Pharmacological and ocular hypotensive properties of topical carbonic anhydrase inhibitors.

There was a time gap of over 40 years between the demonstrated oral effectiveness of acetazolamide in lowering the intraocular pressure (IOP) of glaucoma patients and the introduction of a topical carbonic anhydrase (CA) inhibitor. This is due to the fact that CA-II, the isoenzyme which most likely plays an important role in the production of aqueous humor in humans, must be essentially inhibited by 100% to elicit a pharmacological response. The lack of success with earlier attempts to obtain a topical agent stems from an inability to attain and maintain a sufficiently high intraocular concentration of drug to achieve the required inhibition of CA. Dorzolamide and brinzolamide are two topical CA inhibitors which are currently available to treat ocular hypertension and/or glaucoma. Dorzolamide is a very potent inhibitor of CA-II and its site of action is local within the eye. Like oral CA inhibitors, topically applied dorzolamide lowers IOP by decreasing the production of aqueous humor. The drug is used in monotherapy as a 2% solution administered three times daily. Its ocular hypotensive effect is comparable to that of timolol at peak but is somewhat less at trough. The IOP lowering effect of timolol is enhanced by the twice daily administration of 2% dorzolamide either concomitantly or in combination. Topically applied dorzolamide is generally well tolerated and had a low drop-out rate in clinical studies. The most frequent ocular adverse experience is burning and/or stinging. Corneal and lenticular problems have generally not been encountered with long-term therapy with dorzolamide. Topically applied dorzolamide penetrates directly to the posterior segment of the eye and its presence is consistent with the initial report that dorzolamide increases retinal blood flow velocity in patients with normal tension glaucoma. The most frequent systemic adverse experience is a transient bitter taste. Biochemical changes indicative of the systemic inhibition of CA have not been observed in monotherapy studies lasting up to 2 years. This is in harmony with the inability of dorzolamide at steady-state to saturate CA in the red blood cell and the failure to detect its presence in plasma. A 1% suspension of brinzolamide is comparable to 2% dorzolamide in lowering IOP, both drugs being administered three times daily. Although brinzolamide has a lower incidence of burning/ stinging, it elicits more blurred vision.

Effects of muscarinic cholinergic receptor antagonists on postnatal eye growth of rhesus monkeys.

PURPOSE: To study a potential role for muscarinic receptors in the inhibition of deprivation-induced excessive axial elongation and myopia in a monkey model. METHODS: The right eyes of 20 newborn rhesus monkeys were occluded with a black contact lens. In seven monkeys each, either atropine or pirenzepine was topically applied daily to the occluded eyes. The nonoccluded fellow eyes and both the occluded and nonoccluded fellow eyes of another six monkeys were treated with vehicle solution. RESULTS: After 33 to 39 weeks, in 5 monkeys of the vehicle group, occluded eyes were longer and the myopic shift significantly greater than in the nonoccluded fellow eyes. In six atropine-treated monkeys, axial length and reduction of the initial hyperopia of occluded and nonoccluded fellow eyes were not different statistically. The myopic shift of the occluded eyes was significantly smaller than in the vehicle-treated occluded eyes. In the pirenzepine-treated group, axial length of the occluded eyes was similar to the nonoccluded eyes of controls and the occluded eyes of atropine-treated monkeys. There was a trend of pirenzepine to reduce the myopic shift of the occluded eye. No effect of atropine or pirenzepine was noted on muscarinic receptor density in retina, brain, or heart, but a small increase was observed in iris + ciliary body. CONCLUSIONS: The drug treatment results implicate muscarinic receptors in postnatal eye growth regulation. Because of interanimal differences our data do not indicate whether nonselective or selective muscarinic blockade is more effective in reducing deprivation-induced myopia.

Dorzolamide, a 40-year wait. From an oral to a topical carbonic anhydrase inhibitor for the treatment of glaucoma.

Dorzolamide, on the basis of its pharmacological profile and lack of undesirable side effects in safety assessment studies together with the fact that it could be formulated in solution at 2%, underwent extensive clinical studies. Early clinical studies in the development of dorzolamide have been described elsewhere (Maren, 1995; Serle and Podos, 1995). In a 1-year study in which a comparison was undertaken in patients for intraocular pressure lowering effects between 2% dorzolamide administered three times daily, 0.5% betaxolol twice daily, and 0.5% timolol twice daily, the peak reductions in intraocular pressure were 23, 21, and 25%, respectively. Tachyphylaxis did not develop to dorzolamide nor were electrolyte and/or systemic side effects encountered (Strahlman et al., 1995). The latter is consistent with results of a pharmacokinetic study in humans in which plasma levels of dorzolamide were lower than the limit of detection (5 ng/ml) at a time when the red blood cell content of dorzolamide had reached steady state which was appreciably less than the red blood cell content of the enzyme (Biollaz et al., 1995). Patients taking 0.5% timolol twice daily received either 2% dorzolamide twice daily or 2% pilocarpine four times daily for 6 months and the additional reductions in intraocular pressure elicited by dorzolamide and pilocarpine were very similar. However, pilocarpine usage resulted in a higher discontinuation rate (Strahlman et al., 1996). In a separate study in which dorzolamide and pilocarpine were compared at these dosage schedules, patients preferred dorzolamide to pilocarpine by a ratio of over 7 to 1 in terms of quality of life (Laibovitz et al., 1995). In summary, the quest for a topical, ocular hypotensive, CA inhibitor, though time-consuming, was a successful one with the introduction of dorzolamide into general clinical practice.

The preclinical pharmacology of dorzolamide hydrochloride, a topical carbonic anhydrase inhibitor.

Dorzolamide hydrochloride (S,S-5,6-dihydro-4H-4-ethylamino-6-methylthieno [2,3-b]thiopyran-2-sulfonamide-7,7-dioxide HCl; MK-507; L-671,152) is a water-soluble, potent inhibitor of human carbonic anhydrase isoenzymes II and IV in vitro, the respective IC50 values being 0.18 nM and 6.9 nM. In contrast, it was found to be a much weaker inhibitor of human carbonic anhydrase isoenzyme I (IC50 value of 600 nM). The topical administration of one 50 microliters drop of 0.5%, 1% and 2% solutions of dorzolamide maximally lowered the intraocular pressure (IOP) of glaucomatous monkeys by 22%, 30% and 37%, respectively. Good ocular hypotensive activity was also observed in ocular normotensive and hypertensive rabbits. Its site of action was within the eye, and the reductions in IOP in both species was achieved via decreased aqueous humor inflow. The duration of action of 2% dorzolamide was shorter than that of 0.5% timolol in glaucomatous monkeys. The IOP lowering activity of timolol in this paradigm was enhanced by the concomitant instillation of dorzolamide. Both acutely and repeatedly administered 2% dorzolamide did not decrease regional ocular blood flow in the rabbit, and the topical instillation of the drug had no adverse effects on the eye of rabbits, dogs and monkeys. Dorzolamide has been approved in a number of countries for use in patients with ocular hypertension or open-angle glaucoma.

3-substituted thieno[2,3-b][1,4]thiazine-6-sulfonamides. A novel class of topically active carbonic anhydrase inhibitors.

3-Aminoalkyl derivatives of thieno[2,3-b][1,4]thiazine-6-sulfonamide were prepared for evaluation as topically active ocular hypotensive agents. The compounds described were found to be excellent in vitro inhibitors of carbonic anhydrase II and in vivo to lower intraocular pressure in three rabbit models of ocular hypertension. Compounds 20A, 20B, and 20C met the requirement of formulation as a 1% solution at pH 5.2, but none of the compounds described exhibited greater activity in the normotensive albino rabbit, the alpha-chymotrypsin-treated albino rabbit, or the normotensive pigmented rabbit than MK-927 or MK-507, the present clinical candidates.

Sulfonylmethanesulfonamide inhibitors of carbonic anhydrase.

A series of sulfonylmethanesulfonamide derivatives is described, which are inhibitors of carbonic anhydrase (CA). The most potent of these is the racemic fluoro sulfone 9, which inhibits carbon dioxide hydration catalyzed by human CA II (CA-II) with an IC50 of 3 nM. Binding competition studies versus dansylamide indicate that the enantiomers of 9 have different affinities for CA-II, with equilibrium dissociation constants of 3.6 and 0.6 nM. QSAR analysis suggests that the key factors involved in achieving high affinity in this series are sulfonamide acidity, hydrophobicity, and minimization of steric demands at the carbon atom adjacent to the sulfonamide group.

4-substituted thiophene- and furan-2-sulfonamides as topical carbonic anhydrase inhibitors.

A series of 4-substituted thiophene- and furan-2-sulfonamides was prepared and was found to possess nanomolar-level potency for inhibition of human carbonic anhydrase II in vitro. Selected examples from this group were further evaluated for their potential to act as topically effective ocular hypotensive agents in the ocular normotensive albino rabbit and the ocular alpha-chymotrypsinized rabbit. Solubility studies in water and pH 7.4 buffer were carried out to estimate the ability of compounds to be formulated in solution. The sensitization potential of key representative structures was determined by in vitro glutathione reactivity studies and guinea pig maximization testing.

Thieno[2,3-b]furan-2-sulfonamides as topical carbonic anhydrase inhibitors.

Novel 5-[(alkylamino)methyl]thieno[2,3-b]furan-2-sulfonamides were prepared and evaluated in vitro for inhibition of human carbonic anhydrase II (CA II) and ex vivo for their ability to inhibit Ca II in the albino rabbit eye after topical administration. Compound 11a was found to lower intraocular pressure (IOP) in both the alpha-CT ocular hypertensive albino rabbit and the normal albino rabbit, but was ineffective at lowering IOP in a hypertensive, pigmented monkey model. Since 11a was highly bound to ocular pigment, a series of less basic analogs was prepared. Examples in this series were both less extensively bound to ocular pigment and more active at reducing IOP in pigmented rabbits after topical dosing. Key examples displayed moderate reactivity toward glutathione.

MK-507 (L-671,152), a topically active carbonic anhydrase inhibitor, reduces aqueous humor production in monkeys.

An investigation was carried out to determine the mechanism by which MK-507 (L-671,152), a water-soluble inhibitor of human carbonic anhydrase II in vitro, reduces intraocular pressure when applied topically to monkey eyes. Intraocular pressure, tonographically measured outflow facility, and fluorophotometrically determined aqueous humor flow were measured before and after therapy in eight normal cynomolgus monkeys. Fifty microliters of 2% MK-507 was instilled in one eye and diluent in the contralateral eye. Baseline values for intraocular pressure, outflow facility, and aqueous humor flow were similar in the drug-treated and diluent-treated control eyes. After therapy, intraocular pressure was significantly (P less than .05) reduced from 1 to 7 hours (eg, 14.0 +/- 1.0 and 15.9 +/- 0.9 mm Hg [mean +/- SEM], treated and control eyes, respectively, at 3 hours). Outflow facility was not significantly (P greater than .40) changed at 3 hours, and aqueous humor flow measured over 5 hours was significantly (P less than .05) reduced (38%) in treated (0.9 +/- 0.1 microL/min) as compared with control eyes (1.5 +/- 0.1 microL/min). The results suggest that MK-507 reduces intraocular pressure by decreasing aqueous humor production.

The effect of cyclooxygenase inhibition on the ocular hypotensive action of topical carbonic anhydrase inhibitors in rabbits.

Experiments were undertaken in normal albino rabbits to determine if cyclooxygenase inhibition by nonsteroidal anti-inflammatory drugs modified the ocular hypotensive activities of topically applied MK-507, MK-927 and L-662,583, three water-soluble carbonic anhydrase inhibitors (CAI). Cyclooxygenase was inhibited either by systemic indomethacin or by topically administered flurbiprofen, and epinephrine was included as a positive control. Both a 1-hr pretreatment with indomethacin (5 mg/kg i.p.) and topically applied 0.03% flurbiprofen antagonized the ocular hypotensive effect of one drop (50 microliters) of 1% epinephrine. Two percent solutions of the three CAIs were instilled three times with 10 min between each drop in order to obtain a meaningful and reproducible reduction in intraocular pressure (IOP). This dosage schedule elicited a peak decline in IOP ranging from 4.6 mm Hg to 6.2 mm Hg which was achieved via a local action within the eye. The ocular hypotensive effects of MK-507, MK-927 and L-662,583 were unaltered either by a 1-hr pretreatment with indomethacin (5 mg/kg i.p.) or by topically administered 0.03% flurbiprofen. These studies indicate that the IOP lowering actions of the three CAIs, unlike that of epinephrine, in rabbits are not mediated by endogenous prostaglandins and/or other prostanoids.

A comparison of L-671,152 and MK-927, two topically effective ocular hypotensive carbonic anhydrase inhibitors, in experimental animals.

L-671,152 is a water-soluble, carbonic anhydrase inhibitor structurally similar to MK-927, a carbonic anhydrase inhibitor that, on topical administration, lowers the intraocular pressure (IOP) of experimental animals and humans. L-671,152 was more potent than MK-927 at inhibiting purified, human erythrocyte carbonic anhydrase II in vitro, as reflected in their respective IC50 values of 0.16 nM and 1.19 nM. Both compounds were compared for topical, ocular hypotensive activity in pigmented rabbits and cynomolgus monkeys. Ocular hypertension was induced in the latter by argon laser photocoagulation of the trabecular meshwork. A 2% solution of L-671,152 was more potent than 2% MK-927 in lowering the IOP of ocular hypertensive monkeys, the maximum reductions being 13.8 mm Hg (37%) and 9.6 mm Hg (27%) at 5 hr and 4 hr, respectively. Moreover, the duration of action of L-671,152 was superior to that of MK-927. The ocular hypotensive effect of L-671,152 was greater than that of MK-927 over a range of concentrations (0.5%-2%) in pigmented rabbits whose IOP was inherently elevated. The peak declines in the IOP of these rabbits after the instillation of 2% solutions of L-671,152 and MK-927 were 6.1 mm Hg and 4.8 mm Hg, respectively. L-671,152 was very effective in lowering the elevated IOP of alpha-chymotrypsinized rabbits and the unilateral instillation of 0.5% L-671,152 into the contralateral eye failed to decrease the elevated IOP of the alpha-chymotrypsinized eye. This finding indicates that the site of action of topically applied L-671,152 is local. The enhancement in the potency of L-671,152 over MK-927 is attributed to a greater inhibition of carbonic anhydrase activity.

The ocular hypotensive effect of the topical carbonic anhydrase inhibitor L-671,152 in glaucomatous monkeys.

L-671,152, a new potent water-soluble inhibitor of human carbonic anhydrase II in vitro, was applied topically to cynomolgus monkey eyes in which glaucoma had been produced by argon laser photocoagulation of the trabecular mesh-work. Intraocular pressure was measured at 0 hours, 0.5 hours, and hourly for 8 hours in eight eyes for 2 baseline days, 1 day receiving the vehicle and 5 days receiving therapy with 2% L-671,152 twice a day, after initial single-dose trials of various concentrations. Intraocular pressure was not significantly different comparing baseline and vehicle-treated days. Significant intraocular pressure reductions occurred from 1 to 8 hours after the first dose, and lasted for at least 16 hours after the second dose. The reduction in intraocular pressure became more pronounced from day 1 to day 5 at each time interval. The mean (+/- SEM) maximum reduction in intraocular pressure was 7.8 +/- 2.1 mm Hg on day 1 and 10.1 +/- 2.4 mm Hg on day 5 at 3 hours after administration, comparing the intraocular pressure in drug-treated and vehicle-treated eyes. L-671,152 has a longer duration of action than does previously studied MK-927 in glaucomatous monkeys. It appears to have great clinical potential.

MK-927: a topically active ocular hypotensive carbonic anhydrase inhibitor.

MK-927 (dl-5,6-dihydro-4-(2-methylpropylamino)-4H-thieno(2,3b)thiopyra n-2-sulfonamide-7,7-dioxide hydrochloride) is a water soluble, carbonic anhydrase inhibitor (CAI) possessing a Ki of 12.0 nM against purified human carbonic anhydrase II in vitro. The acute instillation of one drop (50 microliters) of 0.5%, 1% and 2% solutions of MK-927 maximally decreased the intraocular pressure (IOP) of ocular hypertensive, cynomolgus monkeys by 4.7, 5.9 and 9.6 mm Hg, respectively. The decline of 9.6 mm Hg represented a reduction of 27% from the corresponding vehicle-treated value of 35.3 mm Hg. Peak reductions in IOP were present at 2 to 4 hr after the instillation of the three doses and the ocular hypotensive effect was waning at 6 hr. The IOP of normotensive, monkey eyes was significantly lowered by 1% and 2% solutions of MK-927 with the effect being more transient in these eyes than in hypertensive eyes. The elevated IOP of alpha-chymotrypsinized rabbits was dose-dependently decreased by 0.01%, 0.1% and 0.5% solutions of MK-927. MK-927 modestly bound to rabbit ocular pigment in vitro and the concentrations of MK-927 in the iris + ciliary body of pigmented rabbits were higher than those in the same tissue of albino rabbits after dosing with 0.5% MK-927. The ocular hypotensive effect of 2% MK-927 was greater in magnitude and longer in duration in normal pigmented than in albino rabbits. The IOP lowering action of MK-927 was local as evidenced by results of ocular distribution studies and the observation that the unilateral instillation of 0.5% MK-927 into the contralateral eye was devoid of effect on the untreated, hypertensive eye of alpha-chymotrypsinized rabbits. MK-927 has been selected for topical evaluation in glaucoma patients.

L-662,583 is a topically effective ocular hypotensive carbonic anhydrase inhibitor in experimental animals.

1. L-662,583 was a potent inhibitor in vitro of purified, human erythrocyte carbonic anhydrase II, possessing an IC50 of 0.7 nM. The IC50 values for MK-927, acetazolamide and methazolamide were 13.0 nM, 10.8 nM and 21.2 nM, respectively. 2. A 1 h pretreatment with one 50 microliters drop of a 0.1% solution of L-662,583 blocked carbonic anhydrase activity in a homogenate of the iris + ciliary body of albino rabbits by 63%. Similar treatment with 0.1% suspensions of acetazolamide and methazolamide elicited inhibitions of 30% and 20%, respectively. This ex vivo model indirectly assesses the ability of an agent to enter the rabbit eye. 3. Concentrations of L-662,583 in the cornea, aqueous humour and iris + ciliary body of albino rabbits were determined by h.p.l.c. at predetermined times after the instillation (one drop of 50 microliters) of a 2% solution of L-662,583. Peak levels for cornea (47.4 micrograms g-1), aqueous humour (4.51 micrograms ml-1) and iris + ciliary body (9.61 micrograms g-1) occurred at 0.5, 2 and 1 h after instillation, respectively. 4. The experimentally elevated intraocular pressure of the right eye of rabbits, induced by prior intraocular injection of alpha-chymotrypsin, was maximally decreased by 4.5 mmHg, 6.2 mmHg and 9.8 mmHg after the instillation (one drop of 50 microliters) of 0.01%, 0.1% and 0.5% solutions of L-662,583, respectively. All three concentrations lowered intraocular pressure at all time points from 1 h up to and including 5 h, the last recorded time point. The unilateral instillation of L-662,583 (0.5%) into the contralateral, left eye failed to lower the elevated intraocular pressure of the untreated, right eye. This finding indicates that the site of action of topically applied L-662,583 in this paradigm is local. The ocular normotensive, albino rabbit was much less susceptible than the ocular hypertensive rabbit to the intraocular pressure lowering effect of topically applied L-662,583, with a 2% solution maximally decreasing intraocular pressure by 2.3 mmHg. 5. Unilateral ocular hypertension was elicited in the right eye of sedated, cynomolgus monkeys by argon laser-induced photocoagulation of the trabecular meshwork. The instillation (one drop of 50 microL) of L-662, 583 (2%) significantly lowered the elevated intraocular pressure of the right eye at all time points from 1 h up to and including 5 h. The maximum decline was 8.3 mmHg at 3 h and this represented a reduction of 23% from the corresponding baseline value of 36.8 mmHg. The intraocular pressure of the hypertensive, right eye was maximally decreased by 4.1 mmHg and 4.8 mmHg after the instillation of 0.5% and 1% solutions of L-662,583, respectively. Like the rabbit, the normotensive eye of cynomolgus monkeys was more resistant than the hypertensive eye to the ocular hypotensive action of L-662, 583, as indicated by the inability of 0.5% and 1% solutions of the agent to lower intraocular pressure. L-662,583 (2%) maximally reduced the intraocular pressure of normotensive monkey eyes by 2.4 mmHg at 2 h. 6. L-662,583 is structurally different from MK-927, a carbonic anhydrase inhibitor that lowers the intraocular pressure of glaucoma patients following the instillation of a 2% solution. These preclinical observations indicate that L-662,583, like MK-927, is a water-soluble carbonic anhydrase inhibitor which, on topical administration, lowers intraocular pressure by virtue of an action confined to within the eye.

Angiotensin II receptors labelled with 125I-[Sar1, Ile8]-AII in albino rabbit ocular tissues.

High affinity binding sites for the angiotensin II antagonist 125I-[Sar1,Ile8]-AII have been identified and characterized in membrane suspensions of ocular tissues of albino rabbits. Scatchard analysis of the binding indicated a single class of sites with Kd values of 186, 92, 152, 50, 102 pM for the iris + ciliary body, choroid, ciliary process, retina and cornea, respectively. The corresponding concentrations of binding sites were 22, 68, 35, 22 and 4 fmole/mg of protein. The order of potency for several AII analogs to compete with 125I-[Sar1,Ile8]-AII at its binding sites in iris + ciliary body membranes ([Sar1,Leu8]-AII = [Sar1,Ile8]-AII greater than AII = [Sar1, Ala8]-AII greater than AIII greater than AI) resembled the order of potency found for AII receptors in other tissues. The competition curves for this tissue using AII and AIII were best explained by the existence of two populations of binding sites. The addition of the guanine nucleotide, GppNHp, to the assay resulted in a 6.7-fold and 2.3-fold decrease in the respective affinities of AII and AIII for 125I-[Sar1,Ile8]-AII binding sites without a change in the slope of the competition curves. The GppNHp-induced effect was also observed in ciliary process membranes but not in retinal or choroidal membranes. These results indicate the presence of AII receptors regulated by a GTP-binding protein in both the ciliary process and the iris + ciliary body of the rabbit. They also suggest a difference in the guanine nucleotide regulation of AII receptors in different ocular tissues.

Muscarinic receptors of the albino rabbit ciliary process.

Muscarinic receptor binding sites were identified in membranes prepared from albino rabbit ciliary processes, using the muscarinic antagonist [3H]L-quinuclidinyl benzylate as the radioligand. Analysis of saturation binding experiments demonstrated that [3H]L-quinuclidinyl benzylate bound to an apparent homogeneous population of binding sites with a Kd value of 6.4 pm and a Bmax value of 155 fmol mg-1 protein. Seventy percent (70%) of binding sites showed high affinity for pirenzepine, i.e. belonged to the M1 subtype. In contrast, AF-DX 116 was unable to discriminate between subtypes of muscarinic binding sites in this tissue. Carbachol caused a dose-dependent increase in phosphatidylinositol turnover (EC50 = 154 microM) in ciliary processes. A maximum stimulation of 652% of basal activity was obtained following a 45 min incubation with 10 mM carbachol. The potency of muscarinic antagonists to block the carbachol-induced response was comparable to that found for M1 receptors in other tissues. Oxotremorine and pilocarpine behaved like partial agonists in this assay. The carbachol-induced increase in phosphatidylinositol turnover was also observed in a suspension of epithelial cells from ciliary processes and it was blocked by atropine; thus, indicating the presence of muscarinic receptors functionally coupled to phosphatidylinositol turnover in these cells.