Curtis W. Conroy

Carbonic anhydrase inhibitors : synthesis and inhibitory properties of 1,3,4-thiadiazole-2,5-bissulfonamide

Summary 1,3,4-Thiadiazole-2,5-bissulfonamide was prepared from 2,5-dimercapto-1,3,4-thiadiazole, by a modification of the reported literature procedure, ie, via the sulfenamide. This compound is the lead molecule for designing important classes of pharmacological agents, such as benzothiadiazine diuretics, and it seems to behave as a strong inhibitor of the zinc enzyme carbonic anhydrase (CA). Here we prove that the title compound is actually a weak CA inhibitor and try to explain why previous investigators obtained erroneous data.

Chemical and pharmacological properties of MK-927, a sulfonamide carbonic anhydrase inhibitor that lowers intraocular pressure by the topical route

A large number of sulfonamides have now been tested by the topical route for the lowering of intraocular pressure in the normal albino rabbit. Certain compounds with favorable balance between lipid and water solubility, and high activity against carbonic anhydrase, do lower pressure as much as 3 mmHg. MK-927, a thienothiopyrane-2-sulfonamide carrying an alkylamino group of pK 5.8, has desirable physicochemical properties: good water solubility below pH 5.8, a CHCl3/buffer ratio of 0.6 at pH 5.4, and a KI value against carbonic anhydrase of 2-7 nM, depending on assay conditions. Inhibition of CO2 hydration is non-competitive. By comparison with other candidate topically active sulfonamides, it is the most effective in terms of pressure lowering times duration of action. There are no apparent systemic effects or ocular toxicity. The concentration of drug reaching the ciliary process and aqueous humor is of the same order as that following parenteral sulfonamides, so that inhibition of the enzyme exceeds 99%. MK-927 is therefore a candidate for the clinical treatment of glaucoma.

Is cyanate a carbonic anhydrase substracte

A study was undertaken to investigate whether diverse carbonic anhydrase (CA) isozymes (both native Zn as well as cobalt‐substituted) are able to catalyze the hydrolysis of anions such as cyanide, cyanate, and thiocyanate. A controversy exists between the crystallographic and spectroscopic data of CA II‐anion adducts. In the former case it has been shown that “metal poisons” such as CN−and CNO−are not directly coordinated to the active site Zn(II) ion whereas spectroscopic studies indicate otherwise. A theoretical study in the above systems did not resolve this controversy, since it was calculated that all three anions can act as CA substrates. In this paper we prove experimentally that none of them may act as substrates of CA and propose an explanation to the above controversy, discussing the mode of binding of small molecules within the enzyme active site.

Effect of pH on the ocular distribution of a topical carbonic anhydrase inhibitor.

Ampholyte carbonic anhydrase inhibitors (including MK-927) have previously been shown to elicit greater intraocular pressure reduction when applied in ionized form at moderately acidic or alkaline pH compared to application of un-ionized drug at neutral pH. Since ionized solutions should be far more membrane-impermeant than un-ionized solutions, we attempted to solve this apparent paradox. We studied the distribution at 0.5-18 hr of MK-927 in eye tissues and fluid after topical instillation of one drop of a 0.5% solution at pH 4.9, 7.0 and 9.1. Measured drug concentrations at 30 min in corneal epithelium and stroma are approximately 4.5-fold greater after acidic or alkaline application than after neutral application, with the highest levels being found in corneal epithelium. The same pattern is seen in ciliary process, the site of aqueous humor production. Here drug concentration is approximately 60% of that in cornea at 30 min. Free drug concentrations in ciliary process were used to compute the time course of maximal carbonic anhydrase inhibition for the three modes of application. Drug concentration in sclera, uvea and aqueous humor at all times are all low by comparison, suggesting drug movement is from cornea to ciliary process via the corneo-scleral junction. The k(in) for proton movement across the corneal epithelium was measured (k(in) = 12.4 hr-1) from which a permeability coefficient (P = 2.7 x 10(-2) cm sec-1) was computed. Separate analysis was made of the pH status of the cornea 30 min and 1 hr following instillation of 1 drop 2% acidic (pH 4.9) and alkaline (pH 9.1) MK-927, with sodium sulfadiazine and pilocarpine.HCl as pH controls. Stromal bicarbonate at 30 min was approximately halved after acidic drops and doubled after alkaline drops consistent with pH decrease or increase of nearly 0.3 U. The results are in accord with classical schemes for amine permeation of the cornea at acidic pH when consideration is given to movement of acid equivalents and corneal pH. Thus drug inside the eye is in relatively (> 95%) lipophilic form except that trapped in the cornea shortly after acidic or alkaline applications.

The determination of osteopetrotic phenotypes by selective inactivation of red cell carbonic anhydrase isoenzymes

Red cell carbonic anhydrase isoenzyme activities (HCA-I and HCA-II) were quantitated in blood hemolysates of two female siblings affected with autosomal recessive osteopetrosis in addition to family members who were both obligate and potential heterozygotes as well as normal controls. Selective inactivation of red cell hemolysates with 20 mmol/l bromopyruvic acid (pH 7.5) for 50 min at 25 degrees C lead to a near quantitative removal of HCA-I activity without significant loss of HCA-II activity. Comparison of untreated hemolysate HCA activity with residual activity in bromopyruvate treated samples allowed determination of HCA-II/HCA-I activity ratios. Estimation of activity ratios was also attempted by use of the inhibitor I- which is known to discriminate between the two isoenzymes. Hemolysate HCA activity of the two affected individuals (homozygotes) was completely abolished by the procedure, consistent with an absence of the bromopyruvate resistant HCA-II isoenzyme in the red cells of these individuals. HCA-I activities were found to be elevated significantly above normal. Both parents (obligate heterozygotes) were found to have normal HCA-I activities and HCA-II activities 60-80% of normal. Computed HCA-II/HCA-I activity ratios fell into these ranges: 0 for homozygotes, 0.82-0.87 for obligate heterozygotes and 1.08-1.16 for normal controls. KI values for I- inhibition of hemolysate HCA activity gave 0.25-0.30 mmol/l, 0.9-1.5 mmol/l and 2.0 mmol/l for the three categories. These relations should prove useful in the screening of osteopetrotic phenotypes.

The permeability of hydrophobic membranes to 22Na salts and 14CO2 in low dielectric media

The one-way fluxes of 14CO2 and a series of 22Na (Cl, Br, HCO3, ClO4, I) salts across n-hexadecane-impregnated solid-support liquid membranes have been measured in water and low dielectric media (50-90 vol% dioxane/water). One-way fluxes for 14CO2 (J14CO2) were 0.84 and 1.03 x 10(-9) mol cm-2 s-1 in 75% dioxane (aq.) and water, respectively, across both impregnated cellulose and teflon membranes. 22Na fluxes across impregnated cellulose membranes in 75% dioxane (aq.) ranged from 1.8 to 11.4 x 10(-10) mol cm-2 s-1 and had the order NaCl less than NaBr less than NaHCO3 less than NaClO4 less than Nal. 22Na fluxes across impregnated teflon membranes were slightly smaller, 1.5-7.1 x 10(-10) mol cm-2 s-1, but had the same order for the anions tested. No measurable 22Na fluxes were observed in aqueous media. For NaI and NaClO4 there was a 3-6-fold enhancement of fluxes in 90% dioxane (aq.) compared to 75% dioxane (aq.). The corresponding enhancement for fluxes of NaHCO3, NaBr and NaCl was 1.5-fold. The results are discussed in terms of ion-paired salt transport in low dielectric media.