Achim H.-P. Krauss

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.

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.

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

Prostaglandin F2α(PGF2α) receptors are G-protein-coupled receptors consisting of two alternative mRNA splice variants, named FPA and FPB. As compared with the FPA isoform, the FPB isoform lacks the last 46 amino acids of the carboxyl terminus and, therefore, represents a truncated version of the FPA. 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 PGF2α 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 PGF2α the reversal of cell rounding is much slower for cells expressing the FPB isoform as compared with the FPA isoform. Thus, in HEK-293 cells that stably express the FPA isoform, the reversal of cell rounding appears to be complete after 1 h, whereas for FPB-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 FPB-expressing cells than in FPA-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 Ca2+, the FPBisoform resensitizes more slowly than the FPA isoform. These findings suggest that the carboxyl terminus of the FPA is critical for resensitization and that the slower resensitization of the FPB isoform leads to prolonged signaling. This differential signaling distinguishes the FPA and FPB receptor isoforms and could be important toward understanding the physiological actions of PGF2α.

Evidence for human thromboxane receptor heterogeneity using a novel series of 9,11-cyclic carbonate derivatives of prostaglandin F2α

1 The pharmacological activity of a novel series of 9,11‐cyclic carbonate derivatives of prostaglandin F2α (PGF2α) 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 plateletrich 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 μm). 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 μm) was similar to that obtained in PRP. 4 The involvement of TP‐receptors was verified with the potent TP‐antagonist, SQ29548. SQ29548 (0.1 μm in myometrium; 1 μm in aorta; 1 μm and 10 μm 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 PGF2α, 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.

Prostanoid EP4 Receptor Stimulation Produces Ocular Hypotension by a Mechanism That Does Not Appear to Involve Uveoscleral Outflow

PURPOSE As part of a systematic elucidation of the pharmacology of prostaglandins (PG) effects on intraocular pressure in the monkey, the prototypical selective prostanoid EP(4) receptor agonist (3,7-dithia PGE(1)) was examined. It was found to be highly efficacious in nonhuman primates, and its mechanism of ocular hypotensive activity was investigated. METHODS Intraocular pressure (IOP) was measured by pneumatonometry in conscious monkeys restrained in custom-designed chairs. All other animal experiments were performed in animals sedated with ketamine or anesthetized with ketamine/diazepam and given drug or vehicle for various lengths of time. Aqueous flow was determined by fluorophotometry. Total outflow facility was measured by the two-level, constant-pressure method and by 2-minute tonography in both normotensive and hypertensive monkey eyes. Uveoscleral outflow was measured by perfusing the anterior chamber with FITC-labeled dextran for 30 minutes at a fixed IOP of approximately 15 mm Hg. Isometric responses to drugs were measured in longitudinal and radial preparations of monkey and human isolated ciliary smooth muscle specimens. RESULTS The selective EP(4) receptor agonist 3,7-dithia PGE(1) and an isopropyl ester prodrug thereof reduced IOP in monkeys. A single dose of 3,7-dithia PGE(1) isopropyl ester, at a 0.01% or 0.1% dose, decreased IOP in the glaucomatous monkey in the range of 40% to 50%. Studies on total outflow facility by the two-level, constant-pressure perfusion method and tonography indicated that EP(4) receptor stimulation facilitated aqueous humor outflow facility. No effect on aqueous flow was apparent. In contrast to all PGs and prostamides studied to date, 3,7-dithia PGE(1) exerted no effect on uveoscleral outflow measured directly. Moreover, it did not relax longitudinal or radial preparations of isolated human or monkey ciliary muscles. CONCLUSIONS The EP(4) receptor agonist 3,7-dithia PGE(1) is a highly efficacious IOP-lowering drug in monkeys. It has no effect on uveoscleral outflow but does increase total outflow facility, which accounts for a substantial proportion of the ocular hypotensive activity.

Correlation of In Vitro and In Vivo Kinetics of Nitric Oxide Donors in Ocular Tissues

In the eye, nitric oxide (NO) is involved in the regulation of intraocular pressure (IOP) and ocular blood flow. The main purpose of this study was to measure the kinetics of NO release from NO donors in ocular cells and tissues using in vivo and in vitro models and demonstrate the link between the kinetics of NO release with the functional effect, IOP. Nitric oxide release was measured in human ocular cells using a fluorescent dye, diaminofluorescein (DAF), following treatment with short-acting sodium nitroprusside (SNP) and longer-acting S-nitroso-N-acetylpenicillamine (SNAP) NO donors. Both SNP and SNAP were also administered topically to rabbits; IOP was measured and levels of NO and cGMP were assessed as biomarkers over a time course in the aqueous humor (AH) and iris/ciliary body (ICB). Time- and concentration-dependent increases in NO level were produced by SNP and SNAP in human ocular cells. Both NO and cGMP levels appeared to be elevated following treatment with the aforementioned NO donors in rabbit ocular tissues. Transient IOP lowering was accompanied with these biochemical estimations in rabbits, with time of maximal effect being shifted to the right for longer-acting SNAP as compared with short-acting SNP. In vitro and in vivo NO/cGMP assay results displayed a correlation between short- and longer-acting NO donors, discriminating their respective temporal actions in the eye. Due to their translatability, the in vitro DAF assay and in vivo NO fluorometric assay can therefore be potentially useful in screening novel NO donors with different temporal/kinetic profiles.

Bimatoprost effects on aqueous humor dynamics in monkeys

The effects of bimatoprost on aqueous humor dynamics were quantified in monkey eyes. Uveoscleral outflow was measured by the anterior chamber perfusion method, using FITC-dextran. Total outflow facility was determined by the two-level constant pressure method. Aqueous flow was measured with a scanning ocular fluorophotometer. Uveoscleral outflow was 0.96 ± 0.19 μL min−1 in vehicle-treated eyes and 1.37 ± 0.27 μL min−1 (n = 6; P < .05) in eyes that received bimatoprost 0.01% b.i.d. × 5 days. Bimatoprost had no effect on total outflow facility, which was 0.42 ± 0.05 μL min−1 at baseline and 0.42 ± 0.04 μL min−1 after bimatoprost treatment. Bimatoprost had no significant effect on aqueous humor flow. This study demonstrates that bimatoprost increases uveoscleral outflow but not total outflow facility or aqueous humor flow, indicating that it lowers intraocular pressure in ocular normotensive monkeys by a mechanism that exclusively involves uveoscleral outflow.

Prostaglandin ep4 antagonists

Prostaglandin antagonists, with their pharmacological effects, are well-known drugs capable of treating widely diffused illnesses, including pain and inflammation disorders. In recent years, a major research focus has been devoted to the identification of agents able to selectively antagonize each receptor with which prostaglandins interact. This review attempts to give a broad overview of molecules capable of selectively blocking the prostaglandin PGE2 EP4 receptor. Further therapeutic applications and uses have also been disccussed, including the first drug candidate to have reached clinical trials within the last few years.

In Vivo Evaluation of 11β-Hydroxysteroid Dehydrogenase Activity in the Rabbit Eye

PURPOSE Steroids are used in a diverse range of conditions in clinical ophthalmology and one of the most significant complications is corticosteroid-induced glaucoma, which is characterized by an increase in intraocular pressure (IOP). 11beta-Hydroxysteroid dehydrogenase-1 (11beta-HSD1) is known to catalyze the interconversion of hormonally inactive cortisone to hormonally active cortisol and is widely expressed in the eye, particularly ciliary epithelium. Carbenoxolone (CBX), an 11beta-HSD1 inhibitor, has been shown to reduce IOP in healthy volunteers and patients with ocular hypertension (OHT). The purpose of this study was to: (1) develop an in vivo model for the assessment of cortisone to cortisol conversion in the eye, that is, 11beta-HSD1 activity and (2) assess the pharmacokinetic/pharmacodynamic relationship following topical treatment with 11beta-HSD1 inhibitors using an in vivo rabbit model. METHODS Potent and selective 11beta-HSD1 inhibitors were topically administered to the rabbit eye and exogenous cortisone to endogenous cortisol conversion in the eye was assessed in rabbits. Tissues were then evaluated for cortisone, cortisol, and 11beta-HSD1 inhibitor levels by LC/MS/MS. Concomitantly cortisol activity in ocular tissue samples was determined using a secondary mechanistic pLuc-GRE assay. RESULTS Topical treatment with potent and selective 11beta-HSD1 inhibitors resulted in complete inhibition in the conversion of cortisone to cortisol in the rabbit eye as well as decreased pLuc-GRE luciferase activity. The reduction of cortisone conversion was time- and dose-dependent as well as dependent on dosing volume (suggestive of increased spillover and washout with greater dosing volume). CONCLUSIONS In conclusion, topical delivery of 11beta-HSD1 inhibitors can reduce or inhibit the conversion of cortisone to cortisol in the eye, indicating that the rabbit eye possesses an active enzyme for glucocorticoid synthesis. Dosing concentration and volume play an important role in the pharmacokinetic and pharmacodynamic effects of topically delivering an 11beta-HSD1 inhibitor. The rabbit model is useful for mechanistically assessing the conversion of cortisone to cortisol in the eye.

A Comparative Study of Thromboxane (TP) Receptor Mimetics and Antagonists on Isolated Human Umbilical Artery and Myometrium

Pickles (1967) proposed that up to four different prostanoid types existed. Coleman et al. (1984) developed a working classification for the prostanoid receptors and since then there has been some debate concerning heterogeneity of each receptor. Following on from these studies, extensive work has led to systematic prostanoid receptor classification. The present prostanoid receptor classification is a simple systematic working hypothesis whereby each of the natural prostanoids has its own receptor termed a P receptor where it is at least ten times more potent than any of the other natural prostanoids. Thus the prostaglandin E2 (PGE2)-sensitive receptors are termed the EP-receptors, PGF2α FP-receptors, PGD2, DP-receptors, PGI2, IP receptors and the thromboxane A2 (TXA2)-sensitive receptors, TP-receptors (Kennedy et al., 1982). The thromboxane receptor (TP) has been shown to mediate constriction of airway and vascular smooth muscle as well as platelet aggregation but discussion has failed to resolve the number of receptors involved.