David F. Woodward

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.

Analgesic actions of N‐arachidonoyl‐serotonin, a fatty acid amide hydrolase inhibitor with antagonistic activity at vanilloid TRPV1 receptors

N‐arachidonoyl‐serotonin (AA‐5‐HT) is an inhibitor of fatty acid amide hydrolase (FAAH)‐catalysed hydrolysis of the endocannabinoid/ endovanilloid compound, anandamide (AEA). We investigated if AA‐5‐HT antagonizes the transient receptor potential vanilloid‐1 (TRPV1) channel and, as FAAH and TRPV1 are targets for analgesic compounds, if it exerts analgesia in rodent models of hyperalgesia.

Cloning of a Carboxyl-terminal Isoform of the Prostanoid FP Receptor

An FP prostanoid receptor isoform, which appears to arise from alternative mRNA splicing, has been cloned from a mid-cycle ovine large cell corpus luteum library. The isoform, named the FPB receptor, is identical to the original isoform, the FPA, throughout the seven transmembrane domains, but diverges nine amino acids into the carboxyl terminus. In contrast to FPA, whose carboxyl terminus continues for another 46 amino acids beyond the nine shared residues, the FPB terminates after only one amino acid. The FPA isoform appears to arise by the failure to utilize a potential splice site, while a 3.2-kilobase pair intron is spliced out from the FP gene to generate the FPB isoform mRNA. The two isoforms have indistinguishable radioligand binding properties, but seem to differ in functional coupling to phosphatidylinositol hydrolysis. Thus, in COS-7 cells transiently transfected with either the FPA or the FPB receptor cDNAs, prostaglandin F2α stimulates inositol phosphate accumulation to the same absolute maximum, but the basal level of inositol phosphate accumulation is approximately 1.3-fold higher in cells transfected with the FPB as compared with cells transfected with the FPA isoform. Using the polymerase chain reaction, mRNA encoding the FPB isoform was identified in the ovine corpus luteum.

6-isopropoxy-9-oxoxanthene-2-carboxylic acid (AH 6809), a human EP2 receptor antagonist

On studying the interaction of various ligands with the pharmacologically defined, recombinant human EP2 receptor (Regan et al., Mol Pharmacol 46: 213-220, 1994), we discovered that the putative EP1 receptor antagonist 6-isopropoxy-9-oxoxanthene-2-carboxylic acid (AH 6809) also has affinity for the human EP2 receptor. Moreover, AH 6809 behaved as an EP2 receptor antagonist and inhibited prostaglandin E2 (PGE2)-stimulated increases in cyclic AMP. These findings have significant implications for studies that employ AH 6809 to determine the pharmacological basis of PGE2-induced responses in human cells and tissues.

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 F2α has received the greatest research attention. Prostamide F2α 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 F2α. 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 F2α and bimatoprost but not those of PGF2α 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 F2α analogue bimatoprost is the most efficacious ocular hypotensive agent currently available for the treatment of 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.

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

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.

Ultrastructural changes associated with dexamethasone-induced ocular hypertension in mice.

PURPOSE To determine whether dexamethasone (DEX)-induced ocular hypertension (OHT) in mice mimics the hallmarks of steroid-induced glaucoma (SIG) in humans, including reduced conventional outflow facility (C), increased extracellular matrix (ECM), and myofibroblasts within the outflow pathway. METHODS Osmotic mini-pumps were implanted subcutaneously into C57BL/6J mice for systemic delivery of DEX (3-4 mg/kg/d, n = 31 mice) or vehicle (n = 28). IOP was measured weekly by rebound tonometry. After 3 to 4 weeks, mice were euthanized and eyes enucleated for ex vivo perfusion to measure C, for electron microscopy to examine the trabecular meshwork (TM) and Schlemms canal (SC), or for immunohistochemistry to examine type IV collagen and α-smooth muscle actin. The length of basement membrane material (BMM) was measured along the anterior-posterior extent of SC by electron microscopy. Ultrastructural changes in BMM of DEX-treated mice were compared against archived human SIG specimens. RESULTS Dexamethasone increased IOP by 2.6 ± 1.6 mm Hg (mean ± SD) over 3 to 4 weeks and decreased C by 52% ± 17% versus controls. Intraocular pressure elevation correlated with decreased C. Dexamethasone treatment led to increased fibrillar material in the TM, plaque-like sheath material surrounding elastic fibers, and myofibroblasts along SC outer wall. The length of BMM underlying SC was significantly increased in mice with DEX and in humans with SIG, and in mice decreased C correlated with increased BMM. CONCLUSIONS Dexamethasone-induced OHT in mice mimics hallmarks of human SIG within 4 weeks of DEX treatment. The correlation between reduced C and newly formed ECM motivates further study using DEX-treated mice to investigate the pathogenesis of conventional outflow obstruction in glaucoma.

Replacement of the carboxylic acid group of prostaglandin F2α with a hydroxyl or methoxy substituent provides biologically unique compounds

Replacement of the carboxylic acid group of PGF2α with the non‐acidic substituents hydroxyl (‐OH) or methoxy (‐OCH3) resulted in an unexpected activity profile. Although PGF2α 1‐OH and PGF2α 1‐OCH3 exhibited potent contractile effects similar to 17‐phenyl PGF2α in the cat lung parenchymal preparation, they were approximately 1000 times less potent than 17‐phenyl PGF2α in stimulating recombinant feline and human FP receptors. In human dermal fibroblasts and Swiss 3T3 cells PGF2α 1‐OH and PGF2α 1‐OCH3 produced no Ca2+ signal until a 1 μM concentration was exceeded. Pretreatment of Swiss 3T3 cells with either 1 μM PGF2α 1‐OH or PGF2α 1‐OCH3 did not attenuate Ca2+ signal responses produced by PGF2α or fluprostenol. In the rat uterus, PGF2α 1‐OH was about two orders of magnitude less potent than 17‐phenyl PGF2α whereas PGF2α 1‐OCH3 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 PGF2α1‐OH, PGF2α1‐OCH3, and classical FP receptor agonists. Studies on smooth muscle preparations and cells containing DP, EP1, EP2, EP3, EP4, IP, and TP receptors indicated that the activity of PGF2α 1‐OH and PGF2α 1‐OCH3 could not be ascribed to interaction with these receptors. The potent effects of PGF2α 1‐OH and PGF2α 1‐OCH3 on the cat lung parenchyma are difficult to describe in terms of interaction with the FP or any other known prostanoid receptor.

Pharmacologic Manipulation of Conventional Outflow Facility in Ex Vivo Mouse Eyes

PURPOSE Mouse models are useful for glaucoma research, but it is unclear whether intraocular pressure (IOP) regulation in mice operates through mechanisms similar to those in humans. Our goal was to determine whether pharmacologic compounds that affect conventional outflow facility in human eyes exert similar effects in C57BL/6 mice. METHODS A computerized perfusion system was used to measure conventional outflow facility in enucleated mouse eyes ex vivo. Paired eyes were perfused sequentially, either immediately after enucleation or after 3 hours storage at 4°C. Three groups of experiments examined sphingosine 1-phosphate (S1P), S1P with antagonists to S1P(1) and S1P(2) receptors, and the prostanoid EP(4) receptor agonist 3,7-dithia PGE(1). We also examined whether a 24-hour postmortem delay affected the response to 3,7-dithia prostaglandin E(1) (PGE(1)). RESULTS S1P decreased facility by 39%, and was blocked almost completely by an S1P(2), but not S1P(1), receptor antagonist. The S1P(2) receptor antagonist alone increased facility nearly 2-fold. 3,7-dithia PGE(1) increased facility by 106% within 3 hours postmortem. By 24 hours postmortem, the facility increase caused by 3,7-dithia PGE(1) was reduced 3-fold, yet remained statistically detectable. CONCLUSIONS C57BL/6 mice showed opposing effects of S1P(2) and EP(4) receptor activation on conventional outflow facility, as observed in human eyes. Pharmacologic effects on facility were detectable up to 24 hours postmortem in enucleated mouse eyes. Mice are suitable models to examine the pharmacology of S1P and EP(4) receptor stimulation on IOP regulation as occurs within the conventional outflow pathway of human eyes, and are promising for studying other aspects of aqueous outflow dynamics.