REV 2871 (CHBZ): a potent antiallergic agent with a novel mechanism of action. II. Studies on the mechanism of action

REV 2871 (CHBZ) was taken up by rat mast cells and human leukocytes in a specific and saturable manner. The compound can be hydrolyzed by a granule-associated enzyme in the mast cell to an ionic metabolite (REV 3579) whose in vitro profile is identical to that of disodium cromoglycate (DSCG). REV 3579, although achieving millimolar concentrations inside cells incubated with CHBZ, was not itself taken up by rat mast cells or human leukocytes. The unusual in vitro activity of CHBZ is postulated to arise from the fact that it is a prodrug for delivering a DSCG-like drug to the interior of a secretory cell. The internalized drug apparently exerts a more general and longer-lived inhibition of the secretory process than it can by acting on exterior membrane receptors. CHBZ thus represents a novel drug for studying anaphylactic responses in vitro.     

Studies on the mechanism of action of omeprazole

The effects of omeprazole on preparations of pig gastric (H+ + K+)-ATPase have been studied. Omeprazole was found to inhibit the (H+ + K+)-ATPase activity in a time-dependent manner. Inhibition was more pronounced at pH 6.1 compared with pH 7.4 and decreased as the concentration of (H+ + K+)-ATPase preparation increased. The potency of omeprazole was therefore highly dependent upon the conditions used. When pre- incubated with (H+ + K+)-ATPase preparation (30 micrograms protein/ml) for 30 min at 37 degrees and pH 6.1, omeprazole inhibited the (H+ + K+)-ATPase activity with an IC50 of 3.9 microM. This inhibition was shown to be irreversible in nature. Whilst omeprazole itself was not very potent as an inhibitor of the (H+ + K+)-ATPase activity at pH 7.4 (IC50 = 36 microM), transient acidification of omeprazole resulted in the formation of a compound(s) which produced marked inhibition at this pH (IC50 = 5.2 microM). The effects of omeprazole in the absence of acidification may have resulted from the rate-limiting formation of this compound. Radiolabelled omeprazole was shown to incorporate into the (H+ + K+)-ATPase preparation in a time-dependent and pH-dependent manner. Omeprazole, radiolabelled in three separate positions (the sulphur atom and the two adjacent carbon atoms), incorporated with equivalent time courses suggesting that the incorporation did not involve a fragmentation of the omeprazole molecule. Under conditions shown to produce a 50% inhibition of (H+ + K+)-ATPase activity, [14C] omeprazole had incorporated to a level of 4-5 nmoles/mg protein. Incorporation continued beyond the point required to produce 100% inhibition of (H+ + K+)-ATPase activity and reached 30 nmoles/mg protein after 5 hr. Prior acidification of the omeprazole resulted in a more rapid initial rate of incorporation although the final level of incorporation was lower than for omeprazole. Omeprazole was also shown to interact with the (Na+ + K+)-ATPase from dog kidney. Omeprazole inhibited the (Na+ + K+)-ATPase activity (IC50 = 186 microM). Acid-degraded omeprazole inhibited the (Na+ + K+)-ATPase activity with greater potency (IC50 = 19 microM) and was also shown to incorporate into this enzyme preparation.     

Studies on the mechanism of action of LS 1727, a nitrosocarbamate of 19-nortestosterone

LS 1727, a nitrosocarbamate of 19-nortestosterone, was active against lymphoid, antimetabolite-sensitive, cell lines in vitro especially L1210 and Ehrlich ascites tumour lines. It was less effective against alkylating agent-sensitive lines such as the Walker 256 carcinosarcoma. These results suggested a possible antimetabolite mode of action but LS 1727 had no effect on deoxy-or ribonucleotide pool sizes in L1210 cells. Studies on macromolecular synthesis showed an early inhibition of DNA synthesis whilst RNA and protein synthesis continued for 24-48 hours. This was reminiscent of a classic alkylating agent-like effect. In vivo studies with specific alkylating agent-sensitive, or antimetabolite-sensitive tumours showed no antitumour activity although significant inhibition of Ehrlich ascites tumour cell growth was observed at high doses. In vitro cytotoxicity studies showed LS 1727 to be inactivated in the presence of mouse, rat and dog liver supernatants. This would explain the poor antitumour effects in vivo compared with the in vitro observations. Because LS 1727 is a nitrosocarbamate its possible mutagenic activity was investigated. LS 1727 was highly mutagenic but this property was also lost in the presence of a rat liver microsomal fraction. Although an effective cytotoxic agent in vitro LS 1727 is only effective in vivo at toxic doses.     

Studies on the mechanism of action of dantrolene sodium

Summary In rat diaphragm-phrenic nerve preparations, dantrolene sodium has been shown to have no effect on neuromuscular transmission. KCl and acetylcholine contractures in the frog rectus muscle are depressed in the presence of dantrolene sodium. The threshold for caffeine contractures in the frog sartorius and rectus muscles is raised from 2.0 to 4.0 mM. In isolated frog sartorius muscles, the tetanic fusion frequency is increased by 29%, and the twitch response is depressed more than the tetanic.According to these observations it is proposed that the muscle relaxant properties of dantrolene sodium are dependent on its direct inhibitory action on skeletal muscle. It is further hypothesized that this direct action results from an antagonism of calcium release within the muscle.     

Studies on the mechanism of action of amantadine.

1 The effect of amantadine hydrochloride on various aspects of catecholamine metabolism in the rat brain has been investigated. 2 Amantadine failed to have any significant effect on brain concentrations of dopamine or noradrenaline even when administered daily for 9 days. 3 Amantadine had no effect on the rate of decline of noradrenaline and dopamine concentrations after alpha-methyl-p-tyrosine. 4 In vitro amantadine inhibited dopamine uptake into synaptosomes only at high concentrations, and caused little release of dopamine from synaptosomes. 5 There is no evidence from these results to suggest that the anti-Parkinsonian effect of amantadine is related to an action on dopaminergic mechanisms.     

Studies on the mechanism of action of various vasodilators

1 The vascular relaxant effects of histamine, adenosine, isoprenaline nitroglycerine, papaverine and 3-isobutyl-l-methylxanthine (IBMX) were assessed individually, in strips of rabbit renal artery moderately contracted with noradrenaline (NA) in the absence or presence of phosphodiesterase inhibitors (papaverine and IBMX) or verapamil, a Ca²⁺ antagonist.

2 The vasodilator effect of histamine was potentiated by papaverine (6.1 × 10^-7 M) and IBMX (4.4 × 10^-5 M) but inhibited dose-dependently by verapamil (5.1 and 51.0 × 10^-7 M).

3 Adenosine-induced vascular relaxations were greatly increased in the presence of papaverine (6.1 × 10^-7 M) but significantly reduced in the presence of IBMX (4.4 × 10^-5 M) or verapamil (5.1 and 51.0 × 10^-7 M).

4 The vasodilatation produced by isoprenaline was increased in the presence of IBMX (4.4 × 10^-5 M) or papaverine (6.1 × 10^-7 M), but inhibited by verapamil (5.1 and 51.0 × 10^-7 M).

5 The vascular relaxant effects of nitroglycerine and papaverine were inhibited in the presence of IBMX (4.4 × 10^-5 M) or verapamil (5.1 and 51.0 × 10^-7 M). Papaverine (6.1 × 10^-7 M) also antagonized nitroglycerine-induced vascular relaxation.

6 The vasodilator effect of IBMX was greatly reduced in the presence of papaverine (6.1 × 10^-7 M) or verapamil (5.1 and 51.0 × 10^-7 M).

7 The vascular relaxant effect of verapamil was reduced proportionally by raising the extracellular Ca²⁺ concentration from 1.25 to 5.0 mM while those elicited by histamine, adenosine, isoprenaline, nitroglycerine, papaverine and IBMX were not modified by this procedure.

8 These results were taken as an indication that several vasodilators (e.g. histamine, adenosine, isoprenaline, nitroglycerine, papaverine and IBMX), but not a Ca²⁺ antagonist such as verapamil, produce a fraction of their vasodilator effects by promoting Ca²⁺ extrusion from and/or Ca²⁺ sequestration into the vascular smooth muscle cells, via a cyclic adenosine 3′,5′-monophosphate-dependent mechanism.

     

Studies on the goitrogenic mechanism of action of N,N,N',N'-tetramethylthiourea

N,N,N',N'-Tetramethylthiourea (TMTU) is a rat goitrogen inducing thyroid hyperplasia, hypertrophy, and tumor formation. Little is known about the exact underlying mechanism of action. As thyroid peroxidase (TPO) and type I iodothyronine deiodinase (ID-I) have been established as targets of goitrogenic thiourea derivatives, we investigated interactions of TMTU with target enzymes using a partially purified fraction from hog thyroids or solubilized hog thyroid microsomes and 10,000g supernatant from rat liver homogenate, respectively, as enzyme sources. For comparison, comprehensively characterized goitrogenic thiourea derivatives were studied as well. In contrast to propylthiouracil (PTU), and like ethylenethiourea (ETU), TMTU only marginally affected TPO-catalyzed oxidation of guaiacol. TMTU, like ETU, concentration-dependently suppressed TPO-catalyzed iodine formation with concomitant oxidative metabolism. Suppression ceased upon consumption of thiourea derivatives, the rate of the reappearing iodine formation was similar to that of controls. TMTU, like ETU, also suppressed non-enzymatic and TPO-catalyzed monoiodination of l-tyrosine with a stoichiometry of 2:1, i.e., one molecule of thiourea derivative suppressed two times monoiodination. TMTU and ETU were unable to irreversibly inhibit TPO. In contrast to PTU, TMTU did not inhibit ID-I. These findings provide evidence that TMTU interferes with thyroid hormone synthesis at the level of iodination and demonstrate a metabolic route for the oxidative detoxification of TMTU in the thyroid suggesting that low-level or intermittent exposure to TMTU would have only minimal effects on thyroid hormone synthesis. Finally, it can be concluded that meaningful toxicological studies on TPO inhibition can be performed without a need for highly purified TPO.     

Studies on the action and mechanism of action of oxyfedrine on isolated tracheal chains

Oxyfedrine, a β-sympathomimetic drug, did not affect isolated rat and rabbit trachea in concentration from 2.86 × 10^?8 to 2.86 × 10^?4 M, but on the guinea-pig trachea, it caused a dose dependent relaxation of natural tone in lower concentrations (1.79 × 10^?7 to 2.86 × 10^?6 M. In higher concentrations (1.14 × 10^?5 to 2.86 × 10^?4 M), however, a contraction was observed, which was also dose dependent. This contraction was not affected by atropine, lysergic acid diethylamide or by pretreatment with reserpine but was blocked by antihistaminics (isothipendyl and clemastine). Adrenaline, noradrenaline, phenylephrine and isoprenaline did not contract the guinea-pig trachea, whereas contractions were observed after high concentrations of norephedrine, amphetamine, ephedrine and tyramine. These contractions were also unaffected by reserpine pretreatment.It is concluded that the contraction of the guinea-pig trachea by oxyfedrine is related to its structural relationship to the phenylethylamines and might be due to histamine release, an action on histamine receptors or a histamine-like action.     

Studies on the mechanism of action of quinone antitumor agents

The presence of a quinone group in the structure of a compound has been shown to produce cell kill and DNA strand breaks by a mechanism involving free radicals and active oxygen species. The ability of the compound to bind to DNA appeared to increase the DNA damage induced and the cytotoxic activity. A new series of model compounds has been used to investigate further the role of the quinone group in the mechanism of action of quinone antitumor agents. Bis(dimethylamino)benzoquinone, which contains a quinone group, produced significant cell kill of L5178Y lymphoblasts and induced concentration-dependent single-strand and double-strand breaks in the DNA of these cells. Benzoquinone dimustard, which possesses a quinone moiety and active alkylating groups, was approximately 2500 times more cycotoxic to L5178Y cells than was bis(dimethylamino)benzoquinone and was approximately 200-fold more active in inducing DNA double-strand breaks than was the quinone agent. Benzoquinone dimustard induced no apparent DNA single-strand breaks, but produced significant DNA cross-linking, a process which interferes with the assay for single-strand breaks. The cell kill produced by both quinone agents was inhibited by catalase, but not by superoxide dismutase. The cytotoxic activity of bis(dimethylamino)benzoquinone and two other quinone model compounds, hydrolyzed benzoquinone mustard and benzoquinone mustard, appeared to correlate with the induction of DNA strand breaks, while there appeared to be no correlation between cell kill and DNA double-strand breaks induced by benzoquinone dimustard. However, the cytotoxicity of benzoquinone dimustard appeared to be related to the cross-linking activity of this agent. These studies have provided additional evidence that the presence of a quinone group in the structure of a compound can result in significant cell kill by a mechanism that appears to involve active oxygen species. Quinone containing agents can induce DNA strand breaks, and this effect is enhanced when the agent is able to bind to DNA. The induction of DNA strand breaks appeared to correlate with cytotoxic activity for bis(dimethylamino)benzoquinone, hydrolyzed benzoquinone mustard and benzoquinone mustard, but not for benzoquinone dimustard, suggesting that the contribution of quinone-induced strand breaks to the overall cytotoxicity of an agent may vary considerably.     

海兰地嗪对血小板聚集的影响及机理探讨

海兰地嗪(Her)体外对胶原,ADP,A23187和AA诱导的大鼠血小板聚集有明显抑制作用,其IC_(50)分别为14.5,41.6,44.1和48.3μg/ml。Her对AA诱导的大鼠血小板MDA生成不能抑制,但能升高兔血小板cAMP水平和抑制凝血酶诱导的人血小板胞浆内游离钙离子浓度升高。Her的抗血小板聚集作用机理可能与升高血小板内cAMP水平和抑制细胞内游离钙离子浓度升高有关。     

Studies on the mechanism of action of a novel anorectic agent, (−)-threo-chlorocitric acid

(?)-threo-Chlorocitric acid, a novel and potent anorectic agent in rats and dogs, decreased food intake to a comparable extent in rats fed chow, low fat, or high fat diets. This inhibition of food intake was not the result of conditioned aversion. Unlike (?)-threo-hydroxycitric acid, a structurally related compound which inhibits fatty acid synthesis, (?)-threo-chlorocitric acid did not suppress fatty acid synthesis in an isolated hepatocyte system or in vivo. (?)-threo-Chlorocitric acid significantly delayed the rate of gastric emptying. Of the four stereoisomers of chlorocitric acid, (?)-threo-chlorocitric acid was the most active both in delaying gastric emptying and producing anorexia in rats. It is suggested that the mechanism by which (?)-threo-chlorocitric acid may suppress food intake is through a reduction of gastric emptying.     

Studies on the mechanism of action of the tumour inhibitory triazenes

A series of imidazole and phenyl dialkyi triazenes were synthesized and investigated for their anti-cancer activity in experimental animals. Active triazenes had a broad spectrum of anti-tumour action and like bischloroethylnitrosourea (BCNU) were active against tumours not sensitive to conventional alkylating agents. It was confirmed that at least one N-methyl group was necessary for anti-cancer activity but there was no correlation between dealkylation by liver microsomes and activity since a diethyl triazene was readily dealkylated but not active. A factor appears to be present in normal cell cytoplasm which can detoxify triazenes but which is absent from tumours sensitive to these agents.     

Studies on the mechanism of action of the omeprazole-derived cyclic sulphenamide

The inhibitory effects of omeprazole and omeprazole-derived metabolites were studied on Escherichia coli glutaminase activity at pH 2.5 which might represent the conditions present at the target enzyme (K+/H+-ATPase) in the secretory membrane of the intact parietal cell. Omeprazole and the omeprazole-derived cyclic sulphenamide inhibited glutaminase at pH 2.5 with identical potency (IC50 36 microM). The substrate, glutamine as well as the mercaptane, dithiothreitol, protect the enzyme. Furthermore, dithioerythritol was found to reverse inhibition. This indicates that an SH-group localized in the substrate binding center of glutaminase is most likely involved in the reaction leading to enzyme inhibition. Glutaminase inhibition by both compounds was less pronounced at pH 5.0. Omeprazole radical, the metabolite generated from the cyclic sulphenamide at more neutral pH values, failed to affect the enzyme. These findings were in contrast with the properties of the omeprazole-derived cyclic sulphenamide and radical at the K+/H+-ATPase preparation. This enzyme was inhibited by both compounds at pH 7.5 with a high potency, and reversal experiments with dithiothreitol demonstrate that these agents interfere with SH-groups of the K+/H+-ATPase. From these data it is suggested that the cyclic sulphenamide and the radical interfere by different reaction pathways with enzymatic SH-groups.     

The CCR5 receptor-based mechanism of action of 873140, a potent allosteric noncompetitive HIV entry inhibitor

4-{[4-({(3R)-1-Butyl-3-[(R)-cyclohexyl(hydroxy)methyl]-2,5dioxo-1,4,9-triazaspiro[5.5]undec-9-yl}methyl)phenyl]oxy}benzoic acid hydrochloride (873140) is a potent noncompetitive allosteric antagonist of the CCR5 receptor (pK(B) = 8.6 +/- 0.07; 95% CI, 8.5 to 8.8) with concomitantly potent antiviral effects for HIV-1. In this article, the receptor-based mechanism of action of 873140 is compared with four other noncompetitive allosteric antagonists of CCR5. Although (Z)-(4-bromophenyl){1'-[(2,4-dimethyl-1-oxido-3-pyridinyl)carbonyl]-4'-methyl-1,4'-bipiperidin-4-yl}methanone O-ethyloxime (Sch-C; SCH 351125), 4,6-dimethyl-5-{[4-methyl-4-((3S)-3-methyl-4-{(1R)-2-(methyloxy)-1-[4-(trifluoromethyl)phenyl]ethyl}-1-piperazinyl)-1-piperidinyl]carbonyl}pyrimidine (Sch-D; SCH 417,690), 4,4-difluoro-N-((1S)-3-{(3-endo)-3-[3-methyl-5-(1-methylethyl)-4H-1,2,4-triazol-4-yl]-8-azabicyclo[3.2.1]oct-8-yl}-1-phenyl-propyl)cyclohexanecarboxamide (UK-427,857), and N,N-dimethyl-N-[4-[[[2-(4-methylphenyl)-6,7-dihydro-5H-benzocyclo-hepten-8-yl]carbonyl]amino]benzyl]tetrahydro-2H-pyran-4-aminium chloride (TAK779) blocked the binding of both chemokines (125)I-MIP-1alpha (also known as (125)I-CCL3, (125)I-LD78) and (125)I-RANTES ((125)I-CCL5), 873140 was an ineffectual antagonist of (125)I-RANTES (regulated on activation normal T cell expressed and secreted) binding (but did block binding of (125)I-MIP-1alpha). Furthermore, 873140 blocked the calcium response effects of CCR5 activation by CCL5 (RANTES) (as did the other antagonists), indicating a unique divergence of blockade of function and binding with this antagonist. The antagonism of CCR5 by 873140 is saturable and probe-dependent, consistent with an allosteric mechanism of action. The blockade of CCR5 by 873140 was extremely persistent with a rate constant for reversal of <0.004 h(-) (1) (t(1/2) > 136 h). Coadministration studies of 873140 with the four other allosteric antagonists yielded data that are consistent with the notion that all five of these antagonists bind to a common allosteric site on the CCR5 receptor. Although these ligands may have a common binding site, they do not exert the same allosteric effect on the receptor, as indicated by their differential effects on the binding of (125)I-RANTES. This idea is discussed in terms of using these drugs sequentially to overcome HIV viral resistance in the clinic.     

Prasugrel: a novel platelet ADP P2Y12 receptor antagonist. A review on its mechanism of action and clinical development

Antiplatelet therapy is the cornerstone of treatment for patients who present with acute coronary syndrome (ACS) or undergo percutaneous coronary intervention (PCI). Clopidogrel, in combination with aspirin, is associated with improvement in long-term clinical outcomes in these patients and is currently the antiplatelet therapy of choice. However, a significant number of patients experience recurrent ischemic events, which have been in part attributed to variability in individual response profiles to currently recommended treatment regimens. The presence of variable degrees of responsiveness, thus inadequate platelet inhibition in some patients, underscores the need for novel agents with more potency and less variable platelet inhibitory effects. Prasugrel (CS-747; LY640315), a novel third-generation oral thienopyridine, is a specific, irreversible antagonist of the platelet adenosine diphosphate (ADP) P2Y(12) receptor. Pre-clinical and early phase clinical studies have shown prasugrel to be characterized by more potent antiplatelet effects, lower interindividual variability in platelet response, and faster onset of activity compared with clopidogrel. Recent findings from large-scale Phase III testing showed prasugrel to be more efficacious in preventing ischemic events in ACS patients undergoing PCI; however, this is achieved at the expense of an increased risk of bleeding. This article reviews the currently available data regarding the efficacy and safety of prasugrel.