Louis Desantis

Preliminary characterization of a transformed cell strain derived from human trabecular meshwork

Cells isolated from the trabecular meshwork (TM) of a male glaucoma patient were transformed by transfection with an origin defective mutant of SV40 virus. Transformation dramatically increased the growth rate of these cells (designated HTM-3 cells), allowing biochemical and pharmacological characterization. The HTM-3 cells had cytoskeletal components that were reported to be present in TM tissue and non-transformed TM cells. Vimentin, tubulin and smooth muscle specific alpha-actin, but not desmin, were localized in these cells by immunocytochemistry. The extracellular matrix components collagen types I, III and IV, fibronectin and laminin were found in HTM-3 cells as well as their non-transformed parental cells. As predicted, the protein profile of the HTM-3 cells revealed by two-dimensional gel electrophoresis was different from that of the non-transformed cells, probably due to the enhanced growth characteristics of these cells. Furthermore, HTM-3 cells had various intracellular second messenger systems that responded to pharmacological agents. Forskolin, prostaglandin E2, beta-adrenergic and adenosine A2 agonists stimulated the adenylyl cyclase in these cells, whereas muscarinic, serotonergic, dopaminergic and other agonists were ineffective. Sodium nitroprusside increased the intracellular concentration of cGMP, demonstrating the presence of a functional guanylyl cyclase. Phospholipase C activity in these cells was also detected. Muscarinic agonists, histamine and bradykinin, but not adrenergic, serotonergic agonists or prostaglandins, increased phosphoinositide turnover. These drug responses of HTM-3 cells agree with published data on primary TM cells and TM tissues, suggesting that the transformed cells may be a valid substitute for certain pharmacological studies of TM.

Preclinical Overview of Brinzolamide

The development of topically active carbonic anhydrase inhibitors (CAIs) is a significant recent achievement in glaucoma medical treatment. Brinzolamide, the newest topical CAI, exhibits selectivity, high affinity, and potent inhibitory activity for the carbonic anhydrase type II isozyme (CA-II), which is involved in aqueous humor secretion. These characteristics, along with good ocular bioavailability, make brinzolamide maximally effective in lowering intraocular pressure (IOP) by locally inhibiting CA-II in the ciliary processes and suppressing aqueous humor secretion. Notable among its attributes as a safe and efficacious glaucoma drug is brinzolamides superior ocular comfort profile because of its optimized suspension formulation at physiologic pH. The degree of tolerability in the eye is considered an important determinant of a patients willingness to comply with the dosing regimen for a long-term glaucoma medication. Results from the preclinical pharmacologic evaluation of brinzolamide indicated that it acts specifically to inhibit CA without significant other pharmacologic actions that could introduce undesired side effects. Moreover, the typical side effects associated with systemically administered CAIs are expected to occur at a lower incidence or not occur at all with brinzolamide, as its therapeutic dose and low systemic absorption do not produce a problematic level of systemic CA inhibition. Brinzolamides long tissue half-life in the eye, particularly in the iris-ciliary body, favors a prolonged duration of IOP lowering. This was substantiated in clinical trials, which showed that twice-daily brinzolamide provides as significant an IOP reduction as three-times-daily brinzolamide or dorzolamide in a relatively high percentage of patients. Brinzolamide has been shown by the laser Doppler flowmetry technique to improve blood flow to the optic nerve head in pigmented rabbits after topical administration, without producing an increase of blood pCO2, indicating a potential for a local vasodilatory effect involving the optic nerve head circulation. The mean concentration of brinzolamide found in the retina of pigmented rabbits (0.338 microg equivalents/g) after a single dose of 14C-brinzolamide is sufficient to inhibit CA-II. These data suggest that topical brinzolamide could improve the blood flow in the optic nerve head in humans should it inhibit carbonic anhydrase in that vascular bed. Brinzolamide is a new topically active CAI that is safe and efficacious for reducing intraocular pressure. It offers the convenience of topical dose administration and greater freedom from side effects related to the inhibition of CA seen with the systemic administration of CAIs. Its formulation has been optimized to provide greater comfort upon instillation, and this can result in a higher compliance rate by the patient. Results of studies in animals show that brinzolamide has promise for increasing blood flow to the optic nerve head; however, this requires further assessment in the clinic. Brinzolamide represents a significant technical achievement and an important addition to the medical treatment of glaucoma as both a primary and an adjunctive drug.

The direct vascular relaxing action of betaxolol, carteolol and timolol in porcine long posterior ciliary artery

The vascular relaxing properties of three beta adrenoceptor antagonists, betaxolol, carteolol and timolol, currently used in the treatment of glaucoma, were characterized, compared and contrasted in the porcine long posterior ciliary artery. Isolated arterial ring segments precontracted with increased extracellular KCl (plus 40 mM) or the thromboxane analog, U-46619 (3 x 10(-7) M), were relaxed in a concentration-dependent fashion by betaxolol, carteolol, timolol or nitroprusside. In vessel segments depolarized with increased extracellular KCl, EC50 values indicated that the intrinsic relaxant sensitivity to betaxolol was equal to that of nitroprusside, six-fold greater than that of carteolol, and ten-fold greater than that of timolol. Similarly, the maximum relaxation occurring at equimolar concentrations (10(-4) M) for the beta adrenoceptor antagonists was betaxolol > carteolol = timolol. Qualitatively similar results were noted in ring segments of the rabbit external iliac artery precontracted with increased extracellular KCl (plus 30 mM). Under conditions in which specific receptor-linked events are absent and voltage-gated Ca++ entry is maximized, the Ca++ concentration response relationship in porcine long posterior ciliary artery was shifted to the right in an apparent competitive manner by betaxolol, reflecting a 5.6-fold reduction in the sensitivity to Ca++. Conversely, nitroprusside reduced the Ca++ sensitivity three-fold in a noncompetitive fashion; not only shifting the Ca++ concentration response relationship to the right, but also depressing the maximum by 57%. Porcine long posterior ciliary arterial segments precontracted to a similar degree with U-46619, in which voltage-gated Ca++ entry is only one component of many specific cell signalling transduction mechanisms contributing to the precontraction, exhibited a sensitivity to betaxolol that was six-fold less than to nitroprusside, but two-fold greater than to timolol and 20-fold greater than to carteolol. These results are consistent with an obvious direct vascular relaxing capacity for beta adrenoceptor antagonists that primarily represents a capacity for inhibiting voltage-gated Ca++ entry in vascular smooth muscle. Additionally, the differential potencies of these three beta adrenoceptor antagonists characterized in this study suggests that this property is much more likely to contribute to any potentially beneficial effects of betaxolol than carteolol or timolol.

Identification and characterization of the ocular hypotensive efficacy of travoprost, a potent and selective FP prostaglandin receptor agonist, and AL-6598, a DP prostaglandin receptor agonist

The structure-activity studies that led to the identification of travoprost, a highly selective and potent FP prostaglandin analog, and AL-6598, a DP prostaglandin analog, are detailed. In both series, the 1-alcohol analogs are very effective and are thought to be acting as prodrugs for the biologically active carboxylic acids. The efficacy of amide prodrugs depends on the degree of substitution and the size of the substituents. Selected compounds are profiled in vitro and in vivo preclinically. Clinical studies show that travoprost 0.004% (isopropyl ester) provided intraocular pressure control superior to timolol 0.5% when used as monotherapy in patients with open-angle glaucoma or ocular hypertension. In clinical studies, AL-6598 0.01% provided a sustained intraocular pressure reduction with q.d. application; b.i.d. provided greater intraocular pressure control. The acute and, apparently, conjunctival hyperemia associated with topical ocular AL-6598 can be attenuated while maintaining intraocular pressure-lowering efficacy by formulating with brimonidine.

Systemic and Ocular Vascular Roles of the Antiglaucoma Agents b-Adrenergic Antagonists and Ca2+ Entry Blockers ☆

This review addresses whether the antiglaucoma agents beta-adrenergic antagonists and Ca2+ entry blockers cause vasoactive effects in the retinal and other ocular vasculatures, as they do in other tissues. The potent vasodilating effects of Ca2+ entry blockers on ocular vessels have recently been demonstrated in in vivo and in vitro studies, implying that the maintenance of ocular vascular tone relies almost exclusively on extracellular Ca2+. Ca2+ entry blockers may potentially play a role in relaxing the retinal, long posterior ciliary, and ophthalmociliary arteries to improve the ocular circulation in vascular diseases in which there is considerable vascular tone present. The beta-adrenergic antagonists are discussed with reference to their antihypertensive role, their effect on other vascular beds, and finally what is known of their effect in the ocular vasculature. The emerging evidence that particular selective beta-adrenergic antagonists, such as betaxolol, are also potent Ca2+ channel entry blockers in other vascular beds is presented. Betaxolol has been shown to induce vasodilatation in the retinal and other ocular vascular beds, although studies have shown that beta1-adrenergic receptors are sparse in these vascular beds. This implies that an alternative mechanism must be responsible for betaxolol-induced vasodilatation. Evidence is presented that betaxolol vasodilates via its potent Ca2+ channel entry blocking properties, and its potency and ability to vasodilate are compared with those of nimodipine and timolol, as well as with those of other Ca2+ channel entry blockers. Important areas for future research in this area are discussed.

Colour vision anomalies following experimental glaucoma in monkeys

Spectral sensitivity defects, associated with chronic elevated intraocular pressure (IOP) produced by Argon laser trabeculoplasty, were studied in monkeys. Increment‐threshold spectral sensitivity (ITSS) and threshold versus intensity (TVI) functions were measured using a behavioural model. Elevated IOP resulted in slum wavelength (SW) sensitivity losses characteristic of many ocular diseases. The amount of SW sensitivity loss for ITSS functions depended upon the intensity level and chromatic composition of the background field. The optimum condition identifying the greatest SW sensitivity reduction was a yellow background of moderate intensity (100–100 Td). In the early stages of experimental glaucoma. The cone mechanisms and the rod mechanism typically showed decreased test and field sensitivities. The SW cone pathway has slightly greater threshold elevation (∼0.3 log unit) compared to the rod and cone pathways. On the other hand, in the advanced stages of experimental glaucoma, the largest sensitivity losses were in the longer‐wavelength, red‐green opponent mechanism, with the rod and SW cone pathways showing smaller losses. The similarities of the colour vision anomalies in this animal model with those of patients with glaucoma, provides support for its use as an experimental model for human glaucoma.

ATTENUATION OF GANGLION CELL DYSFUNCTION: A Goal for Glaucoma Treatment in the New Millennium

Currently, glaucoma therapy is directed at lowering intraocular pressure (IOP) primarily by pharmacologic means and to a lesser extent by surgery. 2,16,45 Although raised IOP can undoubtedly cause dysfunction to retinal ganglion cells and loss of vision, as in glaucoma, it is not likely to be the major cause for glaucomatous visual loss in every patient. 35 Nevertheless, lowering IOP is presently the only way of treating glaucoma in the clinic. As this millennium draws to a close, however, the inadequacies of this method are increasingly being recognized. The goal for the future must, therefore, be to discover a therapy that preserves visual function irrespective of the cause of glaucomatous optic neuropathy. One possibility is for a drug to interact with intraretinal components to somehow attenuate whatever it is that causes visual field loss in glaucoma. This approach has been termed neuroprotection in glaucoma. 6,21,24,30,33,35 It is known from animal studies that topically applied drugs can reach the retina and other ocular tissues by way of both local and systemic routes. 33,34,35,36

Role of Aldose Reductase and Effects of Aldose Reductase Inhibitors in Ocular Tissue Aging Phenomena in the Diabetic Rat

Studies of the role of aldose reductase in sugar cataractogenesis have been aided by the synthesis and characterization of inhibitors of this enzyme which can prevent lens opacification in galactosemic or diabetic rats1,2. The anti-cataract activity of aldose reductase inhibitors correlates with inhibition of polyol accumulation in the lenses of treated galactosemic or diabetic animals; in untreated diabetic rats, lens sorbitol levels may be elevated 100-fold over the normal value3. Hyperglycemia produces an increase in glucose content of tissues not responsive to insulin. Due to increased substrate availability, the rate of sorbitol formation by action of aldose reductase is greatly accelerated relative to conversion of this metabolite to fructose via sorbitol dehydrogenase1:

Topically applied betaxolol attenuates ischaemia-induced effects to the rat retina and stimulates BDNF mRNA.

Glucose\quad + \quad NADPH\quad + \quad {H^{ + }}\;\xrightarrow{{Aldose\,Reductase}}\;Sorbitol\quad + \quad NAD{P^{ + }}