Thomas H. Maren - US grants

The broad objectives of this research are to increase knowledge of secretory and carbonic anhydrase (CA)-dependent systems in the eye, brain, and inner ear, and apply this to certain medical problems, notably glaucoma, cataract, increased intracranial pressure, & Menieres disease. The physiological work will be done in parallel with enzyme studies on the appropriate tissues. EYE: a) Search will continue for an effective non-toxic carbonic anhydrase inhibitor for treatment of glaucoma by corneal instillation rather than systemically. Current work on principles of corneal penetration show this to be feasible. Studies are also planned on ion-transport from plasma to aqueous following administratin of timolol, to compare with CA inhibitors. b) Major effort will be given to the role of CA in normal lens, which our current works links to dissimilation of HCO3-. It appears likely that this mechanism fails in cataractous lens. BRAIN-CSF: Both as molecular probes and potential drugs, new agents (other than CA inhibitors) remain to be discovered that will reduce CSF flow. Such agents will be sought, with particular attention to their influence on ion transport. ENDOLYMPH: Basic information will be sought on CO2 equilibria and formation rate of endolymph. We shall measure effects of CA inhibitors on ion and fluid movement in inner ear. Because of the high concentration of CA in this locale, analogy to the other fluids and some clinical "impressions", it is possible that these studies may lead to effective treatment of Menieres disease. ENZYMOLOGY: Basic kinetics and inhibition studies of CA will be done on these tissues, with emphasis on the membrane-bound enzyme. These chemical data will be intimately linked to the physiological and pharmacological results, and together form the basis for rational treatment programs.

A five-fold program has been developed in which the central theme is the enzyme carbonic anhydrase (C.A.), its chemistry, inhibition, and role in certain organ systems, presently unknown. The continuity between biochemical and physiological events is stressed. We plan to study the following: I. The role of carbonic anhydrase in muscle, uterus, ova, and secretion of endolymph. This introduces also fundamental questions about the physiology of these organs. Particularly, in muscle we shall study CO2 output after inhibition of the enzyme; in uterus the implantation evironment; in ova the dispersion of the corona radiata; in endolymph the rate and character of secretion. II. We shall study the role of CO2 in neutral secretions (NaCl) and whether there can be renal H+ output independent of the CO2 system. III. New sulfonamide inhibitors combining lipid and water solubility will be developed. Studies of drug-enzyme kinetics will be pursued, with attention ot "uncompetitive" inhibition, and differences between the three C.A. isozymes. IV. We wish to characterize mitochondrial C.A., and also the enzyme in brain myelin, with respect to Kcat, Km, and susceptibility to inhibition by the sulfonamides. V. We plan to study gas exchange and acid-base balance in sub-order of Amphibia, the plethodonts, which lack both lungs and gills, and respire entirely through the skin. They are also the only adult vertebrates that lack red cell C.A.

The proposed work is divided into four related categories, all converging on the problems of aqueous humor formation and treatment of glaucoma: Development of new drugs (topical carbonic anhydrase inhibitors); study of ionic reactions underlying secretion of aqueous; exploration of the effect of A1C13 and other acids on aqueous secretion and pH of ciliary processes; effect of autonomic drugs and Na-K-ATPase inhibitors on electrolyte movement from plasma to aqueous. 1.) We are searching for a topical carbonic anhydrase inhibitor with properties such that 1 drop of solution will lower intraocular pressure in man for 6-12 hours. This demands a continuing effort in organic synthesis, analysis of physico-chemical properties of compounds, and aqueous humor dynamics in animals and man. Feasibility of this goal seems reasonable; it has already been achieved in the rabbit. Almost certainly, such a compound will have no systemic toxicity in man, or any other effect on the eye. 2.) We believe it possible (likely?) that in aqueous humor (and other secretory sites, as cerebrospinal fluid and pancreas), the observed linkage of Na+ and HCO3- has a specific chemical basis, in ion pairing of NaCO3- and NaHCO3-degrees. This has not been considered for epithelial secretion but has been entertained as a basis for HCO3- - Cl- exchange in red cells. In this view Na+ is not the moving force for fluid formation, but Na is incorporated into an anion or undissociated molecule. We shall investigate this; if true, the new concept should profoundly affect studies of ion transport. 3.) We continue to study our finding that protonation by Lewis or Bronsted acids reduces or abolishes aqueous humor secretion. This has theoretical implications for mechanisms of secretion and offers the possibility of reduction of aqueous secretion by new methods. In this context we shall also study the pH of ciliary process and how this may be changed by acids or by carbonic anhydrase inhibitors. 4.) We shall measure the rates of ion movement from plasma to aqueous humor, before and after administration of various classes of drugs known to affect aqueous secretion, as we have done with carbonic anhydrase inhibitors. This is a neglected aspect of ocular pharmacology; we shall study ouabain, timolol, pilocarpine, epinephrine, and several others. Such measurements should throw light on their (presently unknown) mechanism.

The proposed work is divided into four related categories, all converging on the problems of aqueous humor formation and treatment of glaucoma: Development of new drugs (topical carbonic anhydrase inhibitors); study of ionic reactions underlying secretion of aqueous; exploration of the effect of A1C13 and other acids on aqueous secretion and pH of ciliary processes; effect of autonomic drugs and Na-K-ATPase inhibitors on electrolyte movement from plasma to aqueous. 1.) We are searching for a topical carbonic anhydrase inhibitor with properties such that 1 drop of solution will lower intraocular pressure in man for 6-12 hours. This demands a continuing effort in organic synthesis, analysis of physico-chemical properties of compounds, and aqueous humor dynamics in animals and man. Feasibility of this goal seems reasonable; it has already been achieved in the rabbit. Almost certainly, such a compound will have no systemic toxicity in man, or any other effect on the eye. 2.) We believe it possible (likely?) that in aqueous humor (and other secretory sites, as cerebrospinal fluid and pancreas), the observed linkage of Na+ and HCO3- has a specific chemical basis, in ion pairing of NaCO3- and NaHCO3-degrees. This has not been considered for epithelial secretion but has been entertained as a basis for HCO3- - Cl- exchange in red cells. In this view Na+ is not the moving force for fluid formation, but Na is incorporated into an anion or undissociated molecule. We shall investigate this; if true, the new concept should profoundly affect studies of ion transport. 3.) We continue to study our finding that protonation by Lewis or Bronsted acids reduces or abolishes aqueous humor secretion. This has theoretical implications for mechanisms of secretion and offers the possibility of reduction of aqueous secretion by new methods. In this context we shall also study the pH of ciliary process and how this may be changed by acids or by carbonic anhydrase inhibitors. 4.) We shall measure the rates of ion movement from plasma to aqueous humor, before and after administration of various classes of drugs known to affect aqueous secretion, as we have done with carbonic anhydrase inhibitors. This is a neglected aspect of ocular pharmacology; we shall study ouabain, timolol, pilocarpine, epinephrine, and several others. Such measurements should throw light on their (presently unknown) mechanism.