Najam A. Sharif

Comparative effects of topical ocular anti-allergy drugs on human conjunctival mast cells.

BACKGROUNDnThe concept of mast cell heterogeneity is well established. Recent data indicate that human conjunctival tissue mast cells and human connective tissue mast cells respond to various secretagogues in similar fashion. It is now recognized that different mast cell populations respond differently to anti-allergic drugs.nnnOBJECTIVEnThe purpose of the study is to compare the effects of three new ocular anti-allergic drugs (nedocromil, olopatadine, and pemirolast) on mediator release from the target human conjunctival mast cell population with those of cromolyn sodium. The affinity of the compounds for the histamine H1 receptor was also compared.nnnMETHODSnA monodispersed suspension of partially purified human conjunctival mast cells was prepared from cadaver conjunctival tissue. Mast cells (5 x 10(3)) were challenged with anti-human IgE in the presence or absence of test drugs, and histamine content of the cell supernatants was determined using a specific radioimmunoassay. H1 receptor binding activity was assessed using a radioligand binding assay.nnnRESULTSnCromolyn and pemirolast (100 nM to 1 mM) failed to significantly inhibit histamine release from human conjunctival mast cells using exposure times of 1 and 15 minutes prior to challenge. Using identical nedocromil concentrations and exposure times, statistically significant (P < .05) inhibition (28%) of histamine release was observed at only the 100 microM concentration and 1-minute exposure time. In contrast, olopatadine inhibited histamine release in a concentration-dependent fashion (r = 0.891, n = 59, IC50 = 653 microM). Only olopatadine exhibited significant H1 receptor binding activity at relevant concentrations (Ki = 36 nM, n = 13).nnnCONCLUSIONSnThese data indicate that olopatadine possesses anti-allergic activity in the appropriate targets for topical ocular anti-allergic drug therapy, human conjunctival mast cells. Coupled with the compounds antihistaminic activity, this suggests that olopatadine will have efficacy advantages in allergic conjunctivitis patients over the other drugs tested.

[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.

Prostaglandin DP receptors positively coupled to adenylyl cyclase in embryonic bovine tracheal (EBTr) cells: pharmacological characterization using agonists and antagonists

Various prostaglandin agonists representing various classes of receptor subtypes were evaluated for their ability to stimulate adenylyl cyclase via the endogenous DP receptor in embryonic bovine tracheal (EBTr) cells. Two antagonists were used to block the agonist‐induced cyclic AMP production. ZK118182 (EC50=16±4u2003nM), RS‐93520 (EC50=23±4u2003nM), SQ27986 (EC50=33±9u2003nM), ZK110841 (EC50=33±5u2003nM), BW245C (EC50=59±19u2003nM) and PGD2 (EC50=101±10u2003nM) (n=4–70) were the most potent agonists. Whilst most compounds were full agonists (Emax=100% relative to PGD2), BW245C was significantly more efficacious than PGD2 (Emax=121±3%; P<0.001) and RS‐93520 appeared to be a partial agonist (Emax=64±9%; P<0.001). Agonists from the EP (e.g. enprostil; misoprostol; butaprost), FP (e.g. cloprostenol; fluprostenol; PHXA85), IP (iloprost; PGI2) and TP (U46619) prostanoid receptor classes were weak agonists or inactive in the EBTr cell assay system. The DP‐receptor antagonist, BWA868C, showed a competitive antagonist profile with pA2 values of 8.00±0.02 and 8.14±0.13 in Schild analyses with two structurally different agonists, BW245C and ZK118182, respectively (n=3). AH6809, another purported DP‐receptor antagonist, weakly inhibited PGD2‐ and ZK118182‐induced cyclic AMP production (Kis=808±193u2003nM and 782±178u2003nM, respectively). The current studies have characterized the DP receptor positively coupled to adenylyl cyclase in EBTr cells using a wide range of agonist and antagonist prostaglandins. These data support the utility of the EBTr cell line as a useful tool for the evaluation of DP receptor agonists and antagonists and for profiling other classes of prostaglandins.

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 60u2003min 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±0.064u2003min−1, mmol and 0.0042±0.0002u2003min−1, respectively, yielding dissociation constants (KDs) of 5.66±0.44u2003nM (n=4). Specific [3H]‐BWA868C bound to DP receptors with a high affinity (KD=1.45±0.01u2003nM, n=3) and to a finite, saturable number of binding sites (Bmax=21.1±0.6u2003nmol 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.

Use of a semi-automated, robotic radioimmunoassay to measure cAMP generated by activation of DP-, EP2-, and IP-prostaglandin receptors in human ocular and other cell types

The aim of these studies was to compare the effects of several prostaglandin agonists on adenylyl cyclase activity in embryonic bovine tracheal (EBTr) cells, transformed human nonpigmented ciliary epithelial (NPE) cells and National Cancer Bank (NCB-20) cells. These cell types have been shown to express DP, EP2 and IP prostaglandin (PG) receptors, respectively. Cyclic AMP (cAMP) generation was measured by manual and semi-automated radioimmunoassay (RIA) techniques. ZK118182 (EC50 = 10-27 nM), PGE2 (EC50 = 21-27 nM) and PGI2 (EC50 = 3.5-4 nM) had the highest potency at the DP, EP2 and IP receptors, respectively. A plot of potency (EC50) values generated with both techniques showed a high degree of correlation for all three receptors. These studies provide further characterization of prostanoid receptor functional responses in three cell types and demonstrate the advantages of a semi-automated RIA method for the analysis of the second messenger cAMP.

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).

A New Chromatographic Method for Measurement of Rubidium Transport Activities in Cultured Bovine Retinal Pigment Epithelial Cells

A new method for measuring cellular rubidium (Rb+) uptake activities based on cation chromatography was developed and compared with the standard technique, uptake of the radioisotope 86Rb+, using cultured bovine retinal pigment epithelial (RPE) cells. The Rb+ response was strictly linear from 0.25 nmol (detection limit) to 25 nmol. The Na+/K(+)-ATPase inhibitor ouabain inhibited Rb+ uptake with IC50 values of 128.7 +/- 23.5 nM (n = 8; radioactive method) and 56.6 +/- 9.3 nM (n = 9; non-radioactive method, p < 0.01). The latter value is identical to the IC50 value of 54.4 +/- 16.2 nM (n = 3) for ouabain binding to the intact RPE cells. Ouabain and bumetanide, an inhibitor of the Na+/K+/Cl(-)-cotransporter, each inhibited Rb+ uptake maximally by 66 and 30%, respectively. This new technique allows sensitive measurement of intracellular Rb+, as well as K+ and Na+, and, thus, should prove useful for studying the effects of pharmacologic agents and simulated disease conditions on cation transport and cation balance in RPE and other cell types.