Replacement of the carboxylic acid group of prostaglandin f(2alpha) with a hydroxyl or methoxy substituent provides biologically unique compounds.

Replacement of the carboxylic acid group of PGF(2alpha) with the non-acidic substituents hydroxyl (-OH) or methoxy (-OCH(3)) resulted in an unexpected activity profile. Although PGF(2alpha) 1-OH and PGF(2alpha) 1-OCH(3) exhibited potent contractile effects similar to 17-phenyl PGF(2alpha) in the cat lung parenchymal preparation, they were approximately 1000 times less potent than 17-phenyl PGF(2alpha) in stimulating recombinant feline and human FP receptors. In human dermal fibroblasts and Swiss 3T3 cells PGF(2alpha) 1-OH and PGF(2alpha) 1-OCH(3) produced no Ca(2+) signal until a 1 microM concentration was exceeded. Pretreatment of Swiss 3T3 cells with either 1 microM PGF(2alpha) 1-OH or PGF(2alpha) 1-OCH(3) did not attenuate Ca(2+) signal responses produced by PGF(2alpha) or fluprostenol. In the rat uterus, PGF(2alpha) 1-OH was about two orders of magnitude less potent than 17-phenyl PGF(2alpha) whereas PGF(2alpha) 1-OCH(3) produced only a minimal effect. Radioligand binding studies on cat lung parenchymal plasma membrane preparations suggested that the cat lung parenchyma does not contain a homogeneous population of receptors that equally respond to PGF(2alpha)1-OH, PGF(2alpha)1-OCH(3), and classical FP receptor agonists. Studies on smooth muscle preparations and cells containing DP, EP(1), EP(2), EP(3), EP(4), IP, and TP receptors indicated that the activity of PGF(2alpha) 1-OH and PGF(2alpha) 1-OCH(3) could not be ascribed to interaction with these receptors. The potent effects of PGF(2alpha) 1-OH and PGF(2alpha) 1-OCH(3) on the cat lung parenchyma are difficult to describe in terms of interaction with the FP or any other known prostanoid receptor.

Pharmacological evidence for thromboxane receptor heterogeneity--implications for the eye.

The pharmacological activity of two novel thromboxane A2 (TxA2)-mimetics, AGN191976 and AGN192093, was investigated in vitro, using standard organ bath assays and human platelets, to determine potency and selectivity at various prostanoid (PG-) receptors. The effects of these compounds on intraocular pressure in Beagle dogs were then compared with U-46619, a widely employed and structurally different TP-receptor agonist. AGN191976 and AGN192093 were highly potent TP-receptor agonists in the rat aorta (EC50 of 0.32 and 1.3 nM, respectively) and human myometrium. Both compounds were approximately 10 to 50 fold more potent than U-46619. These contractile responses could be blocked with a potent TP-receptor antagonist, SQ29548. In human platelets, AGN191976 (EC50 = 16.3 nM) and U-46619 (EC50 = 538.3 nM) potently stimulated aggregation (TP-receptor mediated effect), whereas AGN192093 was a much weaker agonist (EC50 = 37.9 microM). AGN192093 was not a partial agonist in platelets, since it did not antagonize aggregation induced by AGN191976, U-46619, arachidonic acid or ADP. These results provide evidence for a subdivision of TP-receptors, and AGN192093 appears to be able to distinguish between TP-receptors in smooth muscle and platelets. In the Beagle dog eye, both AGN191976 and AGN192093 were highly potent and efficacious ocular hypotensives. Single 2.5 micrograms doses of drug decreased IOP by 11.4 (AGN191976) and 7.7 mm Hg (AGN192093) relative to the contralateral control eye. In contrast, U-46619 did not lower IOP. AGN191976, but not U-46619, increased outflow facility in these animals, which is consistent with their effects on IOP. Neither compound caused miosis which is FP-receptor mediated in the dog. These studies suggest the existence of heterogeneous populations of TP-receptors. AGN191976 and AGN192093, two novel TP-receptor agonists, appear to be useful tools for the pharmacological distinction of TP-receptor subtypes.

Evidence for human thromboxane receptor heterogeneity using a novel series of 9,11-cyclic carbonate derivatives of prostaglandin F2 alpha.

1. The pharmacological activity of a novel series of 9,11-cyclic carbonate derivatives of prostaglandin F2 alpha (PGF2 alpha) was investigated in various isolated smooth muscle preparations possessing different prostanoid receptor subtypes as well as in human platelets. Since subdivision of thromboxane (TP-) receptors into vascular/smooth muscle and platelet subtypes is a controversial subject, our studies included a human smooth muscle preparation (myometrium) in addition to the widely used rat aorta and human platelets as TP-receptor preparations. 2. Two members of that series, AGN191976 and AGN192093 were found to be highly potent and selective thromboxane-mimetics. AGN191976 and AGN192093 contracted isolated tissues of the rat thoracic aorta with EC50 values of 0.32 +/- 0.08 and 1.30 +/- 0.53 nM, respectively. Both agonists were at least 10 times more potent than the benchmark TP-agonist, U-46619, in this preparation, whilst being at least 500 times less potent at other prostanoid receptors (DP, EP1, EP3, FP, IP) in vitro. 3. In human myometrial strips from pregnant and non-pregnant donors, both AGN191976 and AGN192093 were potent contractile agonists. The rank order of potency in myometrium of AGN191976 > AGN192093 > U-46619 correlated well with that in the rat aorta. In human platelet-rich plasma (PRP), however, AGN191976 had potent proaggregatory activity (EC50 = 16.3 +/- 1.4 nM), which is a TP-receptor-mediated event, whereas AGN192093 was a much weaker agonist (EC50 = 37.9 +/- 2.0 microM). AGN192093 did not behave as an antagonist in the platelets, since it did not antagonize platelet aggregation induced by ADP, arachidonic acid, U-46619 or AGN191976. In human washed platelets, the activity profile of AGN191976 (EC50 = 4.15 +/- 0.52 nM) and AGN192093 (no aggregation up to 10 microM) was similar to that obtained in PRP. 4. The involvement of TP-receptors was verified with the potent TP-antagonist, SQ29548. SQ29548 (0.1 microM in myometrium; 1 microM in aorta; 1 microM and 10 microM in platelets) antagonized responses to U-46619, AGN191976 and AGN192093 as expected. 5. In conclusion, AGN191976 and AGN192093, both 9,11-cyclic carbonate derivatives of PGF2 alpha, were found to be highly potent and selective thromboxane-mimetics in rat vascular and human myometrial smooth muscle. However, only AGN 191976 was a potent agonist at TP-receptors in human platelets. The differential activity of AGN192093 on TP-receptor-mediated events in platelets and smooth muscle provides further evidence for a subdivision of TP-receptors. AGN192093 appears to be a useful tool for the pharmacological distinction of TP-receptor subtypes.

AGN 191976: a novel thromboxane A2-mimetic with ocular hypotensive properties.

The possible subdivision of thromboxane A2-sensitive (TP) receptors is currently a controversial subject. We report herein on a novel thromboxane A2 mimetic, AGN 191976, which has almost identical pharmacological activity to the well-characterized prostaglandin H2/thromboxane A2 (PGH2/TxA2) mimetic U-46619, but its effects on intraocular pressure are quite distinct from U-46619. Prostanoid receptor activity was determined in vitro using different smooth muscle assays and platelets. Intraocular pressure was measured tonometrically in ocular normotensive Beagle dogs and Cynomolgus monkeys. Conjunctival microvascular permeability was determined in guinea pigs. Despite closely resembling U-46619 as a potent and selective TP receptor agonist, AGN 191976 was a potent ocular hypotensive in dogs and monkeys whereas U-46619 did not lower IOP in either species. The ocular hypotensive effect of AGN 191976 in dogs was attenuated by pretreatment with the TP receptor antagonist SQ 29548. Thus, the ocular hypotensive effects of AGN 191976 are consistent with TP receptor stimulation. Both TxA2-mimetics caused plasma leakage in the guinea pig conjunctiva. The disparate activities of U-46619 and AGN 191976 in our studies suggest the existence of heterogeneous populations of TP-receptors in the eye.

Studies on a novel series of acyl ester prodrugs of prostaglandin F2 alpha.

A novel series of prostaglandin F2 alpha (PGF2 alpha) prodrugs, with acyl ester groups at the 9, 11, and 15 positions, was prepared in order to design clinically acceptable prostaglandins for treating glaucoma. Studies involving isolated esterases and ocular tissue homogenates indicated that 9-acyl esters cannot provide a prodrug since PGF2 alpha would not be formed as a product. In contrast, 11-mono, 15-mono, and 11, 15-diesters were converted to PGF2 alpha in ocular tissues and could, therefore, be considered as prodrugs of PGF2 alpha. Carboxylesterase (CE) appeared critically important for the hydrolytic conversion of those PGF2 alpha prodrugs where the 11 or 15-OH group was esterified and such prodrugs were not substrates for acetylcholinesterase (ACHE) or butyrylcholinesterase (BuCHE). The enzymatic hydrolysis of PGF2 alpha-1-isopropyl ester was also investigated for comparative purposes. This PGF2 alpha prodrug was a good substrate for CE, but was also hydrolysed by BuCHE, albeit at a much slower rate. The most striking feature of the enzymatic hydrolysis of PGF2 alpha-1-isopropyl ester in ocular tissue homogenates was that it was much faster than for prodrugs esterified at the 11 and/or 15 positions. In terms of ocular hypotensive activity, all prodrugs which showed detectable conversion to nascent PGF2 alpha were potent ocular hypotensives. Although no separation of ocular hypotensive and ocular surface hyperaemic effects was apparent for PGF2 alpha-1-isopropyl ester, a temporal separation of these effects was apparent for the novel PGF2 alpha ester series. This difference may reflect an unfavourably rapid conversion of PGF2 alpha-1-isopropyl ester in ocular surface tissues compared with anterior segment tissues.

Studies on the ocular hypotensive effects of prostaglandin F2 alpha ester prodrugs and receptor selective prostaglandin analogs.

The use of natural prostaglandins (PG), such as PGD2, PGE2, PGF2 alpha, and PGI2, for treating glaucoma is limited by their ocular side effects. One approach to achieve the required separation of ocular hypotensive activity from side effects is to employ ester prodrugs. From a novel series of 11- and 15-mono and 11,15-diacyl esters of PGF2 alpha we identified prodrugs where PGF2 alpha formation rates in the iris-ciliary body exceeded those in the conjunctiva, sclera, and corneal endothelium. Compared to PGF2 alpha-1-isopropyl ester the ocular tissue hydrolysis rates of the 11-monopivaloyl, the 11,15-dipivaloyl ester and the 1,11-lactone were up to 1000 fold less. Despite this large disparity in hydrolysis rates, the pivaloyl esters and the 1,11-lactone were potent ocular hypotensives in our animal models. In studying prostaglandin analogs, we found that a diverse variety of prostanoid receptor selective agonists lowered intraocular pressure in dogs and/or monkeys. These included DP-, EP1-, EP2-, EP3-, and FP-receptor selective compounds. These findings were surprising and prompted us to re-examine the receptor selectivity of these agonists by radioligand binding studies. Using radiolabelled PGE2, 17-phenyl PGF2 alpha, and sulprostone we were able to confirm the selectivity of the agonists currently used for receptor characterization directly by radioligand binding competition studies. It appears that multiple prostanoid receptor subtypes may be involved in regulating intraocular pressure.