A comparison of imidazole and 9,11-azoprosta-5,13-dienoic acid Two selective thromboxane synthetase inhibitors

Two selective thromboxane A₂ synthetase inhibitors, imidazole and 9,11-azoprosta-5,13-dienoic acid (azo analog I) were compared to determine their effects on the quantitative formation of thromboxane B₂ and prostaglandin E₂ accompanying human platelet aggregation. Azo analog I was at least 200 times more potent, on a molar basis, than imidazole in suppressing thromboxane B₂ formation in either platelet-rich plasma or washed platelet suspensions aggregated with arachidonic acid or prostaglandin H₂. The inhibitors differed in their effect on the aggregation response itself. Azo analog I selectively suppressed thromboxane A₂ formation with an accompanying, parallel, suppression of the platelet aggregation.Imidazole selectively suppressed thromboxane A₂ formation, but only suppressed the accompanying aggregation in platelet rich plasma, and not washed platelet suspensions. The results indicate that azo analog I functions by competitive inhibition of prostaglandin H₂ on the thromboxane synthetase, and that imidazole, while it suppresses thromboxane A₂ formation, may have an associated agonist activity that enhances platelet aggregation. The data presented support this hypothesis, and they emphasize the importance of thromboxane A₂ in arachidonate mediated platelet aggregation.     

Thromboxane A2 is the major arachidonic acid metabolite of human cortical hydronephrotic tissue

Human cortical hydronephrotic microsomes converted [¹⁴C] arachidonic acid to [¹⁴C] thromboxane B₂ as the major metabolic product. Using [¹⁴C] PGH₂ as substrate, similar enzymatic conversions were noted with HHT>TXB₂6KPGF_1αPGE₂PGF_2α as the major products. Inhibition of thromboxane synthetase with imidazole 5 mM reduced thromboxane B₂ production by 60% and the major product then was 6 keto PGF_1α. After addition of imidazole, the metabolic profile showed 6KPGF_1αPGE₂HHT>PGF_2α. Control experiments were carried out using normal cortical tissue obtained from kidneys removed surgically for carcinoma of kidney and rejected for transplantation secondary to fracture as a consequence of blunt trauma. These control kidneys, while they demonstrated an ability to generate thromboxane B₂$\text{in vitro}$, had much less activity than hydronephrotic kidneys and with PGH₂ as substrate PGE₂TxB₂. In addition, inhibition with imidazole produced mainly PGE₂. Thus, like the rabbit and rat, there is enhanced thromboxane and prostacyclin synthesis in human ureteral obstruction and are, therefore, potential vasoactive compounds which may in part be responsible for the hemodynamic alterations occurring in human obstructive uropathy.     

The effect of imidazole and imidazole-analogues on bone resorption in vitro: A suggested role for thromboxane A2

The effects of imidazole, 1-methyl-imidazole and benzimidazole on bone metabolism $\text{in vitro}$ were investigated. The relative potencies of these compounds with respect to the inhibition of bone resorption was found to be comparable to their relative effectiveness as inhibitors of platelet microsome thromboxane synthetase activity. Since studies by others have shown that thromboxanes are produced by resorbing bone $\text{in vitro}$, these results suggest that the inhibition of bone resorption by imidazole is related to the inhibition of thromboxane A₂ formation. This could imply that thromboxane A₂ is an additional arachidonic acid oxidation product that is of importance in the regulation of bone metabolism.     

Imidazole: a selective inhibitor of thromboxane synthetase

Imidazole inhibits the enzymic conversion of the endoperoxides (PGG2 and PGH2) to thromboxane A2 by platelet microsomes (IC50: 22 MICRONG/ML; DETERMINED BY BIOASSAY). The inhibitor is selective, for prostaglandin cyclo-oxygenase is only affected at high doses. Radiochemical data confirms that imidazole blocks the formation of 14C-thromboxane B2 from 14C-PGH2. Several imidazole analogues and other substances were tested but only 1-methyl-imidazole was more potent than imidazole itself. The use of imidazole to inhibit thromboxane formation could help to elucidate the role of thromboxanes in physiology or pathophysiology.     

Inhibitory action of soluble elastin on thromboxane B2 formation in blood platelets

Soluble elastin, prepared from insoluble elastin by treatment with oxalic acid or elastase, was found to inhibit the formation of thromboxane B₂ both from [1-¹⁴C]arachidonic acid added to washed platelets and from [1-¹⁴C]arachidonic acid in prelabeled platelets on stimulation with thrombin. In both systems, the formation of 12-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE) was accelerated. Oxalic acid-treated soluble elastin st 1 and 10 mg/ml inhibited the formation of thromboxane B₂ from exogenously supplied arachidonic acid 21 and 59%, respectively, and the formation of thromboxane B₂ in prelabeled platelets stimulated by thrombin 44 and 94%, respectively. These concentrations of elastin increased the formation of 12-HETE from exogenously supplied arachidonic acid about 3.4- and 7.3-times, respectively. Almost all the added arachidonic acid was converted to metabolites. In prelabeled platelets, soluble elastin at 1 and 10 mg/ml increased the formation of 12-HETE stimulated by thrombin about 1.3- and 2.8-times, respectively, and inhibited the thrombin-induced total productions of thromboxane B₂ (12-hydroxy-5,8,10-heptadecatrienoic acid (12-HETE) and free arachidonic acid by 26 and 25%, respectively. Elastase-treated digested elastin also inhibited the formation of thromboxane B₂ and stimulated the formation of 12-HETE in prelabeled platelets stimulated by thrombin. This inhibitory action of elastin was not replaced by desmosine. The level of cAMP in platelets was not affected by soluble elastin. Soluble elastin was also found to inhibit platelet aggregation induced by thrombin. However, the inhibitory action of soluble elastin on platelet aggregation cannot be explained by inhibition of thromboxane B₂ formation by the elastin.     

TxA2 production by human arteries and veins

Human arterial and venous segments from patients under-going operations when incubated in Tris buffer both alone and with arachidonic acid were able to produce thromboxane B₂ (assessed by radioimmunoassay). Thromoboxane B₂ (TxB₂) production was progressive in time (till 40 min.) and was enhanced by the addition of 1mM norepinephrien. Contamination of tissues by platelet was checked and platelets did not contribute to thromboxane formation. The investigation of the conversions of 1-¹⁴C arachidonic acid by vascular tissue indicated that human vascular tissues produce the metabolites of the cyclooxygenase dependent pathway and that prostacyclin is the main metabolite with a PGI₂/TxA₂ ratio of 4:1. The arterial wall was found to posses an active lipoxygenase dependent pathway. Thromboxane production by intimal cells was neglible and the main source of thromboxane was the media. The production of thromboxane did not change in relation to age, but arterial segments from men produced significantly larger amounts of thromboxane than those from women.     

Prostaglandins and myogenic control of tension in lower esophageal sphincter in vitro

Active tension is produced by the lower esophageal sphincter (LES) of North American opossum $\text{in vitro}$ by a myogenic mechanism. Strips of LES, but not those from the esophageal body, contracted to prostaglandin (PG)F_2α, stable expoxymethano derivatives of PGH₂ and to thromboxane B₂. Stable endoperoxides were more than 500 times more potent than PGF_2α. PGI₂ and 6-keto PGF_1α were weak relaxants of LES strips. LES strips transformed arachidonic acid into contractile substances. This transformation was prevented by agents which interfere with PG synthesis by inhibiting cyclo-oxygenase [indomethacin (IDM), 5,8,11,14-eicosatetraynoic acid (ETA) or thromboxane synthetase [imidazole]. Tranylcypromine 500 μg/ml also inhibited contractions to arachidonic acid. These agents also reduced muscle tone, so that endogenous PG formation may contribute to active tension in the LES. ETA and IDM increased tone before inhibiting it, and this effect was prevented by prior treatment with ETA or imidazole. There may also be an endogenous PG which inhibits LES tone. The possibility that this may be PGI₂ is discussed.     

Synthesis of prostaglandin 6-keto F1α by cultured aortic smooth muscle cells and stimulation of its formation in a coupled system with platelet lysates

Lysed aortic smooth muscle cells, when incubated with [¹⁴C] araachidonate, synthesized only one radioactive product, which was identified as 6-keto-PGF. Formation of this product from smooth muscle cell lysates was stimulated when human platelet extracts were added to the system, and further stimulation was observed when imidazole, as selective inhibitor of thromboxane synthesis, was added to this coupled system. These observations indicate that the cyclooxygenase of the smooth muscle cells was rate-limiting, that the prostacyclin synthetase of these cells can utilize endoperoxides produced by platelets, and that blocking of thromboxane synthesis might, under certain conditions, shunt arachionate metabolism toward prostacyclin formation.     

Production of thromboxane A2 by the kidney in glycerol-induced acute renal failure in the rabbit

The hemodynamic alterations occuring in glycerol induced renal failure are controversial. To date no single humoral substance can fully explain the change in renal resistance observed in this hemodynamic model of acute renal failure. To assess the capacity of the rabbit kidney to produce thromboxane A₂, a potent vasoconstrictor, the following experiments were carried out. Rabbits received 14 ml/kg of 50% glycerol subcutaneously 24 hrs before the study. After 24 hrs., the kidneys were removed and perfused ex vivo in superfusion bioassay cascade. Kidneys from rabbits which developed renal failure, as assessed by elevated serum creatinines, released a substance which produced contraction of rabbit aorta (RCS) in response to bradykinin (BK) and angiotensin II. Microsomes prepared from these kidneys when incubated with [¹⁴C]-arachidonic acid produced a peak of radioactivity which comigrated with thromboxane B₂ on thin layer chromatography and was inhibited by the thromboxane synthetase inhibitor imidazole. Furthermore, an inverse linear relation was found between the BK dose required to release RCS from perfused kidney and the serum creatinine levels. A direct linear relation was found between the percent of TxB₂ produced by renal microsome preparations and the serum creatinine.These studies demonstrate an increased renal capacity of the glycerol-model of acute renal failure to produce TxA₂. The production of TxA₂ a potent vasoconstrictor should therefore be further evaluated as a potential endogenous mediator of the hemodynamic changes occurring in acute renal failure.     

On the inhibitory potency of imidazole and its derivatives on thromboxane synthetase

The relative inhibitory potency of imidazole and derivatives on thromboxane synthetase from human platelets was found to be increased by substitution of the 1-position and abolished in other positions. The potency of 1-substituted imidazoles was increased as the side chain became more hydrophobic. Among the imidazole derivatives tested 1-nonyl-imidazole and 1-(2-isopropyl phenyl)-imidazole showed the highest potency with I₅₀ in the range of 10^?8 M. Inhibition by imidazole and its derivatives appeared to be very specific for thromboxane synthetase since other enzymes in prostaglandin endoperoxide metabolism were not affected. Kinetic studies indicated that inhibition was competitive with respect to prostaglandin endoperoxide substrate.     

Intravenous SRS-induced increases in tracheal mucous gel layer thickness: Evidence for thromboxane involvement

Intravenous (IV) slow reacting substance (SRS) challenge produces bronchoconstriction that can be reduced by cyclooxygenase inhibitors. This report shows that IV SRC challenge also produces significant increases in tracheal mucous gel thickness and that the increases are inhibited by pretreatment with indomethacin (4 mg/kg, PO) or imidazole (10 mg/kg, IV). The increase in gel thickness is preceded by increases in plasma thromboxane B₂ (TXB₂) levels and the inhibition of gel thickening by imidazole is paralleled by decreases in plasma TXB₂ levels. Aerosolized SRS produces increases in tracheal mucous gel thickness which are not inhibited by either indomethacin or imidazole, but are significantly inhibited by FPL-55712. These findings indicate that SRS acts, not only directly to stimulate mucous secretion but also indirectly through an indomethacin and imidazole sensitive mechanism.     

A study of three vasodilating agents as selective inhibitors of thromboxane A2 biosynthesis

Three clinically efficacious vasodilatory drugs were found to be selective inhibitors of thromboxane A₂ biosynthesis. Hydralazine, dipyridamole, and diazoxide inhibited platelet aggregation at 1 × 10^?4 M, 1.75 × 10^?4 M, and 2 × 10^?3 M respectively. Their relative inhibitory potencies on thromboxane B₂ production in human platelet microsomes were examined and found to be similar to that observed for their inhibition on human platelet aggregation. At 10^?3 M, hydralazine, dipyridamole, and diazoxide inhibited thromboxane B₂ formation by 65 percent, 27 percent and 18 percent respectively. These compounds were examined in the sheep vesicular gland system, and they were shown not to be inhibitors of the cyclooxygenase enzyme. Thus, the inhibition of thromboxane A₂ biosynthesis by these three drugs in human platelet microsomes appeared to be specific at the thromboxane synthetase level.     

The effects of dipyridamole on TXA2 formation by horse platelet microsomes

The effects of dipyridamole on thromboxane A₂ formation by horse platelet microsomes were studied in comparison with those of imidazole, a prototype inhibitor of TXA₂ synthetase and nifedipine, a calcium antagonistic vasodilator. Thromboxane A₂ was synthesized by incubating PGH₂ with horse platelet microsomes and was assayed on the superfused rabbit aorta. Dipyridamole induced as strong an inhibition of TXA₂ synthesis as imidazole, while nifedipine was without effects. The possible beneficial clinical outcomes of this effect of dipyridamole are discussed.     

In vitro effects of synthetic antioxidants and vitamin E on arachidonic acid metabolism and thromboxane formation in human platelets and on platelet aggregation

The “in vitro” effects of α-tocopherol, butylhydroxytoluene (BHT) and butylhydroxyanisole (BHA) were studied on aggregation of human platelets induced by collagen and arachidonic acid (AA), on the metabolic conversion of ¹⁴C AA through the cyclooxygenase and lipoxygenase pathways and on the formation of thromboxane B₂ (TXB₂) in washed platelets after stimulation with collagen.Vitamin E completely inhibited AA induced platelet aggregation only at high concentration (mM) and after 10 minutes of preincubation, with limited effects on AA metabolism in platelets and no effect on TXB₂ formation from endogenous substrate. BHA completely inhibited platelet aggregation in the 10⁻⁶M range, gave 50% inhibition of AA metabolism in the 10⁻⁵M range and almost complete inhibition of thromboxane formation in the 10⁻⁴M range. BHT was about 100 times less active on platelet aggregation and AA metabolism. The lipoxygenase and cyclooxygenase pathways were differentially affected at low concentrations of BHA and only at concentrations greater than 5×10⁻⁵M were both pathways depressed.     

Inhibition of GSH-dependent PGH2 isomerase in mammary adenocarcinoma cells by allicin.

We have reported tha allicin, a constituent of garlic oil, has no effect on the activities of platelet cyclooxygenase or thromboxane synthase, or vascular PGI₂ synthase. The effect of allicin on glutathione (GSH) dependent PGH₂ to PGE₂ isomerase is unknown. We therefore studied the effect of allicin on PGE₂ biosynthesis in a murine mammary adenocarcinoma cell line (No 4526). Intact or sonicated cells were incubated with either ¹⁴C-arachidonic acid (AA) or ¹⁴C-PHG₂, respectively. Following metabolism, products were extracted, separated by TLC and analyzed by radiochromatographic scan. PGE₂ was predominantly formed with minimal amounts of PGF_2α and PGD₂. Formation of 6-keto-PGF_1α or TXB₂ was not detected indicating the absence of TXA₂ and PGI₂ synthase activity. Indomethacin and ibuprofen inhibited the PGE₂ formation (p < 0.05). The enzymatic PGE₂ formation in sonicates was blocked by depletion of the cellular non-protein thiols by buthionine sulfoximine and was shown to be dependent on GSH. Allicin, over the range of 10–1000 μM, inhibited the formation of PGE₂ in cells exposed to 2.0 μM ¹⁴C-AA for 20 min. and in sonicated cells incubated with 20.0 μM ¹⁴C-PGH₂ for 2 min (p < 0.05). Allicin did not alter cyclooexygenase-mediated oxygen utilization in ram seminal vessicle microsomes, suggesting that allicin selectively inhibits the GSH-dependent PGH₂ to PGE₂ isomerase in this adenocarcinoma cell line.     

Synthesis of thromboxane A2 by non-aggregating dog platelets challenged with arachidonic acid or with prostaglandin H2

Dog platelets challenged with arachidonic acid fail to aggregate but synthesize a substance which aggregates rabbit and human platelets, this aggregation being suppressed by dibutyryl cyclic AMP. The aggregating substance contracts strips of rabbit aorta and of coeliac and mesenteric arteries, is soluble in diethyl ether, has a half-life of about 40 seconds at 37°C and of 100 seconds at 22°C. Its generation is blocked by various inhibitors of prostaglandin biosynthesis. The thromboxane A₂ synthetase inhibitor imidazole and its analogue benzimidazolamine also suppress generation of vessel contracting activity in incubates of dog platelets and prostaglandin H₂. Since dog platelets also transform prostaglandin H₂ into thromboxane A₂ their failure to aggregate, when stimulated by arachidonic acid or by prostaglandin H₂, is not due to lack of thromboxane synthesizing ability.     

Comparison of the effects of inhibitors of cytochrome P-450-mediated reations on human platelet aggregation and arachidonic acid metabolism

Metyrapone and SKF-525A, together with amphenone B, a structural analogue of metyrapone, which are all inhibitors of cytochrome P-450-mediated reactiors, were shown to inhibit the arachidonic acid-induced aggregation of human platelets. Amphenone B, like metyrapone, exhibited a type II (ligand) binding spectrum with rat liver microsomal cytochrome P-450, in contrast to SKF 525A which is a type I (substrate) binding agent. Independently of their type of binding spectra and of their maximum spectral change, however, the affinity of the three compounds for rat liver cytochrome P-450 showed a close proportional correlation with their platelet aggregation inhibitory potency. All three compounds inhibited the formation of [1?¹⁴C]thromboxane B₂ from [1?¹⁴C]arachidonic acid by human platelets aggregated with collagen. The effect of metyrapone on the remaining labelled products suggested that it is a selective thromboxane synthesis inhibitor, while amphenone B exhibited activity reminiscent of cyclo-oxygenase inhibitors. SKF 525A produced complex effects possibly attributable to cyclo-oxygenase inhibition and enhanced lipid peroxidation, since it also enhanced platelet malonaldehyde formation, which the other two compounds inhibited. These data provide further support for a role of cytochrome P-450 in thromboxane synthesis and platelet aggregation.     

Formation by phospholipase A2 of prostaglandins and thromboxane A2-like activity in the platelets of normal and essential fatty acid deficient rats. Comparison with effect on human and rabbit platelets

When rat platelets are incubated with phospholipase A₂, thromboxane A₂-like activity and prostaglandins are formed. The amounts are approximately similar, whether aggregation is induced after the incubation or not. No aggregation is observed when the platelets are incubated with phospholinase A₂. In the platelets of essential fatty acid deficient rats, only small amounts of thromboxane A₂-like activity and prostaglandins are formed. No formation of these substances occurs when human and rabbit platelets are incubated with phospholipase A₂.The results indicate that formation of thromboxane A₂-like activity enhances aggregation in rat platelets, but that aggregation is not induced.     

Effects of extracellular Ca2+ and the intracellular Ca2+ antagonist 8-(N,N-diethylamino) octyl-3,4,5-trimethoxybenzoate on rabbit platelet conversion of arachidonic acid into thromboxane

The regulatory role of Ca²⁺ on the conversion of arachidonic acid (AA) into thromboxane B₂ (TXB₂) was examined in washed rabbit platelets, whose secretoary processes are known to have requirements for extracellular CA²⁺. Varying the extracellular free Ca²⁺ [Ca_f²⁺] concentration from < 10^?8 to 10^?3 M had no significant effect on the synthesis of immunoreactive TXB₂ by rabbit platelets incubated with 1–4 μM AA. On the other hand, 8-(N,N-diethylamino) octyl-3,4,5- trimethoxybenzoate (TMB-8), a putative antagonist of intracellular Ca²⁺ movement, inhibited AA-stimulated synthesis of TXB₂ in a concentration dependent manner--an effect which could be partially overcome by increasing the AA concentration. The TMB-8 inhibition could not be reversed by increasing the [Ca²⁺_f]. Studies examining platelet metabolism of ¹⁴C-AA and ¹⁴C-prostaglandin H₂ demonstrated that TMB-8 inhibited platelet cyclooxygenase, but not thromboxane synthetase. These studies demonstrate the absence of a requirement for [Ca²⁺_f] but suggest the presence of a TMB-8 sensitive intracellular Ca²⁺ pool in the rabbit platelet synthesis of TXB₂ from AA.     

Manipulation of platelet aggregation by prostaglandins and their fatty acid precursors: Pharmacological basis for a therapeutic approach

Addition of the one-, two- or three- series endoperoxide to human platelet-rich plasma tend to supress aggregation, through the action of their respective non-enzymatic breakdown products PGE₁, PGD₂, or PGD₃ all of which elevate cyclic AMP levels. On the other hand, these stable primary products do not arise in appreciable amounts from intrinsic endoperoxides generated from either endogenous or exogenous free fatty acids. 5,8,11,14,17-Eicosapentaenoic acid (EPA) suppresses arachidonic acid (5,8,11,14-eicosatetraenoic acid) conversion by cycloogygenase (as well as lipoxygenase) to aggregatory metabolites in platelets. Exogenously added EPA was capable of inhibiting PRP aggregation induced either by exogenous or endogenous (released by ADP or collagen) arachidonate. The hypothetical combination of an EPA-rich diet and a thromboxane synthetase inhibitor might abolish production of the pro-aggregatory species, thromboxane A₂, and enhance formation of the anti-aggregatory metabolite, prostacyclin.Whereas EPA is not detectably metabolized by platelets, dihomo-γ-linolenic acid (8,11,14,-eicosatrienoic acid) is primariley converted by cyclooxygenase and thromboxane synthetase into the inactive metabolite, 12-hydroxyheptadecadienoic (HHD) acid. Pretreatment of human platelet suspensions with the thromboxane synthetase inhibitor imidazole unmasks the aggregatory property of PGH₁ and DLL which was partially compromised by the PGE₁ formed. The combination of the thromboxane synthetase inhibitor and an adenylate cyclase inhibitor unmasks a complete irreversible aggregation by DLL or PGH₁. The basis of a dietary strategy that replaces AA with DLL must rely on the production by the platelet of an inactive metabolite (HHD) rather than thromboxane A₂.