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Pulmonary pharmacology of DT-TX 30 SE, a potent selective combined thromboxane synthetase inhibitor and receptor antagonist, in guinea pigs.

A novel chemical compound, DT-TX 30 SE (E-6-(4-2-(4-chlorobenzenesulphonylamino)-ethyl)phenyl)-6-(3-pyrid yl)- hex-5-enoic acid), was studied in various models of guinea pig pulmonary function. The compound was a potent inhibitor (ED50 0.019 mg/kg, i.v.) of bronchospasm induced by the thromboxane receptor agonist U-46619, indicating thromboxane receptor antagonism. At even lower doses (ED50 0.0036 mg/kg, i.v.), it blocked arachidonic acid-induced bronchospasm. Interpretation of the latter results as evidence for additional thromboxane synthetase inhibitory activity was supported by the inhibition of arachidonic acid- or bradykinin-induced thromboxane B2 production in an isolated lung preparation, although prostaglandin E2 and prostaglandin 6-oxo-F(1 alpha) production measured at the same time were not inhibited. The potency of DT-TX 30 SE was compared with thromboxane receptor antagonists and synthetase inhibitors described in the literature. As a receptor antagonist, DT-TX 30 SE was significantly more potent than BM 13505 and BM 13177 (assessed by antagonism of U-46619-induced bronchospasm), but less potent than SQ 29548, while as a thromboxane synthetase inhibitor, it was significantly more potent than OKY 046 and UK 37248 as assessed by antagonism of arachidonic acid-induced bronchospasm or (OKY 046) inhibition of thromboxane production in isolated lung. The compound was active by the oral route as shown by its ability, at 10 mg/kg, p.o., to significantly reduce the immediate allergic response of sensitized guinea pigs to an ovalbumin aerosol.


ABSTRACT-
Thromboxane A2 is a short lived lipid mediator whose biological effects in the lung can include bronchospasm and constriction of the vessels of the pulmonary vascu lature. It is a potent platelet aggregating agent and may therefore play a role in pulmonary thrombosis (1).
In tissues, thromboxane A2 is synthesized from an un stable prostaglandin endoperoxide, prostaglandin H2, by the enzyme thromboxane synthetase. Thromboxane A2 is rapidly hydrolyzed in aqueous media to more stable inac tive metabolites, the most important of which is throm boxane B2. Prostaglandin H2 can also be metabolized via alternative routes: non-enzymic conversion to prostaglan din E2 and prostacyclin synthetase-catalyzed conversion to prostacyclin, another biologically active molecule, many of whose actions (e.g., on platelet aggregation or on smooth muscle) are antagonistic to those of throm boxane A2 (2). Prostacyclin is itself unstable and rapidly becomes converted to prostaglandin 6-oxo-Fl, and other more stable but biologically less active metabolites.
Several thromboxane synthetase inhibitors have al ready been tested clinically for pulmonary diseases. OKY 046 has been reported to provide some benefit in mild to moderate asthma and to reduce bronchial responsiveness to acetylcholine in asthmatic subjects (3, 4).
It has been suggested that one factor that may limit the clinical efficacy of thromboxane synthetase inhibitors is the accumulation, when further metabolism is blocked, of the substrate prostaglandin H2. Prostaglandin H2 can bind and activate the thromboxane A2 receptor almost as well as thromboxane A2 itself (5, 6). This being so, a molecule that can antagonize the prostaglandin H2 /thromboxane A2 receptor as well as being a specific thromboxane synthetase inhibitor would be expected to have advantages over a substance that is a thromboxane synthetase inhibitor alone.
The synthesis and characterization of DT-TX 30 SE in in vitro models of platelet function is described elsewhere (7). Here the substance has been characterized for its ac tions on the lung.
For investigation of thromboxane synthetase inhibi tion, an aqueous solution of arachidonic acid sodium salt (Sigma, St. Louis, MO, USA) was repeatedly injected i.v.
(1 ml/kg) until a dose was found (0.5 2.0 mg/kg) that consistently gave approximately 60% reduction of peak air flow. This dose was then repeated, but 2 min prior to this, DT-TX 30 SE or comparison substance was injected i.v. (1 ml/kg). For investigation of thromboxane receptor antagonism, the procedure was similar, but instead of arachidonic acid, the guinea pigs were challenged with between 2.5 and 25 pg/kg U-46619 (Paesel, Frankfurt am Main, Germany).
Each animal was only tested at one dose level of DT TX 30 SE or comparison substance. At least three and in some cases four doses of drug were tested with at least five animals at each dose level plus the vehicle (saline) controls. ED50 values with 95% confidence limits were calculated by linear regression.

Studies with isolated lungs
Guinea pigs (660-800 g) were killed by cervical dislo cation and the heart and lungs rapidly removed and flushed with a modified Tyrode's solution of the follow ing composition per liter: 8 g NaCI, 0.2 g KCI, 0.24 g CaC12.2H2O, 0.214 g MgC12.6H2O, 1 g NaHCO3, 0.576 g NaH2PO4.2H2O, 1 g glucose. The lung was suspended in a humidified chamber maintained at 37C and ventilated by negative pressure (52 breaths/min, pressure between 0.5 and -16 cmH2O) using a breathing cycle with 0.4 sec between breaths. Modified Tyrode was perfused at a con stant flow rate (0.5 ml/min) via the pulmonary artery and oxygenation ensured by a stream (28 ml/min) of "Carbo gen" (95% oxygen, 5% carbon dioxide).
The flow of air into and out of the lung was continu ously monitored by a "Microbridge" AWM 2100 mass air flow sensor (Honeywell Micro Switch Division, Newtown, CT, USA). Perfusate was collected via a one-way valve for one 2-min interval before and six 2-min intervals after challenge with a 0.1 ml bolus of 3.3 nmol arachidonic acid or 0.02 nmol bradykinin acetate (Serva, Heidelberg, Germany). Volume of perfusate collected during each 2 min period was approximately 1.0 ml. Thromboxane B2, prostaglandin 6-oxo-F,,, and prostaglandin E2 were esti mated in duplicate for each sample by radioimmunoassay using commercially available kits (NEK-024, NEK-025, NEK-020; New England Nuclear, Boston, MA, USA).
The radioimmunoassay of thromboxane B2 and prosta glandin 6-oxo-Fl, was validated by also measuring some of the samples by electron-capture GC-MS as described by Barrow et al. (8).
For the studies with bradykinin, the lungs were challenged twice, DT-TX 30 SE being added to the perfu sate of the test (but not the control) lungs for 30 min be fore until 12 min after the second bradykinin challenge. For studies with arachidonic acid, the lungs were challenged three times, the first challenge being the vehi cle used to place the arachidonic acid in solution (7% methanol, 0.1 % acetic acid in Tyrode's solution), the sec ond and third challenges being arachidonic acid. DT-TX 30 SE was added to the perfusate 30 min before until 12 min after the third challenge of test (but not control) lungs.
A re-equilibration period of at least 1 hr was allowed between challenges.
For studies with bradykinin, DT-TX 30 SE was tested at 6 dose levels (0.001, 0.01, 0.1, 1, 10 and 100 jM) and comparison substance OKY 046 at 4 dose levels (0.1, 1, 10 and 100 pM) with 4-6 lungs at each dose level. All per fusates were measured by radioimmunoassay, and perfu sates from DT-TX 30 SE dose levels 0.01 and 1 pM (plus controls) measured by GC-MS. For the studies using the arachidonic acid challenge, only one dose of DT-TX 30 SE (1 pM) was tested (6 lungs plus 6 control non-sub stance treated lungs).
Statistical analysis of drug effects was performed by non-parametric methods. The measurements 1, 3, 5, 7, 9 and 11 min after stimulus (arachidonic acid or bradyki nin) were summed up for each period for the parameters thromboxane B2, prostaglandin E2 and prostaglandin 6 oxo-Fla and averaged for the change of air flow. The effects of DT-TX 30 SE and (where tested) OKY 046 on the four parameters were examined separately for the two substances. The values for the second challenge with arachidonic acid or bradykinin were taken as a percentage of the values for the first challenge with the same stimuli. With respect to this transformation, the statistical evalu ation was performed in two steps. In order to look for a dose-response relationship (effect increases/decreases with higher doses of the substances) a Jonckheere test was used (9). Wilcoxon tests calculating exact P-values (per mutation test procedure) were added to decide which doses caused different effects compared to the control. Where only two drug doses were examined (bradykinin challenge, GC-MS assay), global effects were tested by the Kruskal-Wallis test. Where only one substance dose was tested (arachidonic acid study), only the Wilcoxon test was applied. In the study with bradykinin challenges, Wilcoxon's test was also used to compare DT-TX 30 SE and OKY 046 within common dose groups. Where the same samples were assayed by two methods (radioim munoassay, GC-MS), correlation of the test results was made by Pearson's test.
Studies of the immediate-reaction in allergized guinea pig Guinea pigs were sensitized by a single i.p. injection containing 40 mg/kg ovalbumin bound to 2 g/10 ml aluminium hydroxide gel (with saline to 0.5 ml/animal). Then, 6 weeks later, they were challenged for 90 sec with an aerosol of 3010 ovalbumin (Technolat type USV 82 nebulizer). Aerosol challenge was preceded by s.c. injec tion, 30 min before, of an antihistamine (mepyramine hydrochloride 1 mg/kg). Ten minutes later, the animals were killed by cervical dislocation and the lungs rapidly dissected free from the heart, blood vessels and associated tissues. The lung volumes were measured by fluid dis placement according to Archimedes' principle, as fol lows: the lung was held by the trachea for immersion in a beaker of water and the decrease in weight on immersion recorded. The volume measured thus corresponded to that of the lung lobes with trapped air, the bronchi and that part of the trachea (standardly 80010 of the total length) which was immersed. Test drug (or vehicle control) at the appropriate doses was administered orally (1 ml/kg) as an aqueous solution containing 0.5% Tylose at 60 min prior to the aerosol challenge.

Controls included
non-sensitized animals treated with drug or vehicle and exposed to ovalbumin aerosol and sensitised animals exposed to saline aerosol.
For all comparisons, Wilcoxon tests calculating exact P-values (permutation test procedure) were estimated. Because of the plurality of the tests, the significance level was not adjusted, and therefore the P-values should be interpreted in a qualitative manner.   DT-TX 30 SE also blocked arachidonic acid-induced systemic hypotension, although the ED50 (between 0.005 and 0.01 mg/kg, i.v.) was somewhat higher than that for inhibition of bronchospasm.

Studies with isolated lungs
Arachidonic acid challenge: The injection of 10 pg arachidonic acid caused a marked rise in the production of all three of the arachidonic acid metabolites examined; in comparison with a previous challenge with vehicle, the effect was highly statistically significant (P<0.0005, Wilcoxon). When a second arachidonic acid challenge was given, thromboxane B2 production differed little from that after the first challenge, but production of prostaglandin E2 and prostaglandin 6-oxo-F,,, was some what decreased (see Fig. 2). The effect of including 1 pM DT-TX 30 SE in the perfusate shortly before and after the second challenge (6 lungs) was to cause a highly sig nificant (P G 0.005) decrease in thromboxane B2 produc tion; relative to the lungs challenged a second time, but not treated with DT-TX 30 SE (N = 6), the addition of DT-TX 30 SE also caused an increase in both prostaglan din E2 and prostaglandin 6-oxo-F,,, but only the increase in prostaglandin E2 reached statistical significance (P < 0.05). All six lungs in the control group reacted to the second arachidonic acid challenge with bronchospasm (>5% fall in mean respiratory flow over the 12 min period), whereas none of the lungs in the DT-TX 30 SE group showed any arachidonic acid-induced broncho spasm.
Bradykinin challenge: Bradykinin also caused a marked rise in the production of all 3 arachidonic acid metabolites examined, but in contrast to the results with arachidonic acid, the amounts of the metabolites were more modest, and the pattern of metabolites did not markedly differ between first and second challenges in the absence of test substance (see Fig. 3).
In the analysis of the effects of DT-TX 30 SE, Jonckheere's test showed a highly significant (P <0.0001) trend of reduction in thromboxane B2 production (radioimmunoassay measurements) with higher DT-TX 30 SE concentrations.
Biologically (median reduction < 25 % control thromboxane production) and statistically (P<0.05, Wilcoxon) significant reduction was present at concentrations of 0.1 pM and above. The comparison substance OKY 046 also showed the expected decrease in thromboxane B2 production with higher concentrations (Jonckheere's test P=0.0001), but statistically (P<0.05) significant reduction was only present at dose levels of 10 and 100 pM. At the 1-pM dose level, the difference be tween DT-TX 30 SE and OKY 046 was statistically sig nificant (P<0.05, Wilcoxon). Figure 4 represents graphi cally the individual results at the dose levels used in both the DT-TX 30 SE and OKY 046 studies. Figure 3 com pares the time course of arachidonic acid metabolite production in the presence and absence of 1 pM DT-TX 30 SE (radioimmunoassay measurements). GC-MS also showed highly significant (P<0.01, Wilcoxon) reduction in thromboxane B2 production by 1 uM DT-TX 30 SE (Fig. 5). Changes in prostaglandin 6-oxo-Fl, were not, either by radioimmunoassay or GC-MS measurements, statistically significant (radioimmunoassay, Jonckheere's test P > 0.05; GC-MS Kruskal-Wallis P > 0.05). There was a reasonable correlation, taking all the studies together (N=17), between GC-MS and radioimmunoassay meas urement (Pearson's correlation coefficients: thromboxane B2 measurements, 0.62; prostaglandin 6-oxo-F,,, measure ments, 0.86). There was a trend for higher doses of either DT-TX 30 SE or OKY 046 to be associated with raised levels of prostaglandin E2 (Jonckheere's test P <0.01).
There was a trend (Jonckheere's test, P <0.05) for in creased doses of DT-TX 30 SE to be associated with reduced bradykinin-induced bronchospasm (reduction in air flow), but the effect was not complete and individual animals, for whom radioimmunoassay and GC-MS both indicated total inhibition of thromboxane B2 production, still showed some bradykinin-induced bronchospasm.
Studies of the immediate reaction in allergized guinea pigs DT-TX 30 SE, dosed orally 60 min before the allergen aerosol, could significantly decrease the allergen-induced increase in lung relaxation volume (Fig. 6), but the effect was not total. That is, at the doses at which there was a significant difference between the drug treated animals and positive control (sensitized animals + ovalbumin aerosol), there was also a significant (P <0.05, Wilcoxon) difference from the negative control (non-sensitized animals + ovalbumin, or sensitized animals + saline).

DISCUSSION
The studies in guinea pigs challenged with arachidonic acid or the thromboxane receptor agonist U-46619 are in agreement with and confirm in vivo studies on human platelets indicating that DT-TX 30 SE can act as both a thromboxane synthetase inhibitor and a receptor an tagonist (7). The dose of DT-TX 30 SE required to inhibit arachidonic acid-induced bronchospasm was significantly (approximately fivefold) lower than the dose required to inhibit U-46619-induced bronchospasm.
This ratio is close to the previously observed difference between the IC50's for thromboxane synthetase inhibition and recep tor binding reported in gel filtered human platelets (7). It has previously been suggested that the thromboxane receptors involved in aggregation of washed human platelets and in guinea pig bronchoconstriction belong to a common homogenous population (10). Interpretation of the in vivo data for inhibition of arachidonic acid-induced bronchospasm as evidence for a thromboxane synthetase inhibitory activity is confirm ed by the specific inhibition of arachidonic acid or bradykinin-induced thromboxane B2 synthesis by rela tively low (0.1 or 1 pM) concentrations of DT-TX 30 SE in the isolated lung preparation. Most of the studies here described measured thromboxane B2 by radioim munoassay, a method whose validity depends on the specificity of the antibodies employed. The correlation be tween GC-MS and radioimmunoassay measurements sug gests the radioimmunoassay results are valid. That the thromboxane B2 measurements made by radioimmuno assay were consistently somewhat higher than those made by GC-MS may be explained by a contribution to the radioimmunoassay results from structurally related alternative metabolites of thromboxane A2 in guinea pig lungs such as 15-oxo-13,14 dehydrothromboxane B2, 11-dehydrothromboxane B2 or 2,3-dinor-thromboxane B2 (11). Since levels of these thromboxane metabolites are likely to vary in a similar way after drug treatment as those of thromboxane B2 itself, cross-reactivity of the radioimmunoassay antibody with these metabolites does not invalidate the conclusions of the study relating to DT-TX 30 SE. The proportion of arachidonic acid collected as metabolites was relatively small (2.3% of the perfused dose), a situation that probably results from ready incor poration of free fatty acids into membrane lipids. This and the relative proportion of the three metabolites (prostaglandin 6-oxo-Fl, > thromboxane B2 > prosta glandin E2) reflects the observations of other workers (12-15).

Inhibition
of thromboxane synthesis was associated with increased prostaglandin E2 synthesis, but as prostaglandin 6-oxo-F,,, synthesis was, even in the ab sence of drug, relatively large in relation to thromboxane synthesis, and varied from challenge to challenge, it is not possible to estimate from these data to what extent inhi bition of prostaglandin H2 conversion to thromboxane A2 was accompanied by metabolism of the unconverted prostaglandin H2 by the alternative route to prostacyclin (and thus prostaglandin 6-oxo-F1 ). What was clear was the inhibition of arachidonic acid metabolism to throm boxane A2 by DT-TX 30 SE was specific, so that the ratio of prostaglandin 6-oxo-F1,, to thromboxane B2 (and therefore presumably the ratio of prostacyclin to throm boxane A2) was greatly increased in the presence of DT TX 30 SE concentrations of 0.1 pM and above.
In comparison with the thromboxane receptor an tagonists reported in the literature, the in vivo studies with U-46619 showed that DT-TX 30 SE had slightly greater potency than BM 13505 and BM 13177, but was some what weaker than SQ 29548. Comparison with standard thromboxane synthetase inhibitors (inhibition of arachi donic acid-induced bronchospasm) showed that it had a potency similar to CGS 13080 and significantly better than UK 37248 or OKY 046. The difference between the ED50's of DT-TX 30 SE and OKY 046 was about 80-fold, which compares with a difference of 100-fold in the con centrations of these drugs required to inhibit throm boxane B2 production in the isolated lung.
To show the biological activity of DT-TX 30 SE in a pulmonary disease model, the effect of the drug against the immediate allergen reactions in guinea pigs was test ed. Other thromboxane synthetase inhibitors have pre viously been reported to reduce the immediate allergic reaction in this species (16, 17). The parameter measured, increase in lung relaxation volume, is a simple method for measuring the immediate reaction in conscious animals, which has been used in a number of laboratories (18, 19). Since the relaxation volume is a static measurement, air way closure (in which contraction of the bronchial mus culature is likely to play an important role) is the only mechanism that can account for the increase in this parameter. The histamine component of the immediate response was excluded by pretreating the animals with mepyramine. DT-TX 30 SE was active in this model. We are cautious in extrapolating these results from guinea pig to humans; the study does, however, suggest that DT-TX 30 SE is orally active and reaches the lung in pharma cologically active amounts.
The combined profile of oral activity, very potent and specific thromboxane synthetase inhibitory activity and potent thromboxane receptor antagonism, suggests that this compound alone, or in combination with other drugs, may prove a useful tool for elucidating the role of thromboxane in pulmonary disease.