Bioassay-guided isolation of fatty acid synthase inhibitory diterpenoids from the roots of <Emphasis Type="Italic">Salvia miltiorrhiza</Emphasis> Bunge

Fatty acid synthase (FAS) is considered as a novel drug target for the development of anticancer and anti-obesity agents. Bioassay-guided fractionation of a n-hexane-soluble extract prepared from the roots of Salvia miltiorrhiza Bunge (Labiatae), using an in vitro enzyme assay, led to the isolation of five abietane diterpenoids: 15,16-dihydrotanshinone I (1), cryptotanshinone (2), tanshinone I (3), tanshinone IIA (4), and dansenspiroketallactone (5). Compounds 1–5 were tested for their in vitro FAS inhibitory activity and, except for compound 5 (IC₅₀ > 100 μM), compounds 1–4 inhibited the enzyme activity with IC₅₀ values ranging from 12.0 to 30.3 μM. Our findings may be partially related to the anticancer activity of abietane diterpenoids from the plant, suggesting a further study on the anticancer potential of tanshinone derivatives.     

Anti-allergic activity of an ethanol extract from <Emphasis Type="Italic">Salviae miltiorrhiza</Emphasis>

As part of an ongoing investigation aimed at the discovery of novel bioactive medicinal herbs with anti-inflammatory properties, the effects of an ethanolic extract from the parts of Salviae miltiorrhiza Bunge (ESM) were evaluated using in vitro and in vivo animal model analysis. ESM inhibited cyclooxygenase-2 (COX-2) and COX-1-dependent phases of prostaglandin D₂ (PGD₂) generation in bone marrow-derived mast cells (BMMC) in a concentration-dependent manner with IC₅₀ values of 3.96 μg/mL and 21.54 μg/mL, respectively. Furthermore, ESM inhibited leukotriene C₄ (LTC₄) production with an IC₅₀ value of 2.6 μg/mL. These results clearly demonstrated the dual COX-2 selective/5-lipoxygenase inhibitory activity that ESM possessed. ESM strongly inhibited a degranulation reaction in a dose dependent manner within a BMMC system, with an IC₅₀ value of 22.4 μg/mL. Additionally, ESM was tested in a rat passive cutaneous anaphylaxis (PCA) reaction assay by oral administration (25 to 100 mg/kg). ESM dose-dependently inhibited the PCA reaction, which was activated by anti-dinirophenyl (DNP) IgE. These results suggested that ESM might be beneficial in regulating various allergic reactions.     

Capsaicin inhibits catecholamine secretion and synthesis by blocking Na+ and Ca2+ influx through a vanilloid receptor-independent pathway in bovine adrenal medullary cells

We report here the effects of capsaicin, a flavoring ingredient in the hot pepper Capsicum family, on catecholamine secretion and synthesis in cultured bovine adrenal medullary cells. Capsaicin inhibited catecholamine secretion (IC₅₀=9.5, 11.8, and 62 μM) stimulated by carbachol, an agonist of the nicotinic acetylcholine receptor, by veratridine, an activator of voltage-dependent Na⁺ channels, and by high K⁺, an activator of voltage-dependent Ca²⁺ channels, respectively. Capsaicin also suppressed carbachol-induced ²²Na⁺ influx (IC₅₀=5.0 μM) and ⁴⁵Ca²⁺ influx (IC₅₀=24.4 μM), veratridine-induced ²²Na⁺ influx (IC₅₀=2.4 μM) and ⁴⁵Ca²⁺ influx (IC₅₀=1.1 μM), and high K⁺-induced ⁴⁵Ca²⁺ influx (IC₅₀=5.8 μM). The reduction in catecholamine secretion caused by capsaicin was not overcome by increasing the concentration of carbachol. Furthermore, capsazepine (10 μM), a competitive antagonist for the transient receptor potential vanilloid 1, and ruthenium red (30 μM), a nonselective cation channel antagonist, did not block the inhibition by capsaicin of catecholamine secretion. Capsaicin also suppressed both basal and carbachol-stimulated ¹⁴C-catecholamine synthesis (IC₅₀=10.6 and 26.4 μM, respectively) from [¹⁴C] tyrosine but not from l-3, 4-dihydroxyphenyl [3-¹⁴C] alanine ([¹⁴C] DOPA) as well as tyrosine hydroxylase activity (IC₅₀=8.4 and 39.0 μM, respectively). The present findings suggest that capsaicin inhibits catecholamine secretion and synthesis via suppression of Na⁺ and Ca²⁺ influx through a vanilloid receptor-independent pathway.     

Effects of the novel amiodarone-like compound SAR114646A on cardiac ion channels and ventricular arrhythmias in rats

Amiodarone is the "gold standard" for current antiarrhythmic therapy because it combines efficacy with good hemodynamic and electrophysiological tolerance. Amiodarone is effective against both atrial and ventricular arrhythmias by intravenous (i.v.) or oral route. However, the i.v. formulation has limitations. Therefore, we identified SAR114646A, an amiodarone-like antiarrhythmic agent with good aqueous solubility suitable for i.v. application. Patch–clamp experiments were performed with isolated cardiomyocytes from guinea pigs and rats. In guinea pig ventricular cardiomyocytes, the fast Na⁺ channel and the L-type Ca²⁺ channels were blocked by SAR114646A with half-maximal concentrations (IC₅₀) of 2.0 and 1.1 μM, respectively. The tail current of the fast activating rectifying potassium channel I_Kr was blocked with an IC₅₀ value of 0.6 μM, whereas the IC₅₀ values for inhibition of the I_Ks and I_K1 channels was >10 μM. ATP-sensitive K⁺ channels were evoked by application of the channel opener rilmakalim (3 μM). SAR114646A blocked this current with an IC₅₀ value of 2.8 μM. In guinea pig atrial cardiomyocytes, carbachol (1 μM) was used to activate the I_KACh and SAR114646A inhibited this current with IC₅₀ of 36.5 nM. The transient outward current I_to and the sustained current I_sus were investigated in rat ventricular myocytes. SAR114646A blocked these currents with IC₅₀ of 1.8 and 1.2 μM, respectively. When expressed in Chinese hamster ovary cells, the currents hKv1.5 and hHCN4 were inhibited with IC₅₀ values of 1.1 and 0.4 μM, respectively. Micropuncture experiments in isolated rabbit left atria revealed that SAR114646A prolonged the 50% repolarization significantly at 3 and 10 μM. In guinea pig papillary muscle, the APD at 90% of repolarization was slightly prolonged at 3 and 10 μM. SAR114646A demonstrates antiarrhythmic activity in anaesthetised rats, subjected to 5 min ischemia followed by 10 min reperfusion, where 1 mg/kg of SAR114646A applied as i.v. bolus 5 min prior to ischemia, decreased mortality to 0% compared to 80% under control conditions. In conclusion, SAR114646A is a multichannel blocker with improved water solubility, compared to amiodarone. In contrast to amiodarone, SAR114646A also blocks the K⁺ channels I_to and I_sus. A potent antiarrhythmic effect as observed in rats can also be expected in other animal models.     

<Emphasis Type="Italic">In vitro</Emphasis> anti-inflammatory activity of carvacrol: Inhibitory effect on COX-2 catalyzed prostaglandin E<Subscript>2</Subscript> biosynthesisb

Possible anti-inflammatory effect of carvacrol was evaluated by in vitro cyclooxygenase-2 (COX-2) assay. Carvacrol inhibited production of prostaglandin E₂ catalysed by COX-2 with an IC₅₀ value of 0.8 μM what is practically the same concentration as the IC₅₀ obtained for the standard inhibitors indomethacin and NS-398 with values of 0.7 μM and 0.8 μM, respectively. The COX-1 was inhibited approximately at the same rate (IC₅₀ of 0.7 μM for carvacrol), which suggests non-selective inhibition of both enzyme isoforms. The results of the study demonstrate possible anti-inflammatory potential of this compound due to the inhibition of inducible COX-2 isoform.     

Inhibition of erythrocyte “apoptosis” by catecholamines

Osmotic shock, oxidative stress and Cl⁻ removal activate a non-selective Ca²⁺-permeable cation conductance in human erythrocytes. The entry of Ca²⁺ leads to activation of a scramblase with subsequent exposure of phosphatidylserine at the cell surface. Phosphatidylserine mediates binding to phosphatidylserine receptors on macrophages which engulf and degrade phosphatidylserine exposing cells. Moreover, phosphatidylserine exposure may lead to adherence of erythrocytes to the vascular wall. In the present study, we explored whether activation of the non-selective cation conductance and subsequent phosphatidylserine exposure might be influenced by catecholamines. Phosphatidylserine exposure has been determined by FITC-annexin V binding while cell volume was estimated from forward scatter in FACS analysis. Removal of Cl⁻ enhanced annexin binding and decreased forward scatter, an effect significantly blunted by the β agonist isoproterenol (IC₅₀ approx. 1 μM). Fluo-3 fluorescence measurements revealed an increase of cytosolic Ca²⁺ activity following Cl⁻ removal, an effect again significantly blunted by isoproterenol exposure (10 μM). Whole-cell patch-clamp experiments performed in Cl⁻ free bath solution indeed disclosed a time-dependent inactivation of a non-selective cation conductance following isoproterenol exposure (10 μM). Phenylephrine (IC₅₀<10 μM), dobutamine (IC₅₀ approx. 1 μM) and dopamine (IC₅₀ approx. 3 μM) similarly inhibited the effect of Cl⁻ removal on annexin binding and forward scatter. In conclusion, several catecholamines inhibit the Cl⁻ removal-activated Ca²⁺ entry into erythrocytes, thus preventing increase of cytosolic Ca²⁺ activity, subsequent cell shrinkage and activation of erythrocyte scramblase. The catecholamines thus counteract erythrocyte phosphatidylserine exposure and subsequent clearance of erythrocytes from circulating blood.     

Inhibition of DNA topoisomerases I and II and cytotoxicity of compounds from <Emphasis Type="Italic">Ulmus davidiana</Emphasis> var. <Emphasis Type="Italic">japonica</Emphasis>

Twenty five compounds including ten triterpenes (1–3, 5–11), six flavonoids (12–15, 24, 25), five lignans (17, 18, 21–23), two butenyl clohexnone glycosides (19–20), one fructofuranoside (16) and one fatty acid (4) were isolated from the roots of Ulmus davidiana var. japonica. The structures of those compounds were identified by comparing their physicochemical and spectral data with those of published in literatures. All the compounds were evaluated for DNA topoisomerase inhibitory activities and cytotoxicities. Among the purified compounds, 4 and 19 showed more potent inhibitory acitivities (IC₅₀: 39 and 19 μM, respectively) than camptothecin, as the positive control (IC₅₀: 46 μM) against topoisomerase I. Compounds, 4, 10, 12, 19, 24 and 25 showed strong inhibitory activities toward DNA topoisomerase II (IC₅₀: 0.1, 0.52, 0.47, 0.42, 0.17 μM and 17 nM, respectively), which were more potent than that of etoposide as positive control (IC₅₀: 20 μM). In A549 cell line, 5 and 6 showed cytotoxicities (IC₅₀: 4 μM and 3 μM, respectively, with IC₅₀ of camptothecin as positive control: 10.3 μM). In the HepG2 cell line, 3, 5 and 7 showed cytotoxicity (IC₅₀: 4, 3 and 4 μM, respectively, with IC₅₀ of camptothecin: 0.3 μM). Compounds 6, 12 and 23 showed cytotoxicities in the HT-29 cell line (IC₅₀: 19, 19 and 15 μM, respectively, with IC₅₀ of camptothecin: 2 μM).     

Potent mechanism-based inhibition of human CYP3A in vitro by amprenavir and ritonavir: comparison with ketoconazole

Objective: Biotransformation of triazolam to its α-hydroxy and 4-hydroxy metabolites by human liver microsomes in vitro was used as an index of human cytochrome P ₄₅₀ 3A (CYP3A) activity. Results: The reaction was strongly inhibited by co-incubation with the viral protease inhibitors ritonavir (IC₅₀=0.14 μM) and amprenavir (IC₅₀=2.5–2.9 μM), and by the azole derivative ketoconazole (IC₅₀ = 0.07 μM). Pre-incubation of microsomes with ritonavir or amprenavir increased inhibitory potency (IC₅₀ reduced to 0.07 μM and 1.4 μM, respectively). This was not the case with ketoconazole. Conclusions: Thus, ritonavir and amprenavir are highly potent mechanism-based inhibitors of human CYP3A isoforms. Received: 11 January 2000 / Accepted in revised form: 9 March 2000     

Anti-inflammatory activity of 6-hydroxy-2,7-dimethoxy-1,4-henanthraquinone from tuberous roots of yam (Dioscorea batatas) through inhibition of prostaglandin D₂ and leukotriene C₄ production in mouse bone marrow-derived mast cells

6-Hydroxy-2,7-dimethoxy-1,4-phenanthraquinone (PAQ) isolated from the tuberous roots of Yam (Dioscorea batatas) inhibited cyclooxygenase-2 (COX-2) and cyclooxygenase-1 (COX-1) dependent prostaglandin D₂ (PGD₂) generation in mouse bone marrow-derived mast cells in a concentration-dependent manner with IC₅₀ values of 0.08 μM and 0.27 μM, respectively. In the Western blotting with specific anti-COX-2 antibodies, the decrease of the quantity of PGD₂ was accompanied by a decrease in the COX-2 protein level. But PAQ did not affect COX-1 protein level. In addition, this compound inhibited 5-lipoxygenase (5-LOX) dependent production of leukotriene C₄ in a dose-dependent manner, with an IC₅₀ of 0.032 μM. These results demonstrate that PAQ has a dual COX-2/5-LOX inhibitory activity. This compound also inhibited the degranulation reaction in a dose-dependent manner with an IC₅₀ of 2.7 μM. Thus, these results suggest that PAQ may be useful in regulating mast cell-mediated inflammatory diseases.     

Effects of propofol on the leukocyte nitric oxide pathway: in vitro and ex vivo studies in surgical patients

This study was designed to evaluate the mechanism by which propofol modifies leukocyte production of nitric oxide (NO) in humans. In vitro experiments used whole blood from healthy volunteers (n = 10 samples/experiment). Ex vivo experiments studied the effects of an intravenous dose of 2.5 mg propofol per kilogram body weight followed by intravenous infusion of 4 mg kg⁻¹ h⁻¹ in surgical patients in ASA class I or II (n = 20). In whole blood, neutrophils and plasma, we measured NO production and the activities of the enzymes nitric oxide synthase [inducible (iNOS) and constitutive (cNOS)] and cyclooxygenase [constitutive (COX-1) and inducible (COX-2)]. Concentrations of interleukins (IL-1β, IL−6, and IL−10) and tumor necrosis factor-alpha (TNFα) were measured in plasma. In blood from healthy donors, propofol increased NO production and cNOS activity. The concentration of propofol that increased NO production by 50% (EC₅₀) was 23.5 μM, and the EC₅₀ of propofol for cNOS was 18.6 μM. In blood from surgical patients, propofol increased NO production by 52% and cNOS activity by 57%. Propofol inhibited iNOS activity in vitro; the concentration that reduced activity by 50% (IC₅₀) was 19.9 μM. In surgical patients propofol inhibited iNOS activity by 53%. COX-1 and COX-2 activities were inhibited in vitro (IC₅₀ 32.6 and 187 μM, respectively) and in surgical patients (53 and 81% inhibition, respectively). Plasma concentrations of IL-1β, IL-6, and TNFα were significantly reduced in surgical patients (32, 23, and 21% inhibition, respectively). None of these parameters were modified in a group of patients (n = 10) anesthetized with sevoflurane. We conclude that propofol stimulated constitutive NO production and inhibited inducible NO production, possibly by curtailing the stimulation of iNOS by inflammatory mediators in surgical patients.     

Involvement of different calcium channels in K+- and veratridine-induced increases of cytosolic calcium concentration in rat cerebral cortical synaptosomes

Intracellular calcium ion concentrations ([Ca²⁺]_i) in rat cerebral cortical synaptosomes were measured, using the calcium chelating fluorescence dye fura-2. The synaptosomes were depolarized by elevation of the extracellular K⁺ concentration or by addition of veratridine, which opens voltage-dependent Na⁺-channels and prevents their inactivation. Both enhancement of the concentration of extracellular K⁺ (up to 60 mM) and veratridine (1–100 μM) increased the [Ca²⁺]_i in a concentration-dependent manner. In the absence of extracellular Ca²⁺, the K⁺- and veratridine-induced increases in [Ca²⁺]_i were abolished, indicating that the increase in [Ca²⁺]_i was due to an influx of extracellular Ca²⁺. Tetrodotoxin (TTX), a blocker of the voltage-dependent Na⁺ channel, inhibited the veratridine-induced (10 μM) Ca²⁺ influx by more than 80%, while the K⁺-evoked (30 mM) increase of [Ca²⁺]_i was TTX-resistant. Both the K⁺- and the veratridine-induced Ca²⁺ influx were not reduced by nifedipine (1 μM), a blocker of L-type Ca²⁺ channels. Blockade of the voltage dependent N-type Ca²⁺ channels with ω-conotoxin GVIA (ω-CTx GVIA; 0.1 μM) and of the voltage-dependent P/Q-type channels with ω-agatoxin IVA (ω-AgaTx IVA; 0.2 μM) inhibited the K⁺-induced increase in [Ca²⁺]_i by about 30 and 55%, respectively; these effects were additive. ω-Conotoxin MVIIC (ω-CTx MVIIC) at a concentration of 0.2 μM, which may be assumed to block predominantly the Q-type Ca²⁺ channel, inhibited the K⁺-induced increase in [Ca²⁺]_i by 50%. The veratridine-induced increase in [Ca²⁺]_i was reduced by about 25% by ω-CTx GVIA (0.1 μM), but was resistant to ω-AgaTx IVA (0.2 μM) and ω-CTx MVIIC (0.2 μM). Mibefradil (former designation Ro 40-5967), a Ca²⁺ antagonist which blocks all types of voltage-dependent Ca²⁺ channels including the T and R channels, led to a concentration-dependent inhibition of the K⁺- and veratridine-induced increase in [Ca²⁺]_i (abolition at 10 μM mibefradil). Ifenprodil, another non-specific blocker of voltage-dependent Ca²⁺ channels, also inhibited the K⁺- and veratridine-induced increase in [Ca²⁺]_i in concentration-dependent manner and abolished it at 320 μM ifenprodil. In contrast, KB-R 7943 (2-[2-[4-(4-nitrobenzyloxy)phenyl]ethyl]isothiourea methanesulphonate; 1 and 3 μM), a highly potent and selective inhibitor of the Na⁺/Ca²⁺ exchanger (NCX1), failed to inhibit the K⁺- and veratridine-induced increase in [Ca²⁺]_i. It is concluded that the K⁺-induced increase in free cytosolic Ca²⁺ results from Ca²⁺ influx through voltage-dependent N- and, above all, Q-type Ca²⁺ channels. N-type Ca²⁺ channels also play a minor role in the veratridine-induced increase in [Ca²⁺]_i, but P/Q-type channels do not appear to be involved at all. The inhibition of the veratridine-induced, ω-CTx GVIA- and ω-AgaTx IVA-resistant increase in [Ca²⁺]_i by mibefradil and the failure of KB-R 7943 to inhibit this response are compatible with the suggestion that in rat cerebral cortical synaptosomes, Ca²⁺ influx via the R-type Ca²⁺ channel and/or another so far uncharacterized Ca²⁺ channel may substantially contribute to the veratridine-induced increase in [Ca²⁺]_i. Received: 7 March 1997 / Accepted: 9 September 1997     

Cordycepin-Enriched WIB801C from Cordyceps militaris Inhibits Collagen-Induced [Ca2+]i Mobilization via cAMP-Dependent Phosphorylation of Inositol 1, 4, 5-Trisphosphate Receptor in Human Platelets

In this study, we prepared cordycepin-enriched (CE)-WIB801C, a n-butanol extract of Cordyceps militaris-hypha, and investigated the effect of CE-WIB801C on collagen-induced human platelet aggregation. CE-WIB801C dose-dependently inhibited collagen-induced platelet aggregation, and its IC₅₀ value was 175 μg/ml. CE-WIB801C increased cAMP level more than cGMP level, but inhibited collagen-elevated [Ca²⁺]_i mobilization and thromboxane A₂ (TXA₂) production. cAMP-dependent protein kinase (A-kinase) inhibitor Rp-8-Br-cAMPS increased the CE-WIB801C-downregulated [Ca²⁺]_i level in a dose dependent manner, and strongly inhibited CE-WIB801C-induced inositol 1, 4, 5-trisphosphate receptor (IP₃R) phosphorylation. These results suggest that the inhibition of [Ca²⁺]_i mobilization by CE-WIB801C is resulted from the cAMP/A-kinase-dependent phosphorylation of IP₃R. CE-WIB801C suppressed TXA₂ production, but did not inhibit the activities of cyclooxygenase-1 (COX-1) and TXA₂ synthase (TXAS). These results suggest that the inhibition of TXA₂ production by WIB801C is not resulted from the direct inhibition of COX-1 and TXAS. In this study, we demonstrate that CE-WIB801C with cAMP-dependent Ca²⁺-antagonistic antiplatelet effects may have preventive or therapeutic potential for platelet aggregation-mediated diseases, such as thrombosis, myocardial infarction, atherosclerosis, and ischemic cerebrovascular disease.     

Effect of prenylated flavonoids and chalcones isolated from <Emphasis Type="Italic">Artocarpus</Emphasis> species on platelet aggregation in human whole blood

Five prenylflavonoids and two prenylchalcones from Artocarpus lowii King, A. scortechinii King and A. teysmanii Miq., and acetylated derivatives of cycloheterophyllin and artonin E were investigated for their ability to inhibit arachidonic acid (AA), collagen and adenosine diphosphate (ADP)-induced platelet aggregation in human whole blood by using an electrical impedance method. Among the tested compounds, only cycloheterophyllin inhibited AA-induced platelet aggregation with an IC₅₀ value of 100.9 μM. It also showed strong inhibition against ADP-induced aggregation, with an IC₅₀ value of 57.1 μM. Isobavachalcone, 2′,4′-dihydroxy-4-methoxy-3′-prenyldihydrochalcone, cycloartobiloxanthone, artonin E and artonin E triacetate showed selective inhibition against ADP-induced aggregation, with IC₅₀ values ranging from 55.3 to 192.0 μM, but did not show such effect against other inducers.     

Triazenoindazoles and triazenopyrazolopyridines: Design,synthesis, and cytotoxic activity

Several triazenoindazoles 3a–e and triazinopyrazolopyridines 6a–i were prepared through the reaction of the corresponding 3-amino-4-chloroindazole and 3-aminopyrazolopyridine diazonium salts 2 and 5 with a number of secondary amines. All compounds were evaluated for their in vitro cytotoxic activity on three cell lines, HepG2, MCF7, and HeLa. Most compounds inhibited cell growth with IC₅₀ less than 0.1 μM. Compound 6d was the most potent, with an IC₅₀ of 0.03 μM against HepG2 and 0.05 μM against MCF7 and HeLa cells.     

Preclinical characterization of atiprimod,a novel JAK2 AND JAK3 inhibitor

We herein report on the activity of the JAK2/JAK3 small molecule inhibitor atiprimod on mouse FDCP-EpoR cells carrying either wild-type (JAK2 ^WT) or mutant (JAK2 ^V617F) JAK2, human acute megakaryoblastic leukemia cells carrying JAK2 ^V617F (SET-2 cell line), and human acute megakaryocytic leukemia carrying mutated JAK3 (CMK cells). Atiprimod inhibited more efficaciously the proliferation of FDCP-EpoR JAK2 ^V617F (IC₅₀ 0.42 μM) and SET-2 cells (IC₅₀ 0.53 μM) than that of CMK (IC₅₀ 0.79 μM) or FDCP-EpoR JAK2 ^WT cells (IC₅₀ 0.69 μM). This activity was accompanied by inhibition of the phosphorylation of JAK2 and downstream signaling proteins STAT3, STAT5, and AKT in a dose- and time-dependent manner. Atiprimod-induced cell growth inhibition of JAK2 ^V617F–positive cells was coupled with induction of apoptosis, as evidenced by heightened mitochondrial membrane potential and caspase-3 activity, as well as PARP cleavage, increased turnover of the anti-apoptotic X-linked mammalian inhibitor of apoptosis (XIAP) protein, and inhibition of the pro-apoptotic protein BCL-2 in a time- and dose-dependent manner. Furthermore, atiprimod was more effective at inhibiting the proliferation of peripheral blood hematopoietic progenitors obtained from patients with JAK2 ^V617F-positive polycythemia vera than at inhibiting hematopoietic progenitors from normal individuals (p = 0.001). The effect on primary expanded erythroid progenitors was paralleled by a decrease in JAK2^V617F mutant allele burden in single microaspirated BFU-E and CFU-GM colonies. Taken together, our data supports the clinical testing of atiprimod in patients with hematologic malignancies driven by constitutive activation of JAK2 or JAK3 kinases.     

Inhibition of human liver phenol sulfotransferase by nonsteroidal anti-inflammatory drugs

Objective: The aim of this investigation was to study the inhibition of 11 nonsteroidal anti-inflammatory drugs (NSAIDs) on the human liver phenol sulfotransferases (HL-PST) and catechol sulfotransferase (HL-CST). Methods: The activities of HL-PST and HL-CST were measured with 4 μM 4-nitrophenol and 60 μM dopamine (the sulfate acceptors) and 0.4 μM 3′-phosphoadenosine-5′-phosphosulfate [³⁵S] (the sulfate donor). Samples of liver were obtained from five patients, aged 55–79 years, undergoing clinically indicated hepatectomy. The inhibition curves were constructed with at least five concentrations of the inhibitor. Results: With the exception of piroxicam, NSAIDs inhibited HL-PST, and the estimates of the inhibitory concentration for 50% of responses (IC₅₀; μM) were: 0.02 (mefenamic acid), 3.7 (diflunisal), 5.4 (nimesulide), 9.5 (diclofenac), 30 (salicylic acid), 41 (ketoprofen), 74 (indomethacin), 159 (ibuprofen), 245 (ketoralac) and 473 (naproxen). With 4-nitrophenol as the variable substrate, the inhibition of salicylic acid on HL-PST was non-competitive and the K_i and K_ies were 18 μM and 21 μM (n = 5; P = 0.548), respectively. HL-CST was less susceptible than HL-PST to inhibition by NSAIDs, with only five drugs inhibiting this enzyme. The IC₅₀ estimates for these drugs (μM) were 76 (mefenamic acid), 79 (diflunisal), 103 (indomethacin), 609 (salicylic acid) and 753 (diclofenac). Conclusion: The comparison of the IC₅₀ estimates of HL-PST with the therapeutic plasma concentrations of NSAIDs corrected for the plasma unbound fraction was consistent with the view that mefenamic acid and salicylic acid, when administered at therapeutic doses, should impair the hepatic sulfation of those compounds that are substrates of phenol sulfotransferase. Received: 7 June 1999 / Accepted in revised form: 13 January 2000     

In vitro inhibition screening of human hepatic P 450 enzymes by five angiotensin-II receptor antagonists

Objective: Metabolic interactions at the level of drug-metabolising enzymes are important for drug therapy. We investigated potential interactions of losartan, irbesartan, valsartan, eprosartan and candesartan with cytochrome P ₄₅₀ (CYP) enzymes in human liver microsomes. Methods: In incubations with human liver microsomes in vitro, the inhibitory potency of angiotensin-II receptor antagonists (sartans) on CYP-specific model activities were compared by measuring the IC₅₀ and, with respect to more potent inhibition, K _i values. Results: None of the five sartans inhibited CYP2A6-, CYP2D6- or CYP2E1-associated activities (coumarin 7-hydroxylation, dextromethorphan O-demethylation and chlorzoxazone 6-hydroxylation, respectively) to any significant extent. Losartan and irbesartan inhibited the CYP2C9-associated tolbutamide methylhydroxylation more potently (K _i values 4.1 μM and 24.5 μM), than valsartan, candesartan or eprosartan (K _i values 135 μM, 155 μM and >1000 μM, respectively). Losartan and irbesartan inhibited CYP1A2- and CYP3A4-associated activities (ethoxyresorufin O-deethylation and testosterone 6β-hydroxylation) with relatively weak affinities (IC₅₀ values between 200 μM and 500 μM). CYP2C19-associated S-mephenytoin 4′-hydroxylation activity was inhibited by losartan (IC₅₀ value 138 μM) and much less or not at all by the other sartans tested. Conclusion: All sartans except eprosartan have at least some affinity for CYP2C9, but only losartan has an affinity for CYP2C19. Losartan and irbesartan have modest affinity for CYP1A2 and CYP3A4. This would suggest that the theoretical potential for drug interactions is likely to be quite low, with the possible exceptions of losartan and irbesartan for CYP2C9. Based on these findings, further studies on the interaction potential of losartan and irbesartan are warranted. Received: 27 October 1999 / Accepted in revised form: 10 February 2000     

Inhibitory effects of NS-21, a novel drug for urinary incontinence, and its active metabolite, RCC-36, on L-type calcium currents in isolated guinea pig detrusor smooth muscle cells

The inhibitory effects of NS-21, a newly developed drug for the treatment of urinary frequency and urinary incontinence, and its active metabolite, RCC-36, on L-type Ca²⁺ currents (I_Ca) in guinea pig detrusor smooth muscle cells have been compared to those of terodiline by a whole-cell patch-clamp technique. Like terodiline (10 μM), both NS-21 (10 μM) and RCC-36 (10 μM) induced a sizeable decrease in I_Ca elicited from a holding potential of -60 mV without changing the current-voltage relationship. The three drugs shifted the inactivation curves for I_Ca in the hyperpolarizing direction by 13 to 20 mV but had no effect on the activation curves for I_Ca, resulting in a decrease in the calcium window current. The inhibitory effects of NS-21 and RCC-36 were greater than those of terodiline. The three drugs inhibited I_Ca in a concentration- and holding-potential-dependent manner. The IC₅₀ values at a holding potential of -60 mV were 7.9 μM for NS-21, 6.4 μM for RCC-36, and 5.9 μM for terodiline, and at -40 mV they were 1.3, 1.2, and 3.5 μM, respectively. The ratio calculated by dividing the IC₅₀ value at -60 mV by the value at -40 mV was 6.1, 5.3 and 1.7, respectively, indicating that the inhibitory effects of NS-21 and RCC-36 on I_Ca were more sensitive to voltage than those of terodiline. These results suggest that NS-21 and RCC-36 could be more effective in the treatment of urinary bladder ailments, such as urinary frequency and urinary incontinence. Received: 27 August 1996 / Accepted: 13 February 1997     

Interaction between thiol-modifying agents and P1075, a KATP channel opener, in rat isolated aorta

In vascular smooth muscle, openers of ATP-dependent potassium channels (K _ATP channels), such as P1075 (N-cyano-N’-(1,1-dimethylpropyl)-N’’-3-pyridylguanidine), produce relaxation. In this study we have investigated the effects of thiol-modifying agents on the binding of P1075 and on the ⁸⁶Rb⁺ efflux stimulating and vasorelaxant effects of the opener in rat aortic rings. The increase in ⁸⁶Rb⁺ efflux induced by P1075 was taken as a qualitative measure of K⁺ channel opening. The hydrophilic SH-group-oxidizing substance, thimerosal (1 to 100μM), abolished specific binding of [³H]-P1075 with an IC₅₀ value of 7.6±1.2μM; at 30μM, the half time for inhibition was 38min. Two other thiol-oxidizing agents, PMB (4-hydroxy-mercuribenzoic acid) and DTBNP (2,2’-dithio-bis(5-nitropyridine)), inhibited binding up to 86% and 44%, respectively. The disulphide bond reducing substance, DTT (1,4-dithiothreitol, 0.1 to 1mM), reduced [³H]-P1075 binding by up to 20% and partially reversed the inhibitory effect of thimerosal. In ⁸⁶Rb⁺ efflux experiments, thimerosal (3 to 100μM) concentration-dependently increased basal efflux but inhibited P1075-stimulated tracer efflux with an IC₅₀ value of 7±1μM. The inhibitory effect occurred with a half-time of approximately 8min and was essentially reversed by DTT. In rings precontracted with noradrenaline, thimerosal inhibited the vasorelaxant effect in a noncompetitive manner, shifting the concentration-relaxation curves to the right and reducing maximum relaxation.The data show that oxidation of thiol groups interferes with the binding of the K _ATP channel opener, P1075; concomitantly, the ⁸⁶Rb⁺ efflux stimulating and the vasorelaxant effects are inhibited. Reduction of disulphide bonds by DTT has only minor effects on the action of P1075. Collectively, the results suggest that intact thiol groups are essential for the functioning of the K_ATP channel in rat aorta. The different kinetics governing the inhibition of opener binding and of opener-stimulated ⁸⁶Rb⁺ efflux suggest that the SH-groups involved in the two processes differ in their accessibility to thimerosal and/or in their reactivity. Received: 7 April / Accepted: 9 July 1997     

γ-Mangostin, a novel type of 5-hydroxytryptamine 2A receptor antagonist

γ-Mangostin, purified from the fruit hull of the medicinal plant Garcinia mangostana caused a parallel rightwards shift of the concentration/response curve for the contraction elicited by 5-hydroxytryptamine (5-HT) in the rabbit aorta (pA₂ = 8.2) without affecting the contractile responses to KCl, phenylephrine (α₁) or histamine (H₁). The perfusion pressure response of rat coronary artery to 5-HT (5-HT_2A) was reduced concentration dependently by γ-mangostin (IC₅₀ = 0.32 μM). 5-HT amplified, ADP-induced aggregation of rabbit platelets (5-HT_2A) was inhibited by γ-mangostin (IC₅₀ = 0.29 μM), whereas that induced by thrombin was not affected, nor did γ-mangostin affect 5-HT-induced contraction of the guinea-pig ileum (5-HT₃)in the presence of 5-HT₁, 5-HT₂ and 5-HT₄ receptor antagonists. Furthermore, 5-HT-induced contraction of the rat fundus (5-HT_2B) and 5-HT-induced relaxation of the rabbit aorta in the presence of ketanserin (5-HT₁) and carbachol-induced contraction of the guinea-pig ileum (muscarinic M₃) were not affected by γ-mangostin (5 μM). γ-Mangostin inhibited [³H]spiperone binding to cultured rat aortic myocytes (IC₅₀ = 3.5 nM). The K _d for [³H]spiperone binding was increased by γ-mangostin (3 nM) from 11.7 to 27.4 nM without affecting B _max. These results suggest that γ-mangostin is a novel competitive antagonist, free from a nitrogen atom, for the 5-HT_2A receptors in vascular smooth muscles and platelets. Received: 10 June 1997 / Accepted: 11 September 1997