A dual acting compound with latanoprost amide and nitric oxide releasing properties, shows ocular hypotensive effects in rabbits and dogs.

The IOP lowering effects of NCX 139, a new chemical entity comprising latanoprost amide and a NO-donating moiety, were compared to those of the respective des-nitro analog in in vitro assays and in rabbit and dog models of ocular hypertension. The NO donor, molsidomine as well as the prostamide bimatoprost (Lumigan(®)) and the prostaglandin agonist, latanoprost (Xalatan(®)) were also investigated for comparison. NCX 139 but not its des-nitro analog resulted in NO-mediated vascular relaxant effect in pre-contracted rabbit aortic rings (EC(50)=0.70±0.06 µM; E(max)=80.6±2.9%). Like bimatoprost (IC(50)=3.07±1.3 µM) or latanoprost (IC(50)=0.48±0.15 µM), NCX 139 displaced (3)H-PGF2α binding on recombinant human prostaglandin-F (FP) receptors with an estimated potency of 0.77±0.13 µM. In transient ocular hypertensive rabbits, bimatoprost and latanoprost were not effective while molsidomine elicited a dose-dependent reduction of IOP confirming the responsiveness of rabbits to NO but not to FP receptor agonists. NCX 139 tested at a therapeutically relevant dose, significantly lowered IOP while the des-nitro analog was not effective (0.03% NCX 139, Δ(max)=-12.8±2.0 mmHg). In glaucomatous dogs, 0.03% NCX 139 decreased IOP to a greater extent compared to an equimolar dose of the respective des-nitro derivative (Δ(max)=-4.6±1.0 and -2.7±1.3 mmHg, respectively for NCX 139 and its des-nitro analog). Albeit with low potency, NCX 139 also resulted effective in normotensive dogs while it did not reduce IOP in normotensive rabbits. NCX 139, a compound targeting two different and important mechanisms, is endowed with ocular hypotensive effects more evident in hypertensive conditions which may be of interest in the search of more effective treatments for hypertensive glaucoma.

Identification and pharmacological characterization of the prostaglandin FP receptor and FP receptor variant complexes.

BACKGROUND AND PURPOSE: A prostamide analogue, bimatoprost, has been shown to be effective in reducing intraocular pressure, but its precise mechanism of action remains unclear. Hence, to elucidate the molecular mechanisms of this effect of bimatoprost, we focused on pharmacologically characterizing prostaglandin FP receptor (FP) and FP receptor variant (altFP) complexes. EXPERIMENTAL APPROACH: FP receptor mRNA variants were identified by reverse transcription-polymerase chain reaction. The FP-altFP4 heterodimers were established in HEK293/EBNA cells co-expressing FP and altFP4 receptor variants. A fluorometric imaging plate reader was used to study Ca2+ mobilization. Upregulation of cysteine-rich angiogenic protein 61 (Cyr61) mRNA was measured by Northern blot analysis, and phosphorylation of myosin light chain (MLC) by western analysis. KEY RESULTS: Six splicing variants of FP receptor mRNA were identified in human ocular tissues. Immunoprecipitation confirmed that the FP receptor is dimerized with altFP4 receptors in HEK293/EBNA cells co-expressing FP and altFP4 receptors. In the studies of the kinetic profile for Ca2+ mobilization, prostaglandin F2alpha (PGF2alpha) elicited a rapid increase in intracellular Ca2+ followed by a steady state phase. In contrast, bimatoprost elicited an immediate increase in intracellular Ca2+ followed by a second phase. The prostamide antagonist, AGN211335, selectively and dose-dependently inhibited the bimatoprost-initiated second phase of Ca2+ mobilization, Cyr61 mRNA upregulation and MLC phosphorylation, but did not block the action of PGF2alpha. CONCLUSION AND IMPLICATIONS: Bimatoprost lacks effects on the FP receptor but may interact with the FP-altFP receptor heterodimer to induce alterations in second messenger signalling. Hence, FP-altFP complexes may represent the underlying basis of bimatoprost pharmacology.

Prostamides (prostaglandin-ethanolamides) and their pharmacology.

The prostamides are part of a large and continually expanding series of pharmacologically unique neutral lipids. They are COX-2 derived oxidation products of the endocannabinoid/endovanniloid anandamide. Prostamide pharmacology is unique and, as in the case of the endocannabinoids anandamide and 2-arachidonylglycerol, bears little resemblance to that of the corresponding free acids. By virtue of its close relationship to the anti-glaucoma drug bimatoprost, prostamide F(2alpha) has received the greatest research attention. Prostamide F(2alpha) and bimatoprost effects appear independent of prostanoid FP receptor activation, according to a litany of agonist studies. Studies involving freshly isolated and separate feline iridial smooth muscle cells revealed that bimatoprost and FP receptor agonists stimulated different cells, without exception. This suggests the existence of receptors that preferentially recognize prostamide F(2alpha). The recent discovery of prostamide antagonists has provided further support for prostamide receptors as discrete entities. The prototypical prostamide antagonists, AGN 204396 and 7, blocked the effects of prostamide F(2alpha) and bimatoprost but not those of PGF(2alpha) and FP receptor agonists in the feline iris. Second generation more potent prostamide antagonists, such as AGN 211334, should allow the role of prostamides in health and disease to be elucidated. From the therapeutics standpoint, the prostamide F(2alpha) analogue bimatoprost is the most efficacious ocular hypotensive agent currently available for the treatment of glaucoma.

Identification of an antagonist that selectively blocks the activity of prostamides (prostaglandin-ethanolamides) in the feline iris.

BACKGROUND AND PURPOSE: The prostamides (prostaglandin-ethanolamides) and prostaglandin (PG) glyceryl esters are biosynthesized by COX-2 from the respective endocannabinoids anandamide and 2-arachidonyl glycerol. Agonist studies suggest that their pharmacologies are unique and unrelated to prostanoid receptors. This concept was further investigated using antagonists. EXPERIMENTAL APPROACH: The isolated feline iris was used as a key preparation, where prostanoid FP receptors and prostamide activity co-exist. Activity at human recombinant FP and other prostanoid receptors was determined using stable transfectants. KEY RESULTS: In the feline iris, AGN 204396 produced a rightward shift of the dose-response curves for prostamide F2alpha and the prostamide F2alpha analog bimatoprost but did not block the effects of PGF2alpha and synthetic FP receptor agonists. Studies on human recombinant prostanoid receptors confirmed that AGN 204396 did not behave as a prostanoid FP receptor antagonist. AGN 204396 exhibited no antagonism at DP and EP1-4, but was a highly effective TP receptor antagonist. Contrary to expectation, the FP receptor antagonist AL-8810 efficaciously contracted the cat iris. AGN 204396 did not affect AL-8810 induced contractions, demonstrating that AL-8810 and AGN 204396 are pharmacologically distinct. Unlike AL-8810, the ethylamide derivate of AL-8810 was not an agonist. Al-8810 did not block prostamide F2alpha activity. Finally, AGN 204396 did not block PGE2-glyceryl ester activity. CONCLUSIONS AND IMPLICATIONS: The ability of AGN 204396 to selectively block prostamide responses suggests the existence of prostamide sensitive receptors as entities distinct from receptors recognizing PGF2alpha and PGE2-glyceryl ester.

Bimatoprost: a novel antiglaucoma agent.

The aim of glaucoma therapy is to preserve vision by reducing intraocular pressure (IOP). Following recent National Eye Institute sponsored studies, it is becoming increasingly apparent that every mmHg of extra IOP lowering counts. Bimatoprost is the newest and most effective addition to the physician's armamentarium of ocular hypotensive drugs. Direct clinical comparisons have demonstrated that it is more efficacious than the prostaglandin (PG) FP receptor agonist prodrugs, latanoprost and travoprost, as well as a beta-adrenoceptor antagonist, timolol, alone or in fixed combination with the carbonic anhydrase inhibitor, dorzolamide. Moreover, patients that are refractory to latanoprost therapy may be successfully treated with bimatoprost. Such evidence provides support, at the clinical level, for the contention that bimatoprost is pharmacologically distinct from PG FP receptor agonist prodrugs. Bimatoprost is a structural analog of PGF2alpha-ethanolamide (prostamide F2alpha), which is formed from the endocannabinoid anandamide by a biosynthetic pathway involving cyclooxygenase-2 (COX-2). Their pharmacology is remarkably similar, such that bimatoprost may be regarded as a prostamide mimetic. The target receptor for bimatoprost and the prostamides appears unique and unrelated to PG- and endocannabinoid-sensitive receptors. Extensive ocular distribution/metabolism studies in non-human primates demonstrate that bimatoprost is not a prodrug, it remains essentially intact. Its profound ocular hypotensive effects may, therefore, be attributed to its prostamide-mimetic properties.

Latanoprost-induced pigmentation in human iridial melanocytes is fibroblast dependent.

The prostaglandin F2alpha derivative, latanoprost (LT), used in glaucoma treatment, can induce pigmentation in irises of patients with hazel or heterochromatic eye colour. The mechanism by which LT induces pigmentation in the iris is not yet established, although it does not appear to induce proliferation of iridial melanocytes. The purpose of this study was to develop an in vitro model in which to investigate this mechanism. The pigmentary responses to LT and prostaglandin F(2alpha) (PGF(2alpha)) were examined in human iridial melanocytes alone or in co-culture with epithelial cells (non-ocular human epidermal keratinocytes and iris pigment epithelial cells) or mesenchymal cells (non-ocular dermal fibroblasts or iridial fibroblasts). Melanogenesis was assessed after 4 days culture with prostanoids, using dopa oxidase activity. Prostaglandin FP expression on human iridial fibroblasts and melanocytes was investigated using an immunofluorescent technique employing antibody to PGF(2alpha) receptor and RT-PCR. Iridial melanocytes did not show a convincing increase in dopa oxidase when cultured alone but in the presence of fibroblasts (ocular or non-ocular) there was a significant increase (25-30%) in dopa oxidase activity in response to 10(-7)-10(-5)m LT and PGF(2alpha). Co-culture of melanocytes with epithelial cells, while leading to increased dopa oxidase activity, did not lead to any melanogenic response to LT or PGF(2alpha). FP receptor expression was detected on fibroblasts but not iridial melanocytes by immunocytochemistry and RT-PCR. The melanocyte/fibroblast co-culture model developed in this study also showed that LT and PGF(2alpha) increased dopa oxidase activity in melanocytes from donors with brown but not blue eyes. These results suggest that LT may be inducing pigmentation in the human iris indirectly through the FP receptor on adjacent fibroblasts.

Prostaglandin ethanolamides (prostamides): in vitro pharmacology and metabolism.

We investigated whether prostaglandin ethanolamides (prostamides) E(2), F(2alpha), and D(2) exert some of their effects by 1) activating prostanoid receptors either per se or after conversion into the corresponding prostaglandins; 2) interacting with proteins for the inactivation of the endocannabinoid N-arachidonoylethanolamide (AEA), for example fatty acid amide hydrolase (FAAH), thereby enhancing AEA endogenous levels; or 3) activating the vanilloid receptor type-1 (TRPV1). Prostamides potently stimulated cat iris contraction with potency approaching that of the corresponding prostaglandins. However, prostamides D(2), E(2), and F(2alpha) exhibited no meaningful interaction with the cat recombinant FP receptor, nor with human recombinant DP, EP(1-4), FP, IP, and TP prostanoid receptors. Prostamide F(2alpha) was also very weak or inactive in a panel of bioassays specific for the various prostanoid receptors. None of the prostamides inhibited AEA enzymatic hydrolysis by FAAH in cell homogenates, or AEA cellular uptake in intact cells. Furthermore, less than 3% of the compounds were hydrolyzed to the corresponding prostaglandins when incubated for 4 h with homogenates of rat brain, lung, or liver, and cat iris or ciliary body. Very little temperature-dependent uptake of prostamides was observed after incubation with rat brain synaptosomes or RBL-2H3 cells. We suggest that prostamides' most prominent pharmacological actions are not due to transformation into prostaglandins, activation of prostanoid receptors, enhancement of AEA levels, or gating of TRPV1 receptors, but possibly to interaction with novel receptors that seem to be functional in the cat iris.

Pharmacological characterization of a novel antiglaucoma agent, Bimatoprost (AGN 192024).

Replacement of the carboxylic acid group of prostaglandin (PG) F(2alpha) with a nonacidic moiety, such as hydroxyl, methoxy, or amido, results in compounds with unique pharmacology. Bimatoprost (AGN 192024) is also a pharmacologically novel PGF(2alpha) analog, where the carboxylic acid is replaced by a neutral ethylamide substituent. Bimatoprost potently contracted the feline lung parenchymal preparation (EC(50) value of 35-55 nM) but exhibited no meaningful activity in a variety of PG-sensitive tissue and cell preparations. Its activity seemed unrelated to FP receptor stimulation according to the following evidence. 1) Bimatoprost exhibited no meaningful activity in tissues and cells containing functional FP receptors. 2) Bimatoprost activity in the cat lung parenchyma is not species-specific because its potent activity in this preparation could not be reproduced in cells stably expressing the feline FP receptor. 3) Radioligand binding studies using feline and human recombinant FP receptors exhibited minimal competition versus [(3)H]17-phenyl PGF(2a) for Bimatoprost. 4) Bimatoprost pretreatment did not attenuate PGF(2alpha)-induced Ca(2+) signals in Swiss 3T3 cells. 5) Regional differences were apparent for Bimatoprost but not FP agonist effects in the cat lung. Bimatoprost reduced intraocular pressure in ocular normotensive and hypertensive monkeys over a 0.001 to 0.1% dose range. A single-dose and multiple-dose ocular distribution/metabolism studies using [(3)H]Bimatoprost (0.1%) were performed. Within the globe, bimatoprost concentrations were 10- to 100-fold higher in anterior segment tissues compared with the aqueous humor. Bimatoprost was overwhelmingly the predominant molecular species identified at all time points in ocular tissues, indicating that the intact molecule reduces intraocular pressure.

Cellular and hormonal regulation of pigmentation in human ocular melanocytes.

The purpose of this study was to examine some of the factors that may be relevant to regulating pigmentation in the human eye, specifically whether choroidal and iridial melanocytes are sensitive to regulation by epithelial and stromal cells and alpha-melanocyte stimulating hormone (alpha-MSH). Human choroidal and iridial melanocytes were established in culture and co-cultured with epithelial cells and stromal cells derived both from skin and from eye in order to determine their influence on choroidal and iridial melanocyte dopa oxidase activity. In all cases, co-culture of melanocytes with either epithelial cells or fibroblasts led to an increase in dopa oxidase activity during 5 days of co-culture. The extent of the increase ranged from 60% (non-significant) to as much as 185% when both fibroblasts and keratinocytes were present. The optimal ratio of fibroblasts to melanocytes was 1:10 (for dermal fibroblasts) or 1:2 (for iridial fibroblasts) and 1:1 for all epithelial cells to melanocytes. Both choroidal (three out of three cultures) and iridial (two out of three cultures) melanocytes showed increases in dopa oxidase activity to alpha-MSH when cultured in Green's media but the same cells cultured in MCDB153 were unresponsive to alpha-MSH. These in vitro studies suggest that ocular melanocytes have the capacity to be influenced by adjacent epithelial and stromal cells with respect to pigmentation.

The pharmacology of bimatoprost (Lumigan).

Bimatoprost (Lumigan) is a pharmacologically unique and highly efficacious ocular hypotensive agent. It appears to mimic the activity of a newly discovered family of fatty acid amides, termed prostamides. One biosynthetic route to the prostamides involves anandamide as the precursor. Bimatoprost pharmacology has been extensively characterized by binding and functional studies at more than 100 drug targets, which comprise a diverse variety of receptors, ion channels, and transporters. Bimatoprost exhibited no meaningful activity at receptors known to include antiglaucoma drug targets as follows: adenosine (A(1-3)), adrenergic (alpha(1), alpha(2), beta(1), beta(2)), cannabinoid (CB(1), CB(2)), dopamine (D(1-5)), muscarinic (M(1-5)), prostanoid (DP, EP(1-4), FP, IP, TP), and serotonin (5HT(1-7)). Bimatoprost does, however, exhibit potent inherent pharmacological activity in the feline iris sphincter preparation, which is prostamide-sensitive. Bimatoprost also resembles the prostamides in that it is a potent and highly efficacious ocular hypotensive agent. A single dose of bimatoprost markedly reduces intraocular pressure in dogs and laser-induced ocular hypertensive monkeys. Decreases in intraocular pressure are well maintained for at least 24 hr post-dose. Human studies have demonstrated that systemic exposure to bimatoprost is low and that accumulation does not occur. The sclera is the preferred route of accession to the eye. The high scleral permeability coefficient Papp is a likely contributing factor to the rapid onset and long-acting ocular hypotensive profile of bimatoprost.

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.

Delayed reversal of shape change in cells expressing FP(B) prostanoid receptors. Possible role of receptor resensitization.

Prostaglandin F(2 alpha) (PGF(2 alpha)) receptors are G-protein-coupled receptors consisting of two alternative mRNA splice variants, named FP(A) and FP(B). As compared with the FP(A) isoform, the FP(B) isoform lacks the last 46 amino acids of the carboxyl terminus and, therefore, represents a truncated version of the FP(A). We recently found (Pierce, K. L., Fujino, H., Srinivasan, D., and Regan, J. W. (1999) J. Biol. Chem. 274, 35944-35949) that stimulation of both isoforms with PGF(2 alpha) leads to activation of a Rho signaling pathway, resulting in tyrosine phosphorylation of p125 focal adhesion kinase, formation of actin stress fibers, and cell rounding. Although the activation of Rho and subsequent cell rounding occur at a similar rate for both isoforms, we now report that following the removal of PGF(2 alpha) the reversal of cell rounding is much slower for cells expressing the FP(B) isoform as compared with the FP(A) isoform. Thus, in HEK-293 cells that stably express the FP(A) isoform, the reversal of cell rounding appears to be complete after 1 h, whereas for FP(B)-expressing cells there is essentially no reversal even after 2 h. Similarly, the disappearance of stress fibers and dephosphorylation of p125 focal adhesion kinase following removal of agonist are much slower in FP(B)-expressing cells than in FP(A)-expressing cells. The mechanism of this differential reversal appears to involve a difference in receptor resensitization following the removal of agonist. Based upon whole cell radioligand binding, agonist-induced stimulation of inositol phosphate formation, and mobilization of intracellular Ca(2+), the FP(B) isoform resensitizes more slowly than the FP(A) isoform. These findings suggest that the carboxyl terminus of the FP(A) is critical for resensitization and that the slower resensitization of the FP(B) isoform leads to prolonged signaling. This differential signaling distinguishes the FP(A) and FP(B) receptor isoforms and could be important toward understanding the physiological actions of PGF(2 alpha).

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.

Prostaglandin effects on the contractility of bovine trabecular meshwork and ciliary muscle.

The ocular hypotensive activity of prostaglandins (PGs) has previously been demonstrated in various species including man. The underlying mechanism of action of prostanoids other than PGF2 alpha remains contentious. Because the trabecular meshwork and ciliary muscle are believed to have a role in the regulation of aqueous humor outflow, the aim of this study was to identify the PG-receptor subtypes present in these tissues using receptor-selective agonists. Contractions of isolated strips of bovine trabecular meshwork and ciliary muscle were recorded isometrically in continuously perfused tissue chambers. Contractile activity of PGs was determined relative to a maximally effective concentration of carbachol (1 microM) as a standard agonist. The following prostanoids were employed: PGF2 alpha, 17-phenyl PGF2 alpha (FP-receptor agonists), sulprostone (EP3 > EP1-agonist), AH13205 (EP2-agonist), 11-deoxy PGE1 (non-selective EP-agonist), and U-46619 (TP-agonist). The thromboxane-mimetic U-46619 elicited a strong contraction of the trabecular meshwork with the highest concentration (1 microM) being almost twice as efficacious (186.6%) as the maximal carbachol concentration, whereas the effect on the ciliary muscle was small. The U-46619 induced trabecular meshwork contraction could be blocked with a potent and selective TP-receptor antagonist, 1 microM SQ29548, indicating the involvement of TP-receptors. The other PG-analogs studied had either no or a small but statistically significant effect. Thus, 17-phenyl PGF2 alpha (1 microM) weakly contracted the ciliary muscle (4.8%), sulprostone (1 microM) the trabecular meshwork (10.1%), 11-deoxy PGE1 (1 microM) and AH13205 (10 microM) elicited relaxations in both tissue precontracted with carbachol (1 microM). The relaxant effects were more pronounced in trabecular meshwork (15.6% for 11-deoxy PG1 and 21.4% for AH13205) than ciliary muscle (6.8 and 7.4% respectively). PGF2 alpha did not elicit a significant response in either tissue. Our studies suggest the existence of TP- and EP2-receptors in the bovine trabecular meshwork and potentially FP- and EP2-receptors in the ciliary muscle. In conclusion, thromboxane-mimetics and EP2-agonists have opposing activities on contractile elements in the meshwork and may modulate trabecular outflow in a functionally antagonistic manner. Prostanoid effects on ciliary muscle appear rather modest compared to parasympathomimetic drugs. It is conceivable that TP-agonists may substantially affect trabecular outflow.

Effects of leukotrienes B4 (LTB4) and D4 (LTD4) on motility of isolated normodense human eosinophils and neutrophils.

The effects of exogenous leukotrienes B4 and E4 (LTB4, LTD4) on the under-agarose motility of isolated normodense human eosinophils and neutrophils were examined using a novel sampling strategy for quantitation of leukocyte migration distance and vectorial orientation. Eosinophil chemotaxis to LTD4 was evident at a 10(-10)M threshold. The selective peptide-LT antagonist, SK&F 104353, abolished LTD4-induced eosinophil migration, indicating pharmacological specificity of the response. Neutrophil chemotaxis was apparent only with a very high (10(-5)M LTD4 concentration. LTB4 was a potent eosinophil and neutrophil chemoattractant over a 10(-9)M to 10(-4)M dose range. Analysis of leukocyte orientations provided evidence that chemokinetic responses were not being interpreted as indications of chemotactic behavior. LTB4 and LTD4 significantly altered neutrophil vectorial orientation. Comparison of migration distance and orientation at the leading edge and at the periphery of the well seeded with cells suggested that cell polarization appeared to be the earliest response to chemoattractive LTs. These results indicate that chemoattractant responses to LTs may be identified by utilizing the under-agarose technique and computer assisted analysis of cell orientation.

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.

Radioligand binding analysis of receptor subtypes in two FP receptor preparations that exhibit different functional rank orders of potency in response to prostaglandins.

The rat colon and Swiss 3T3 cells have been proposed as FP receptor preparations. However, the rank orders of potency for contraction of the rat colon and Ca++ signaling in Swiss 3T3 cells were found to be disparate. Although both appeared to be FP receptor preparations in that PGF2 alpha and FP receptor selective analogs were the most potent agonists, the potency ranking for other PGs and their analogs differed markedly. This presented two alternative major hypotheses for interpreting these data: (1) Swiss 3T3 cells and the rat colon possess different FP receptor subtypes and (2) the rat colon contains a heterogeneous population of prostanoid receptors. To further characterize prostanoid receptor populations in these two preparations, radioligand binding studies were performed with 3H-PGE2 and 3H-17-phenyl-PGF2 alpha. The rank order of potency for inhibition of 3H-PGE2 binding in the rat colon was consistent with EP3 receptor pharmacology. Thus, MB 28767, sulprostone and PGE2 were potent inhibitors, whereas PGF2 alpha, PGD2 and other analogs were substantially less potent. The rank order of potency for inhibition of 3H-17-phenyl-PGF2 alpha binding in the rat colon was consistent with the presence of an FP receptor. Thus, the potency rank order for the natural PGs was PGF2 alpha > PGD2 > PGE2 and among the synthetic analogs only PGF2 alpha analogs were potent competitors. In Swiss 3T3 cells an identical rank order of potency for eliciting a Ca++ transient signal and inhibition of 3H-17-phenyl-PGF2 alpha binding was obtained.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Comparison of leukotriene B4 and D4 effects on human eosinophil and neutrophil motility in vitro.

The motility of isolated normal human peripheral blood eosinophils and neutrophils in response to exogenous leukotrienes B4 and D4 was examined by means of a modified under-agarose technique and a novel quantitative sampling strategy. Leukotriene D4 was a potent chemoattractant for eosinophils, with a significant threshold chemotactic effect evident at 10(-10) M. The abolition of eosinophil chemotaxis by the potent and selective peptide-leukotriene-antagonist SK&F 104353 indicated the pharmacological specificity of the leukotriene D4-induced response. The chemokinetic response of eosinophils to leukotriene D4 generally did not differ significantly from spontaneous migratory activity of unstimulated cells. Leukotriene D4 did not, however, alter directed neutrophil motility until a very high concentration (10(-5) M) was achieved, although significant neutrophil chemokinesis relative to unstimulated movement was observed over the tested concentration range. Directional emigration of both eosinophils and neutrophils was induced by leukotriene B4 at concentrations as low as 10(-8) M. Analysis of leukocyte orientations provided evidence that chemokinetic responses were not being interpreted as indications of chemotactic behavior. These studies suggest that leukotriene D4 may behave as a potent and selective chemoattractant for human eosinophils at physiologically relevant concentrations.