Therapy for glaucoma is generally individualized for each glaucoma patient, with several factors, including intraocular pressure (IOP) and the degree of optic nerve damage, given consideration. In the treatment of glaucoma, several types of agents such as beta-blockers, prostaglandin analogues and carbonic anhydrase (CA) inhibitors are clinically used at present in pharmacotherapy.1-2) Beta-blockers or prostaglandin F2-alpha analogues have been shown to be effective ocular hypotensive agents when used alone in ocular hypertensive or open-angle glaucoma patients.3,4) These two types of agents are currently recognized as the first-line medications for treating glaucoma.5) However, mono-therapy with timolol, a beta-blocker, or latanoprost, a prostaglandin analogue, is often insufficient to maintain a desirable IOP over the long term in many patients. 6) In these cases, CA inhibitors are often used as an adjunctive therapy to timolol or to latanoprost in order to achieve a desirable IOP. 7) Although it has been shown that monotherapy with timolol results in a smaller IOP reduction than monotherapy with latanoprost, the combination therapies of either timolol or latanoprost with CA inhibitors show comparable IOP reduction activity in glaucoma patients. 8) These clinical observations suggest that there exist different interactions between CA inhibitors and latanoprost or timolol that affect IOP reduction.In our previous paper, 9) latanoprost was shown in an in vitro study to be a weak noncompetitive inhibitor of human carbonic anhydrase (HCA) I and II and to bind to the zinc ion, which plays an important role in the expression of enzyme activity according to the AutoDocking simulation method.In the present study, to elucidate the interaction mechanism between timolol and CA inhibitors, the reaction mechanism and binding mode of timolol with respect to CA were investigated by enzyme-kinetic studies and an AutoDocking simulation method. MATERIALS AND METHODS MaterialsHuman CA I and II (HCA I and HCA II) were purchased from Sigma Co., Ltd. Since the purities of HCA I and II were shown to be more than 95% by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), these enzymes were used without further purification. Timolol hemihydrate (formal name:,5-thiadiazol-3-yl}oxy]-2-propanol 1/2H 2 O) was provided by Santen Pharmaceutical Co. Ltd. All other reagents used in this experiment were of analytical grade and were purchased from Wako Pure Chemical Co., Ltd. (Tokyo, Japan).Determination of the Concentration of CO 2 The CO 2 concentration of pure water saturated with CO 2 was measured using a CO 2 electrode (Orion 9502BN) connected to an Orion Ion-meter 720A. The standard solutions of CO 2 were prepared using 0.5-5 ml of 0.1 M NaHCO 3 solutions mixed with 5 ml of 1 M citric acid buffer (pH 4.5) and filled up to 50 ml with pure water.Determination of the Enzyme Activity of Carbonic Anhydrase Determination of the enzyme activity was based on the hydration of CO 2 catalyzed by HCA I and II. The technique used for...

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In the treatment of glaucoma several types of agents such as beta-adrenoceptor blockers, carbonic anhydrase inhibitors and prostaglandin F2alpha analogues are clinically used at present.1) These agents are given alone and/or in combination.2) Beta-adrenoceptor blockers such as timolol have been used most commonly and reduce intraocular pressure (IOP) through the reduction of aqueous humor formation by blocking beta-adrenergic receptors in the ciliary body.3) The inhibition of carbonic anhydrase (CA) in the ciliary body decreases aqueous humor secretion presumably by slowing the formation of bicarbonate ions with a subsequent reduction in sodium and fluid transport. Thus, the inhibition of CA results in a reduction of IOP through the reduction of aqueous humor production and dorzolamide, a CA inhibitor, is used clinically to treat glaucoma. 4)Monotherapy patients treated with dorzolamide, a CA inhibitor, show a smaller intraocular pressure (IOP) reduction than those treated with timolol, a beta-adrenergic blocker. 5)This suggests that beta-adrenergic blockers have stronger effect for IOP reduction than CA inhibitors. Latanoprost is the only available prostaglandin F2alpha analogue and has been shown to be an effective ocular hypotensive agent when used for the treatment of ocular-hypertensive or open-angle glaucoma patients. 6,7) In spite of the weaker IOP reduction activity of dorzolamide compared to timolol in monotharapy, the combination treatment of dorzolamide with latanoprost showed a comparable IOP reduction activity with the combination use of timolol and latanoprost.8) Puscas and Coltau have shown that prostaglandin F2alpha, enhances CA activity, resulting in an elevation of blood pressure.9) Therefore, latanoprost is supposed to have some influence on CA activity in the eyes. CA is an enzyme found in many tissues of the body.10) It catalyzes the reversible reaction involving the hydration of carbon dioxide and the dehydration of carbonic acid. There exist a number of isoenzymes of CA, and the most active one is CA II (CA-II) in humans, found primarily in red blood cells but also in other tissues. In eye tissue, types I and II of CA exist. 10)Latanoprost is an isopropyl ester of latanoprost free acid, a form of pro-drug, which is enzymatically converted to latanoprost free acid by endogenous esterase. The free acid is a 200 times more potent ligand than latanoprost for the human recombinant prostaglandin F (FP) receptor. The free acid acts as a selective prostanoid FP receptor agonist which reduces IOP by increasing the outflow of aqueous humour.12) On the other hand, it has been shown that the aqueous inflow is not affected. 13)The structure of latanoprost free acid, which is an effective ocular hypotensive agent, is very similar to that of prostaglandin F2alpha. The purpose of this report is to clarify the effect and mechanism of latanoprost free acid on the hydration activity of HCA I or II in an in vitro study. In our previous paper, we showed that docking-simulation of the ligand molecule with the e...

We conducted a study of the annual cost of various ophthalmic products used in Japan for treating glaucoma including six of brands and generic ocular beta-adrenergic blockers (38 products). The total number of drops in one bottle of each solution was counted drop by drop. The cost per drop was calculated by dividing the government-controlled standard prices by the total number of drops in one bottle. The annual cost of ophthalmic solution was calculated by multiplying the cost per drop by the number of drops typically used per day. The total number of drops of the ophthalmic solutions in one bottle ranged from 108 to 168. The yearly cost of the beta-adrenergic blockers studied ranged widely, from ¥5392 to ¥27236. DiŠerences in the total number of drops and the usage eŠect on the annual cost of ophthalmic solutions were found. The annual cost depended on not only the price of the products but also on the total number of drops in one bottle and the usage. Annual cost data may be helpful in selecting ophthalmic products for treating glaucoma in Japan.

Alkaloids U 0600 Diastereoselective Fischer-Type Pyrroloindole Synthesis and Its Application to the Synthesis of Chiral Pyrroloindole Alkaloids. -The facile preparation of optically active pyrroloindoles by diastereoselective Fischer-type pyrroloindole synthesis and short-step conversions of the cycloadducts to pyrroloindole alkaloids [cf. (XII), (XV)] are described. -(NISHIDA*, A.; USHIGOME, S.; SUGIMOTO, A.; ARAI, S.; Heterocycles 66 (2005) 181-185; Grad. Sch. Pharm. Sci., Chiba Univ., Inage, Chiba 263, Japan; Eng.) -H. Toeppel 21-192