2 and Huh7 cells. Pretreatment with 5-10 µM HsA significantly decreased the luciferase activity of the LXR response element, which was transactivated by T0901317, GW 3965, and LXRα/retinoid X receptor α overexpression. In addition, it significantly inhibited the mRNA expression of LXRα in HepG2 and Huh7 cells. It also suppressed the expression of sterol regulatory element-binding protein-1c and lipogenic genes and reduced the triglyceride accumulation triggered by T0901317. Intraperitoneal injection of HsA (5 and 10 mg/kg) in mice significantly alleviated the T0901317-mediated increases in hepatocyte diameter and the percentage of regions in hepatic parenchyma occupied by lipid droplets. Furthermore, HsA significantly attenuated hepatic triglyceride accumulation by restoring the impaired expression of LXRα-dependent lipogenic genes caused by T0901317. Therefore, based on its inhibition of the LXRα-dependent signaling pathway, HsA has prophylactic potential for steatosis. [BMB Reports 2021; 54(2): 106-111].

Protein Kinase A Inhibitor H89 Attenuates Experimental Proliferative Vitreoretinopathy.

Purpose: This study aimed to explore the role of the protein kinase A (PKA) pathway in proliferative vitreoretinopathy (PVR) and the effect of the PKA inhibitor H89 on experimental PVR. Methods: Epiretinal membranes (ERMs) were acquired from PVR patients and analyzed by frozen-section immunofluorescence. An in vivo model was developed by intravitreal injecting rat eyes with ARPE-19 cells and platelet-rich plasma, and changes in eye structures and vision function were observed. An in vitro epithelial-mesenchymal transition (EMT) cell model was established by stimulating ARPE-19 cells with transforming growth factor (TGF)-ß. Alterations in EMT-related genes and cell function were detected. Mechanistically, PKA activation and activity were explored to assess the relationship between TGF-ß1 stimulation and the PKA pathway. The effect of H89 on the TGF-ß-Smad2/3 pathway was detected. RNA sequencing was used to analyze gene expression profile changes after H89 treatment. Results: PKA was activated in human PVR membranes. In vivo, H89 treatment protected against structural changes in the retina and prevented decreases in electroretinogram b-wave amplitudes. In vitro, H89 treatment inhibited EMT-related gene alterations and partially reversed the functions of the cells. TGF-ß-induced PKA activation was blocked by H89 pretreatment. H89 did not affect the phosphorylation or nuclear translocation of regulatory Smad2/3 but increased the expression of inhibitory Smad6. Conclusions: PKA pathway activation is involved in PVR pathogenesis, and the PKA inhibitor H89 can effectively inhibit PVR, both in vivo and in vitro. Furthermore, the protective effect of H89 is related to an increase in inhibitory Smad6.

Design and Synthesis of TASIN Analogues Specifically Targeting Colorectal Cancer Cell Lines with Mutant Adenomatous Polyposis Coli (APC).

Despite advances in targeted anticancer therapies, there are still no small-molecule-based therapies available that specifically target colorectal cancer (CRC) development and progression, the second leading cause of cancer deaths. We previously disclosed the discovery of truncating adenomatous polyposis coli (APC)-selective inhibitor 1 (TASIN-1), a small molecule that specifically targets colorectal cancer cells lines with truncating mutations in the adenomatous polyposis coli (APC) tumor suppressor gene through inhibition of cholesterol biosynthesis. Here, we report a medicinal chemistry evaluation of a collection of TASIN analogues and activity against colon cancer cell lines and an isogenic cell line pair reporting on the status of APC-dependent selectivity. A number of potent and selective analogues were identified, including compounds with good metabolic stability and pharmacokinetic properties. The compounds reported herein represent a first-in-class genotype-selective series that specifically target apc mutations present in the majority of CRC patients and serve as a translational platform toward a targeted therapy for colon cancer.

Muscarinic receptor signaling contributes to atypical antipsychotic drug reversal of the phencyclidine-induced deficit in novel object recognition in rats.

Enhancement of cholinergic function via muscarinic acetylcholine receptor M1 agonism improves cognition in some schizophrenia patients. Most atypical antipsychotic drugs, including clozapine and its active metabolite, N-desmethylclozapine, and lurasidone, enhance the release of acetylcholine in key brain regions involved in cognition (e.g. hippocampus). We determined the effect of muscarinic acetylcholine receptor M1 stimulation on novel object recognition and its contribution to the ability of atypical antipsychotic drugs to reverse the novel object recognition deficit in rats withdrawn from subchronic phencyclidine, a rodent model of cognitive impairment in schizophrenia. In control rats, the non-specific muscarinic acetylcholine receptor antagonist, scopolamine, and the M1 selective antagonist, VU0255035, induced a novel object recognition deficit, which was reversed by the M1 agonist, AC260584. Scopolamine fully blocked the effect of clozapine and N-desmethylclozapine, but not lurasidone, to restore novel object recognition in subchronic phencyclidine-treated rats. VU0255035 also blocked these effects of clozapine and N-desmethylclozapine, but not lurasidone; however, the blockade was not as complete as that achieved with scopolamine. Furthermore, subchronic phencyclidine increased hippocampal M1 mRNA expression. These data suggest that M1 agonism is required for clozapine and N-desmethylclozapine to ameliorate the phencyclidine-induced deficit in novel object recognition, additional evidence that M1 agonism is a potential target for treating cognitive impairment in schizophrenia.

Attenuating Nicotine Reinforcement and Relapse by Enhancing Endogenous Brain Levels of Kynurenic Acid in Rats and Squirrel Monkeys.

The currently available antismoking medications have limited efficacy and often fail to prevent relapse. Thus, there is a pressing need for newer, more effective treatment strategies. Recently, we demonstrated that enhancing endogenous levels of kynurenic acid (KYNA, a neuroinhibitory product of tryptophan metabolism) counteracts the rewarding effects of cannabinoids by acting as a negative allosteric modulator of α7 nicotinic receptors (α7nAChRs). As the effects of KYNA on cannabinoid reward involve nicotinic receptors, in the present study we used rat and squirrel monkey models of reward and relapse to examine the possibility that enhancing KYNA can counteract the effects of nicotine. To assess specificity, we also examined models of cocaine reward and relapse in monkeys. KYNA levels were enhanced by administering the kynurenine 3-monooxygenase (KMO) inhibitor, Ro 61-8048. Treatment with Ro 61-8048 decreased nicotine self-administration in rats and monkeys, but did not affect cocaine self-administration. In rats, Ro 61-8048 reduced the ability of nicotine to induce dopamine release in the nucleus accumbens shell, a brain area believed to underlie nicotine reward. Perhaps most importantly, Ro 61-8048 prevented relapse-like behavior when abstinent rats or monkeys were reexposed to nicotine and/or cues that had previously been associated with nicotine. Ro 61-8048 was also effective in monkey models of cocaine relapse. All of these effects of Ro 61-8048 in monkeys, but not in rats, were reversed by pretreatment with a positive allosteric modulator of α7nAChRs. These findings suggest that KMO inhibition may be a promising new approach for the treatment of nicotine addiction.

NO synthase inhibition attenuates EDHF-mediated relaxation induced by TRPV4 channel agonist GSK1016790A in the rat pulmonary artery: Role of TxA2.

BACKGROUND: The aim of the present study was to observe the concomitant activation of nitric oxide (NO) and endothelium-derived hyperpolarizing factor (EDHF) pathways by TRPV4 channel agonist GSK1016790A in the rat pulmonary artery and explore the mechanism by which NO synthase inhibition attenuates EDHF-mediated relaxation in endothelium-intact rat pulmonary artery. METHODS: Tension experiments were conducted on the pulmonary artery from male Wistar rats. RESULTS: TRPV4 channel agonist GSK1016790A (GSK) caused concentration-dependent relaxation (Emax 86.9±4.6%; pD2 8.7±0.24) of the endothelium-intact rat pulmonary artery. Combined presence of apamin and TRAM-34 significantly attenuated the relaxation (Emax 61.1±6.0%) to GSK. l-NAME (100µM) significantly attenuated (8.2±2.9%) the relaxation response to GSK that was resistant to apamin plus TRAM-34. However, presence of ICI192605 or furegrelate alongwith l-NAME revealed the GSK-mediated EDHF-response (Emax of 28.5±5.2%; Emax 24.5±4.3%) in this vessel, respectively. Further, these two TxA2 modulators (ICI/furegrelate) alongwith l-NAME had no effect on SNP-induced endothelium-independent relaxation in comparison to l-NAME alone. This EDHF-mediated relaxation was sensitive to inhibition by K(+) channel blockers apamin and TRAM-34 or 60mMK(+) depolarizing solution. Further, combined presence of apamin and TRAM-34 in U46619 pre-contracted pulmonary arterial rings significantly reduced the maximal relaxation (Emax 71.6±6.9%) elicited by GSK, but had no effect on the pD2 (8.1±0.03) of the TRPV4 channel agonist in comparison to controls (Emax, 92.4±4.3% and pD2, 8.3±0.06). CONCLUSION: The present study suggests that NO and EDHF are released concomitantly and NO synthase inhibition attenuates GSK-induced EDHF response through thromboxane pathway in the rat pulmonary artery.

Fluorescent sulfonamide carbonic anhydrase inhibitors incorporating 1,2,3-triazole moieties: Kinetic and X-ray crystallographic studies.

Fluorescent sulfonamide carbonic anhydrase (CA, EC 4.2.1.1) inhibitors (CAIs) were essential for demonstrating the role played by the tumor-associated isoform CA IX in acidification of tumors, cancer progression towards metastasis and for the development of imaging and therapeutic strategies for the management of hypoxic tumors which overexpress CA IX. However, the presently available such compounds are poorly water soluble which limits their use. Here we report new fluorescent sulfonamides 7, 8 and 10 with increased water solubility. The new derivatives showed poor hCA I inhibitory properties, but were effective inhibitors against the hCA II (KIs of 366-127 nM), CA IX (KIs of 8.1-36.9 nM), CA XII (KIs of 4.1-20.5 nM) and CA XIV (KIs of 12.8-53.6 nM). A high resolution X-ray crystal structure of one of these compounds bound to hCA II revealed the factors associated with the good inhibitory properties. Furthermore, this compound showed a three-fold increase of water solubility compared to a similar derivative devoid of the triazole moiety, making it an interesting candidate for ex vivo/in vivo studies.

PTEN upregulation may explain the development of insulin resistance and type 2 diabetes with high dose statins.

PURPOSE: Statins increase the incidence of new onset diabetes. Prolonged statin therapy upregulates PTEN expression. PTEN levels are also elevated in diabetic animals. Activation of protein kinase A by cAMP decreases PTEN expression. We assessed whether prolonged treatment with rosuvastatin (ROS) induces glucose intolerance by upregulating Phosphatase and Tensin Homologue on Chromosome 10 (PTEN) in mice receiving normal (ND) or Western Diet (WD) and whether concomitant treatment with cilostazol (CIL, a phosphodiesterase-3 inhibitor) attenuates the effects. METHODS: PTEN(loxp/cre) or PTEN(+/-) mice received ND or WD without or with ROS (10 mg/kg/day). Wild-type mice received ND or WD without or with ROS, CIL (10 mg/kg/day), or ROS+CIL for 30 days. Fasting insulin and glucose tolerance test were measured as well as PTEN and P-AKT levels in skeletal muscle. RESULTS: Serum glucose after intraperitoneal injection of glucose was higher in PTEN(loxp/cre) mice receiving WD or ROS and especially WD+ROS. Levels were lower in PTEN(+/-) mice compared to PTEN(loxp/cre) in each treatment group. CIL decreased glucose levels in mice receiving WD, ROS and their combination. Insulin levels were higher in the WD+ROS group. CIL decreased insulin in mice receiving WD+ROS. WD, ROS and especially their combination increased PTEN and decreased P-AKT levels. CIL attenuated the effect of WD, ROS and their combination. CONCLUSIONS: Long-term ROS can induce diabetes by upregulating PTEN. CIL attenuates these changes. Partial knockdown of PTEN also ameliorates ROS-induced insulin resistance. Further studies are needed to assess the effects of increasing cAMP levels to prevent the induction of diabetes by statins.

A BSL-4 high-throughput screen identifies sulfonamide inhibitors of Nipah virus.

Nipah virus is a biosafety level 4 (BSL-4) pathogen that causes severe respiratory illness and encephalitis in humans. To identify novel small molecules that target Nipah virus replication as potential therapeutics, Southern Research Institute and Galveston National Laboratory jointly developed an automated high-throughput screening platform that is capable of testing 10,000 compounds per day within BSL-4 biocontainment. Using this platform, we screened a 10,080-compound library using a cell-based, high-throughput screen for compounds that inhibited the virus-induced cytopathic effect. From this pilot effort, 23 compounds were identified with EC50 values ranging from 3.9 to 20.0 µM and selectivities >10. Three sulfonamide compounds with EC50 values <12 µM were further characterized for their point of intervention in the viral replication cycle and for broad antiviral efficacy. Development of HTS capability under BSL-4 containment changes the paradigm for drug discovery for highly pathogenic agents because this platform can be readily modified to identify prophylactic and postexposure therapeutic candidates against other BSL-4 pathogens, particularly Ebola, Marburg, and Lassa viruses.

Fluorescence amplified detection of proteases by the catalytic activation of a semisynthetic sensor.

A general approach to mimic the sensing scheme of allosteric enzymes is developed. Through the covalent labeling of a sulfonamide inhibitor to the enzyme HCAII via SNAP-tag protein, the enzyme is rendered inactive. Catalytic activation is triggered only when a protease is present to cleave the recognized peptide sequence.

Resveratrol suppresses T0901317-induced hepatic fat accumulation in mice.

Liver X receptor (LXR) has been identified as a potential target for treatment of atherosclerosis and diabetes. Activation of LXR, however, is associated with increased lipogenesis and fat accumulation in the liver. The objective of the current study was to examine the effect of resveratrol on LXR activator-induced fat accumulation in liver using mice as an animal model. Three groups of C57BL/6 mice were studied. Animals in group 1 were treated with T0901317, a potent activator of LXR in mice. Animals in group 2 served as the control and were treated with carrier solution and those in group 3 were treated with T0901317/resveratrol combination. Using histochemical and biochemical methods, we demonstrate that resveratrol treatment significantly suppressed fat accumulation in the liver induced by T0901317. In addition, resveratrol completely blocked elevation of blood levels of triglyceride and cholesterol and reduced blood glucose level. Quantitative PCR analysis revealed that resveratrol treatment did not change the mRNA levels of abca1, abcg1, cyp7a1, srebp-1c, chrebp, and acc genes compared to that of animals treated with T0901317 alone but reduced pepck and g6p gene expressions. Immunohistochemistry and Western blot analyses show resveratrol treatment activated AMP-activated protein kinase (AMPK) and increased phosphorylation of acetyl-CoA carboxylase. Treatment with T0901317 on hepatocytes increased intracellular fat accumulation and this increase was suppressed by resveratrol; the suppressive effect of resveratrol was greatly repressed by Compound C which is an inhibitor of AMPK. Collectively, these data suggest that resveratrol blocks T0901317-induced lipid accumulation in the liver and can be considered for inclusion into the treatment of diseases involving activation of liver X receptor.

Proton-in-flight mechanism for the spontaneous hydrolysis of N-methyl O-phenyl sulfamate: implications for the design of steroid sulfatase inhibitors.

The hydrolysis of N-methyl O-phenyl sulfamate (1) has been studied as a model for steroid sulfatase inhibitors such as Coumate, 667 Coumate, and EMATE. At neutral pH, simulating physiological conditions, hydrolysis of 1 involves an intramolecular proton transfer from nitrogen to the bridging oxygen atom of the leaving group. Remarkably, this proton transfer is estimated to accelerate the decomposition of 1 by a factor of 10(11). Examination of existing kinetic data reveals that the sulfatase PaAstA catalyzes the hydrolysis of sulfamate esters with catalytic rate accelerations of ~10(4), whereas the catalytic rate acceleration generated by the enzyme for its cognate substrate is on the order of ~10(15). Rate constants for hydrolysis of a wide range of sulfuryl esters, ArOSO(2)X(-), are shown to be correlated by a two-parameter equation based on pK(a)(ArOH) and pK(a)(ArOSO2XH).

Cloning, characterization and sulfonamide inhibition studies of an α-carbonic anhydrase from the living fossil sponge Astrosclera willeyana.

The α-carbonic anhydrase (CA, EC 4.2.1.1) Astrosclerin-3 previously isolated from the living fossil sponge Astrosclera willeyana (Jackson et al., Science 2007, 316, 1893), was cloned, kinetically characterized and investigated for its inhibition properties with sulfonamides and sulfamates. Astrosclerin-3 has a high catalytic activity for the CO(2) hydration reaction to bicarbonate and protons (k(cat) of 9.0×10(5) s(-1) and k(cat)/K(m) of 1.1×10(8) M(-1) × s(-1)), and is inhibited by various aromatic/heterocyclic sulfonamides and sulfamates with inhibition constants in the range of 2.9 nM-8.85 µM. Astrosclerin, and the human isoform CA II, display similar kinetic properties and affinities for sulfonamide inhibitors, despite more than 550 million years of independent evolution. Because Astrosclerin-3 is involved in biocalcification, the inhibitors characterized here may be used to gain insights into such processes in other metazoans.

The Akt inhibitor MK2206 synergizes, but perifosine antagonizes, the BRAF(V600E) inhibitor PLX4032 and the MEK1/2 inhibitor AZD6244 in the inhibition of thyroid cancer cells.

PURPOSE: The purpose of the study was to explore optimal combinations of currently actively developed drugs for dually targeting the Ras → Raf → MAPK kinase (MEK) → MAPK/ERK (MAPK) and the phosphatidylinositol 3-kinase/Akt pathways as effective treatments for thyroid cancer. EXPERIMENTAL DESIGN: We tested the combinations of the Akt inhibitors MK2206 or perifosine with the BRAF(V600E) inhibitor PLX4032 or the MEK1/2 inhibitor AZD6244 in thyroid cancer cells harboring both the BRAF(V600E) and PIK3CA mutations. RESULTS: We found that MK2206 could potently, when used alone, and synergistically, when combined with either PLX4032 or AZD6244, inhibit thyroid cancer cell growth with all the combination index values lower than 1. Perifosine could potently inhibit thyroid cancer cell growth when used alone, but a strong antagonism occurred between this drug and PLX4032 or AZD6244 in the inhibition of thyroid cancer cell growth with all combination index values higher than 1. Combinations of MK2206 with PLX4032 or AZD6244 dramatically enhanced G1 cell cycle arrest induced by each drug alone. However, G2 cell cycle arrest uniquely induced by perifosine alone and G1 cell cycle arrest induced by PLX4032 or AZD6244 were both reversed by combination treatments, providing a mechanism for their antagonism. All these drugs could correspondingly inhibit the MAPK and phosphatidylinositol 3-kinase/Akt signalings, confirming their expected target effects. CONCLUSIONS: We demonstrated, unexpectedly, opposite outcomes of MK2206 and perifosine in their combinational treatments with BRAF(V600E)/MEK inhibitors in thyroid cancer cells. The data may help appropriate selection of these prominent drugs for clinical trials of combination therapies for thyroid cancer.

Soybean glycinin improves HDL-C and suppresses the effects of rosuvastatin on hypercholesterolemic rats.

BACKGROUND: This study was an investigation of the effects of ingesting a daily dose of isolated glycinin soy protein (11S globulin), in association with rosuvastatin, on the control of hypercholesterolemia in experimental animals. METHODS: Male Wistar rats were kept in individual cages under appropriate controlled conditions of temperature, light and humidity. The animals were divided into five groups (n = 9): 1) standard (STD): fed on casein as protein source; 2) hypercholesterolemic (HC): STD plus 1% cholesterol and 0.5% cholic acid; 3) HC+11S: hypercholesterolemic + glycinin (300 mg/kg/day); 4) HC+ROS: hypercholesterolemic + rosuvastatin (10 mg/kg/day); 5) HC+11S+ROS: HC diet, the 11S protein and the drug in the doses given in (3) and (4). The protein and the drug were administered by gavage for 28 days. The results indicated that the addition of 1% cholesterol and 0.5% cholic acid induced hypercholesterolemia in the animals without interfering with their weight gain. RESULTS: A single daily dose of glycinin contributed an additional 2.8% of dietary protein intake and demonstrated its functional role, particularly in raising HDL-C, decreasing triglycerides in the liver and improving the atherogenic index in animals exposed to a hypercholesterolemic diet. CONCLUSION: Most of the beneficial effects of the isolated treatments disappeared when the drug (rosuvastatin) and the protein (glycinin) were taken simultaneously. The association was shown not to interact additively, as noted in the plasma levels of total cholesterol and non-HDL cholesterol, and in the significant increase of cholesterol in the liver. Studies are in progress to identify the effects of peptides derived from the 11S globulin and their role in cholesterol metabolism.

Inhibition of LXRα-dependent steatosis and oxidative injury by liquiritigenin, a licorice flavonoid, as mediated with Nrf2 activation.

Liver X receptor-α (LXRα) functions as a major regulator of lipid homeostasis through activation of sterol regulatory element binding protein-1c (SREBP-1c), which promotes hepatic steatosis and steatohepatitis. NF-E2-related factor 2 (Nrf2) is the crucial transcription factor that is necessary for the induction of antioxidant enzymes. This study investigated the potential of liquiritigenin (LQ), a hepatoprotective flavonoid in licorice, to inhibit LXRα-induced hepatic steatosis, and the underlying mechanism of the action. LQ treatment attenuated fat accumulation and lipogenic gene induction in the liver of mice fed a high fat diet. Also, LQ had the ability to inhibit oxidative liver injury, as shown by decreases in thiobarbituric acid reactive substances formation and nitrotyrosinylation. Moreover, LQ treatment antagonized LXRα agonist (T0901317)-mediated SREBP-1c activation, and transactivation of the lipogenic target genes. LQ was found to activate Nrf2, and the ability of LQ to inhibit LXRα-mediated SREBP-1c activation was reversed by Nrf2 deficiency, which supports the inhibitory role of Nrf2 in LXRα-dependent lipogenesis. Consistently, treatment with other Nrf2 activators or forced expression of Nrf2 also inhibited LXRα-mediated SREBP-1c activation. Our results demonstrate that LQ has an efficacy to activate Nrf2, which contributes to inhibiting the activity of LXRα that leads to SREBP-1c induction and hepatic steatosis.

A novel selective muscarinic acetylcholine receptor subtype 1 antagonist reduces seizures without impairing hippocampus-dependent learning.

Previous studies suggest that selective antagonists of specific subtypes of muscarinic acetylcholine receptors (mAChRs) may provide a novel approach for the treatment of certain central nervous system (CNS) disorders, including epileptic disorders, Parkinson's disease, and dystonia. Unfortunately, previously reported antagonists are not highly selective for specific mAChR subtypes, making it difficult to definitively establish the functional roles and therapeutic potential for individual subtypes of this receptor subfamily. The M(1) mAChR is of particular interest as a potential target for treatment of CNS disorders. We now report the discovery of a novel selective antagonist of M(1) mAChRs, termed VU0255035 [N-(3-oxo-3-(4-(pyridine-4-yl)piperazin-1-yl)propyl)-benzo[c][1,2,5]thiadiazole-4 sulfonamide]. Equilibrium radioligand binding and functional studies demonstrate a greater than 75-fold selectivity of VU0255035 for M(1) mAChRs relative to M(2)-M(5). Molecular pharmacology and mutagenesis studies indicate that VU0255035 is a competitive orthosteric antagonist of M(1) mAChRs, a surprising finding given the high level of M(1) mAChR selectivity relative to other orthosteric antagonists. Whole-cell patch-clamp recordings demonstrate that VU0255035 inhibits potentiation of N-methyl-D-aspartate receptor currents by the muscarinic agonist carbachol in hippocampal pyramidal cells. VU0255035 has excellent brain penetration in vivo and is efficacious in reducing pilocarpine-induced seizures in mice. We were surprised to find that doses of VU0255035 that reduce pilocarpine-induced seizures do not induce deficits in contextual freezing, a measure of hippocampus-dependent learning that is disrupted by nonselective mAChR antagonists. Taken together, these data suggest that selective antagonists of M(1) mAChRs do not induce the severe cognitive deficits seen with nonselective mAChR antagonists and could provide a novel approach for the treatment certain of CNS disorders.

Inhibition of neutrophil elastase reduces lung injury and bacterial count in hamsters.

Neutrophil elastase (NE) has been recognized as a double-edge sword as it can be both host-defensive and pro-inflammatory depending on its tissue level and microenvironment. The present study was designed to examine whether inhibition of NE activity by sivelestat, a specific NE inhibitor with low cellular permeability, is beneficial for acute lung injury induced by Streptococcus pneumoniae in hamsters. Intratracheal inoculation of live S. pneumoniae (1.5 x 10(7) CFU/Lung) into hamsters caused acute lung injury characterized by an increase in lung alveolar permeability and leukocytes accumulation in the lungs. These pathophysiological changes, which were associated with elevation of NE activity in the bronchoalveolar lavage fluid (BALF), were transient but remained high 4-22 h post-bacterial inoculation. Intravenous infusion of sivelestat at 3mg/kg/h 0-22 h after bacterial inoculation reduced the increase in NE activity and lung alveolar permeability, as indicated by leakage of pre-injected human serum albumin into BALF, without affecting the number of leukocytes in BALF and lung interstitial tissue. Interestingly, the number of bacteria in BALF and lung interstitial tissue was also reduced by sivelestat to less than 10% of the control values. Furthermore, S. pneumoniae-induced decrease in the level of surfactant protein D (SP-D), a protein that protects against bacterial infection, was preserved by sivelestat treatment with a significant negative correlation between NE activity and SP-D content in BALF. These results suggest that sivelestat is beneficial in acute lung injury associated with lung bacterial infection and that this inhibitor rather decreases infection by preserving host defense system.

A novel antagonist of prostaglandin D2 blocks the locomotion of eosinophils and basophils.

BACKGROUND: Chemoattractant receptor homologous molecule of Th2 cells (CRTH2) has been shown to mediate the chemotaxis of eosinophils, basophils and Th2-type T lymphocytes. The major mast cell product prostaglandin (PG) D(2) is considered to be the principal ligand of CRTH2. MATERIALS AND METHODS: We developed a novel CRTH2 antagonist, AZ11665362 [2,5-dimethyl-3-(8-methylquinolin-4-yl)-1H-indole-1-yl]acetic acid, and characterized its efficacy in binding assay in HEK293 cells, eosinophil and basophil shape change assay and migration assay, platelet aggregation and eosinophil release from guinea pig bone marrow. The effects were compared with ramatroban, the sole CRTH2 antagonist clinically available to date. RESULTS: AZ11665362 bound with high affinity to human and guinea pig CRTH2 expressed in HEK293 cells and antagonized eosinophil and basophil shape change responses to PGD(2). AZ11665362 was without effect on the PGD(2)-induced inhibition of platelet aggregation. In contrast, AZ11665362 effectively inhibited the in vitro migration of human eosinophils and basophils towards PGD(2). The release of eosinophils from the isolated perfused hind limb of the guinea pig was potently stimulated by PGD(2), and this effect was prevented by AZ11665362. In all assays tested, AZ11665362 was at least 10 times more potent than ramatroban. CONCLUSIONS: AZ11665362 is a potent CRTH2 antagonist that is capable of blocking the migration of eosinophils and basophils, and the rapid mobilization of eosinophils from bone marrow. AZ11665362 might hence be useful for the treatment of allergic diseases.