Terry L. Davis

[3H]AL-5848 ([3H]9β-(+)-Fluprostenol). Carboxylic Acid of Travoprost (AL-6221), a Novel FP Prostaglandin o study the Pharmacology and Autoradiographic Localization of the FP Receptor

AL‐5848 (5Z,13E)‐(9 S,11R,15S)‐9,11,15‐trihydroxy‐5,13‐prostadienoic acid) is the carboxylic acid of travoprost (AL‐6221), a single (+)‐isomer of (±)‐fluprostenol, an FP‐class prostaglandin agonist which lowers intraocular pressure. We have prepared a radioligand from this selective prostaglandin and demonstrated its utility for studying the pharmacology and autoradiographic location of the FP‐receptor. Specific [3H]AL‐5848 binding (84% of total) was linearly related to bovine corpus luteum tissue concentration and reached equilibrium within 275 min at 23°C. Scatchard analysis of saturation isotherms indicated interaction of [3H]AL‐5848 with a single class of high‐affinity (dissociation constant, Kd, = 33.8 ± 2.9 nM, n = 4) and saturable (Bmax = 37.3 ± 3.0 pmol (g wet weight tissue)−1) FP receptor‐binding sites in bovine corpus luteum. Specific [3H]AL‐5848 binding was potently inhibited by the FP‐receptor ligands 16‐phenoxyPGF2α (inhibition constant Ki = 17.3 nM); cloprostenol (Ki = 56.8 nM); 17‐phenyl PGF2α (Ki = 87.0 nM); AL‐5848 (Ki = 52.1 nM); PGF2α (Ki = 195 nM); PHXA85 (Ki = 223 nM); (n = 3–11) but very weakly by PGD2, ZK118182, BW245C, PGE2, PGI2 and U‐46619. The pharmacology of specific [3H]AL‐5848 binding correlated well with the pharmacology of [3H]PGF2α binding in the bovine corpus luteum preparation (r = 0–98, n = 14, P < 0.0001) and also with functional responses in Swiss 3T3 and rat vascular smooth muscle cells (A7r5) (r = 0.96) expressing FP receptors. Autoradiographic studies revealed high levels of specific FP‐receptor binding with [3H]AL‐5848 on granulosa cells in the bovine corpus luteum sections, and on longitudinal ciliary muscle, the ciliary process, the iris sphincter and the retina in eye sections from man.

Levobetaxolol (Betaxon™) and Other β-Adrenergic Antagonists: Preclinical Pharmacology, IOP-Lowering Activity and Sites of Action in Human Eyes

The pharmacological characteristics of levobetaxolol, a single active isomer of betaxolol, were determined and compared with activities of other beta-adrenoceptor antagonists. Levobetaxolol (43-fold beta1-selective) exhibited a higher affinity at cloned human beta1 (Ki = 0.76 nM) than at beta2 (Ki = 32.6 nM) receptors, while dextrobetaxolol was much weaker at both receptors. Levobetaxolol potently antagonized functional activities at cloned human beta1 and beta2 receptors, and also at guinea pig atrial beta1, tracheal beta2 and rat colonic beta3 receptors (IC50s = 33.2 nM, 2970 nM and 709 nM, respectively). Thus, levobetaxolol was 89-times beta1-selective (vs beta2). Levobetaxolol (Ki = 16.4 nM) was more potent than dextrobetaxolol (Ki = 2.97 microM) at inhibiting isoproterenol-induced cAMP production in human non-pigmented ciliary epithelial cells. Levobunolol and (l)-timolol had high affinities at beta1 and beta2 receptors but were considerably less beta1-selective than levobetaxolol. Levo-, dextro- and racemic-betaxolol exhibited little or no affinity, except at sigma sites and Ca2+-channels (IC50s > 1 microM), at 89 other receptor/ligand binding sites. Levobetaxolol exhibited a micromolar affinity for L-type Ca2+-channels. In conscious ocular hypertensive cynomolgus monkeys, levobetaxolol was more potent than dextrobetaxolol, reducing intraocular pressure by 25.9+/-3.2% at a dose of 150 microg/eye (n = 15-30). Quantitative [3H]-levobetaxolol autoradiography revealed high levels of binding to human ciliary processes, iris, choroid/retina, and ciliary muscles. In conclusion, levobetaxolol is a potent, high affinity and beta1-selective IOP-lowering beta-adrenoceptor antagonist.

Effects of bradykinin on signal transduction, cell proliferation, and cytokine, prostaglandin E2 and collagenase-1 release from human corneal epithelial cells

1 We recently demonstrated the presence of phospholipase C‐coupled bradykinin (BK) B2‐receptors in human primary and SV40 virus‐immortalized corneal epithelial (CEPI) cells. 2 The aims of the present studies were to demonstrate the specific binding of [3H]‐BK to CEPI cell membranes and to study its pharmacological characteristics. In addition, we wished to study the functional coupling of the BK receptors to various physiological and pathological mechanisms in the CEPI cells, including phosphoinositide (PI) turnover, intracellular Ca2+‐mobilization ([Ca2+]i), cell proliferation (via [3H]‐thymidine incorporation), and the release of various cytokines, collagenase‐1 (matrix metalloproteinase‐1) and prostaglandin E2 (PGE2). 3 Specific [3H]‐BK binding comprised 83±2% of the total binding, and was of high affinity (Kd=1.66±0.52 nM, n=5), saturable (Bmax=640±154 fmol g−1 wet weight) and reversible. Competition studies yielded the following affinity values for BK and a number of BK‐related peptides: Hoe‐140 (D‐Arg‐[Hyp3,Thi5,D‐Tic7,Oic8]BK; icatibant): Ki=0.17±0.07 nM; BK: Ki=1.0±0.11 nM; [Tyr8]‐BK: Ki=12.9±2.3 nM; [des‐Arg9]‐BK: Ki>9,200 nM (all n=3–5)). 4 BK potently stimulated PI turnover (EC50=2.3±0.3 nM; n=7) and [Ca2+]i mobilization (EC50=8–20 nM) in CEPI cells and both responses were inhibited in a concentration‐dependent manner by 100 nM–10 μM Hoe‐140, a selective B2‐receptor antagonist, and also inhibited by the selective phospholipase C (PLC) inhibitor, U73122 (1‐(6‐((17β‐3‐methoxyestra‐1,3,5(10)‐trien‐17‐yl)amino)hexyl)‐1H‐pyrrole‐2,5‐dione) (IC50=3.0±1.6 μM). BK‐induced [Ca2+]i mobilization was reduced by about 30% in the presence of 4 mM EGTA, but was not significantly affected by 100 nM nifedipine. 5 BK (0.1 nM–10 μM) significantly (P<0.05–0.001) stimulated [3H]‐thymidine incorporation into CEPI cellular DNA. However, while interleukin‐1α (IL‐1α; 10 ng ml−1) potently stimulated the release of IL‐6, IL‐8 and granulocyte macrophage colony‐stimulating factor from CEPI cells, BK (0.1 nM–10 μM) was without effect. 6 Whilst phorbol‐12‐myristate‐13‐acetate (PMA; 3 μg ml−1) and 10% foetal bovine serum (positive control agents) significantly stimulated the release of both MMP‐1 and PGE2 from CEPI cells, BK (0.1 nM–10 μM) was without any significant effect under these conditions. 7 In conclusion, these data indicate that the CEPI cells express high‐affinity [3H]‐BK binding sites representing B2‐subtype BK receptors coupled to PI turnover and [Ca2+]i mobilization which appear to stimulate [3H]‐thymidine incorporation into cellular DNA. In contrast, BK failed to elicit the release of PGE2, various cytokines and MMP‐1 from CEPI cells. These results suggest that BK may have a potential role in corneal epithelium wound healing by stimulating cell proliferation.

Pharmacology and autoradiography of human DP prostanoid receptors using [3H]-BWA868C, a DP receptor-selective antagonist radioligand

A potent and highly selective DP prostanoid receptor antagonist radioligand, [3H]‐cyclohexyl‐N‐BWA868C (3‐benzyl‐5‐(6‐carboxyhexyl)‐1‐(2‐cyclohexyl‐2‐hydroxyethyl‐amino) hydantoin, ([3H]‐BWA868C)), has been generated for receptor binding and autoradiographic studies. Specific [3H]‐BWA868C binding to human platelet membranes achieved equilibrium within 60 min at 23°C and constituted up to 95% of the total binding. The association (K+1) and dissociation (K−1) rate constants of binding were 0.758±0.064 min−1, mmol and 0.0042±0.0002 min−1, respectively, yielding dissociation constants (KDs) of 5.66±0.44 nM (n=4). Specific [3H]‐BWA868C bound to DP receptors with a high affinity (KD=1.45±0.01 nM, n=3) and to a finite, saturable number of binding sites (Bmax=21.1±0.6 nmol g−1 wet weight). DP receptor class prostanoids (e.g. ZK118182, BW245C, BWA868C, PGD2) exhibited high (nanomolar) affinities for [3H]‐BWA868C binding, while prostanoids selective for EP, FP, IP and TP receptors showed a low (micromolar) affinity. Specific DP receptor binding sites were autoradiographically localized on the ciliary epithelium/process, longitudinal and circular ciliary muscles, retinal choroid and iris in human eye sections using [3H]‐BWA868C. While [3H]‐PGD2 yielded similar quantitative distribution of DP receptors as [3H]‐BWA868C, the level of non‐specific binding observed with [3H]‐PGD2 was significantly greater than that observed with [3H]‐BWA868C. It is concluded that [3H]‐BWA868C is a high‐affinity and very specific DP receptor radioligand capable of selectively labelling the DP receptor. [3H]‐BWA868C may prove useful for future homogenate‐based and autoradiographic studies on the DP receptor.

Pharmacology of [3H]-pyrilamine binding and of the histamine-induced inositol phosphates generation, intracellular Ca2+-mobilization and cytokine release from human corneal epithelial cells

1 We recently reported on the successful generation of immortalized (CEPI‐17‐CL4) cells from primary human corneal epithelial (P‐CEPI) cells which exhibited phenotypic, immunohistochemical and metabolic characteristics akin to the P‐CEPI cells. 2 The aims of the present studies were to investigate the ligand binding and functional coupling of the histamine receptors to various biochemical and physiological systems in the P‐CEPI and CEPI‐17‐CL4 cells and to relate these findings to the normal and/or pathophysiological role of histamine on the human ocular surface. 3 Specific [3H]‐pyrilamine binding to CEPI‐17‐CL4 cell homogenates comprised >93% of the total binding and represented interaction with an apparent single population of high affinity (Kd=3.76±0.78 nm; n=4) and saturable (Bmax=1582±161 fmol g−1 tissue) number of histamine‐1 (H1) receptor binding sites on CEPI‐17‐CL4 cell homogenates. The H1‐receptor selective antagonists, pyrilamine (Ki=3.6±0.84 nm, n = 4) and triprolidine (Ki=7.7±2.6 nm, n = 3), potently displaced [3H]‐pyrilamine binding, while the H2‐ and H3‐receptor selective antagonists, ranitidine and clobenpropit, were weak inhibitors (Kis>13 μm). 4 Histamine induced phosphoinositide (PI) hydrolysis 2.7–4.4 fold above basal levels and with a potency of 14.9±4.9 μm (n = 9) and 4.7±0.2 μm (n = 9) in P‐CEPI and CEPI‐17‐CL4 cells, respectively. Histamine‐induced PI turnover was antagonized by H1‐receptor selective antagonist, triprolidine, with a potency (Ki) of 3.2±0.66 nm (n = 10) and 3.03±0.8 nm (n = 4) in P‐CEPI and CEPI‐17‐CL4 cells, respectively, but weakly effected by 10 μm cimetidine and clobenpropit, H2‐ and H3‐receptor antagonists. The PI turnover response was attenuated by pre‐treatment of the cells with the selective phospholipase C inhibitor, U73122 (1‐(6‐((17β‐3‐methoxyestra‐1,3,5(10)‐trien‐17‐yl)amino)hexyl)‐1H‐pyrrole‐2,5‐dione) (IC50=4.8±2.4 μm, n = 3). 5 Histamine stimulated intracellular Ca2+ ([Ca2+]i) mobilization in CEPI‐17‐CL4 cells with a potency of 6.3±1.5 μm (n = 4). The histamine‐induced [Ca2+]i mobilization was reduced by about 28% following pre‐incubation of the cells with 4 mm EGTA. While triprolidine completely inhibited histamine‐induced [Ca2+]i mobilization, it did not influence the bradykinin‐induced [Ca2+]i mobilization response. 6 Histamine (EC50s=1.28–2.77 μm, n = 3–4) concentration‐dependently stimulated the release of interleukin‐6 (IL‐6), IL‐8 and granulocyte macrophage colony‐stimulating factor, but it did not significantly alter release of tumour necrosis factor‐α, PGE2 or collagenase‐1 (matrix metalloproteinase‐1; MMP‐1) from CEPI cells. However, IL‐1 (10 ng ml−1), foetal bovine serum (10%) and phorbol‐12‐myristate‐13‐acetate (3 μg ml−1) were effective positive control secretagogues of all the cytokines, PGE2 and MMP‐1, respectively, from these cells. 7 It is concluded that the CEPI cells express H1‐histamine receptors which are positively coupled to PI turnover and [Ca2+]i mobilization which may be directly or indirectly responsible for the release of various cytokines from these cells at physiologically and/or pathologically relevant concentrations.

Cloned human EP1 prostanoid receptor pharmacology characterized using radioligand binding techniques.

Prostaglandins such as prostaglandin E2 (PGE2) interact with EP‐class prostanoid receptors including EP1, EP2, EP3 and EP4 subtypes. We have conducted a detailed pharmacological characterization of the binding of [3H]‐PGE2 to recombinant human EP1 prostanoid receptors expressed in human embryonic kidney (HEk‐293) cells using a broad panel of natural and synthetic prostanoids. The receptor displayed high affinity (kd = 16.0 ± 0.69 nM; n = 3) for [3H]‐PGE2, and was expressed at high levels (Bmax = 3.69 ± 0.30 pmol (mg protein)−1 in cell membranes of HEk‐293 cells. Specific binding constituted 97.5 ± 1.4% (n = 12) of the total binding. In competition assays, the rank order of affinities of natural prostanoids for the receptor was PGE2 > PGE1 > PGF2 > PGI2 > PGD2. PGE2 was more effective than PGE1 at displacing bound [3H]‐PGE2 (ki for PGE2 = 14.9 ± 2.2 nM; ki for PGE1 = 165 ± 29 nM). The affinities of enprostil (ki = 14.5 ± 3.1 nM) and 17‐phenyl‐ω‐trinor‐PGE2 (ki = 7.3 ± 2.7 nM) for the receptor were quite similar to that of PGE2, while that of sulprostone (ki = 137 ± 13 nM) more closely resembled PGE1. Some compounds historically classified as specific for DP prostanoid receptors bound with relatively high affinity to the recombinant human EP1 receptor (e.g. Zk118182 (ki = 73.4 ± 8.6 nM) and Zk110841 (ki = 166 ± 20 nM)). All FP (e.g. travoprost acid, fluprostenol), IP (iloprost) and TP (SQ29548) receptor‐specific ligands exhibited low affinity (ki ≥ 1μM).