Dorzolamide targets carbonic anhydrase (CA), a zinc-containing enzyme that catalyzes the reversible hydration of carbon dioxide to carbonic acid. The compound is a potent inhibitor of carbonic anhydrase II (CA-II) with an IC50 of 0.18 nM for red blood cell CA-II, and exhibits significantly lower potency against CA-I (IC50 = 600 nM). Carbonic anhydrase II is the primary isoenzyme involved in aqueous humor production in the ciliary body of the eye. By inhibiting CA-II, dorzolamide reduces the formation of bicarbonate ions, thereby decreasing fluid secretion and lowering intraocular pressure.

Dorzolamide blocks Component A, which is generated by CO2 influx and hydration by CA-II, in a dose-dependent manner with an IC50 of 2.4 μM (95% confidence interval: 0.5-10.85 μM) for 50% inhibitory concentration [2].
In vitro studies demonstrate that dorzolamide is a potent carbonic anhydrase II inhibitor with IC50 values of 0.18 nM for CA-II and 600 nM for CA-I. The compound blocks component A (caused by an inward flux of CO2 and its subsequent hydration by CA-II) in a dose-dependent manner with an IC50 of 2.4 μM (95% confidence interval: 0.5-10.85 μM). Dorzolamide has demonstrated anti-tumor activity in preclinical studies. The compound shows selectivity for CA-II over other CA isoenzymes, making it suitable for targeted therapy of conditions involving CA-II.

In an EAC solid tumor model, dorzolamide (3, 10, or 30 mg/kg/day, intraperitoneally) has anticancer efficacy when combined with mitomycin C. The computed ratios (relative values 57.3±1, 25.5±1.8, and 24.3±0.7%, respectively) decline with dorzolamide dosage [3].
In vivo studies show that dorzolamide (3, 10, or 30 mg/kg/day, IP) synergizes with mitomycin C to exhibit anti-tumor activity in Ehrlich ascites carcinoma (EAC) solid tumor models. The compound produces a dose-dependent decrease in the calculated ratio (relative values of 57.3±1, 25.5±1.8, and 24.3±0.7%, respectively). In animal models, dorzolamide upregulates TXNIP and p53 while downregulating Bcl-2. In clinical use, dorzolamide effectively lowers intraocular pressure in patients with open-angle glaucoma and ocular hypertension. It can be used alone or in combination with timolol for patients insufficiently responsive to beta-blockers.

The in vitro enzyme assay for dorzolamide involves measuring carbonic anhydrase activity using the CO2 hydration or p-nitrophenyl acetate (pNPA) esterase assay. Enzyme (CA-I or CA-II) is incubated with varying concentrations of dorzolamide (0.01 nM-10 μM) and substrate. For the CO2 hydration assay, the reaction is monitored spectrophotometrically using a pH indicator. For the pNPA assay, the release of p-nitrophenol is measured at 405 nm. IC50 values are determined from dose-response curves using non-linear regression analysis. Inhibition constants (Ki) are calculated using the appropriate kinetic models. The compound's selectivity for CA-II over CA-I is confirmed by comparing IC50 values.

In vitro cellular assays for dorzolamide typically use human trabecular meshwork cells, ciliary body epithelial cells, or cancer cell lines. Cells are cultured in appropriate media and treated with the compound at concentrations of 0.1-100 μM for 24-72 hours. Carbonic anhydrase activity in cell lysates is measured using the pNPA or CO2 hydration assay. Cell viability is assessed using MTT or CCK-8 assays. For anti-tumor studies, apoptosis is evaluated using caspase-3/7 activity assays and Annexin V staining. Gene expression changes (TXNIP, p53, Bcl-2) are analyzed by qPCR and Western blot.

Animal/Disease Models: Female Swiss albino mouse (EAC solid tumor) [3].
Doses: 3, 10 or 30 mg/kg/day (with mitomycin C).
Doses: IP, one time/day for 3 weeks.
Experimental Results: TXNIP and p53 were up-regulated, and bcl-2 was down-regulated. Effectively delays the growth of EAC in mice.
In vivo animal studies for dorzolamide involve female Swiss albino mice bearing EAC solid tumors. The compound is administered intraperitoneally at doses of 3, 10, or 30 mg/kg/day (in combination with mitomycin C) for 3 weeks. Tumor volume is measured regularly and tumor weight is determined at necropsy. Histological analysis is performed on tumor tissues. For glaucoma studies, animal models of ocular hypertension are used. Intraocular pressure is measured using a tonometer before and after treatment. The compound's effect on aqueous humor production is assessed by fluorophotometry.

Absorption, Distribution and Excretion
Animal studies have shown that dazolamide readily penetrates the eye. Following ophthalmic administration, dazolamide is absorbed via the cornea and stroma. Systemic absorption of dazolamide has been reported following topical administration. A study in healthy subjects evaluated systemic exposure to dazolamide after long-term administration. Subjects received 2 mg of dazolamide orally twice daily, equivalent to three times daily use of 2% dazolamide eye drops. Steady-state was reached within 8 weeks after 20 weeks of treatment. Dazolamide is primarily excreted unchanged in the urine; however, N-desethyldazolamide can also be detected in urine. Limited information is available regarding the volume of distribution of dazolamide; however, plasma concentrations of dazolamide and its major metabolites are typically below the limit of quantitation (15 nM). With prolonged use, dazolamide accumulates within erythrocytes (RBCs) due to its binding to carbonic anhydrase II (CA-II). Limited information is available regarding the clearance rate of dazolamide.

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Metabolism/Metabolites
Dzozolamine is slowly metabolized to N-deethyldzozolamine, which has weaker pharmacological activity against CA-II but some inhibitory effect on CA-I. Like the parent drug, N-deethyldzozolamine is also stored in erythrocytes and binds to CA-I. An in vitro study using Sprague-Dawley rat liver microsomes showed that CYP2B1, CYP2E1, and CYP3A2 are involved in the metabolism of dzozolamine in rat liver.

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Biological Half-Life
After drug administration is discontinued, dzozolamine stored in erythrocytes is cleared from erythrocytes in a non-linear manner, with a terminal elimination half-life in erythrocytes ≥120 days. After an initial rapid decline in drug concentration, a slow elimination phase begins, in which the elimination half-life is approximately >4 months.
Pharmacokinetic properties of dorzolamide indicate good tissue penetration when applied topically to the eye. The compound has a molecular weight of 324.44 and is a sulfonamide derivative. It is typically administered as an ophthalmic solution (2% dorzolamide hydrochloride). Following topical application, dorzolamide is absorbed through the cornea and reaches the ciliary body where it inhibits carbonic anhydrase. Systemic absorption is minimal but detectable. The compound is partially metabolized in the liver and excreted in urine. The duration of action is approximately 8-12 hours after a single dose, and the compound is typically administered three times daily for optimal intraocular pressure control.

Effects During Pregnancy and Lactation
◉ Overview of Medication Use During Lactation
Limited experience with dzodamine eye drops suggests it is unlikely to have adverse effects on breastfed infants. French guidelines recommend that carbonic anhydrase inhibitor eye drops be the first-line treatment for glaucoma during lactation. To significantly reduce the amount of medication entering breast milk after using eye drops, press the tear duct near the corner of the eye with your finger for at least 1 minute, then blot away any excess medication with absorbent tissue. ◉ Effects on Breastfed Infants
A newborn was breastfed while the mother received different combinations of ophthalmic timolol, dipinevelnine, dzodamine, brimonidine, and several doses of acetazolamide. Ultimately, the mother received 0.5% timolol gel solution and 2% dzodamine eye drops. The medication was administered immediately after breastfeeding, and punctal occlusion was performed; no apnea or bradycardia was observed in the infant.
◉ Effects on lactation and breast milk
As of the revision date, no relevant published information was found.

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Protein binding
Dolzodamine binds to plasma proteins at approximately 33%.
Toxicological data for dorzolamide indicates it is generally well-tolerated for ophthalmic use. Common adverse effects include ocular burning, stinging, blurred vision, and taste disturbances (bitter taste). Systemic side effects are rare but may include headache, nausea, and fatigue. The compound is contraindicated in patients with sulfonamide hypersensitivity, severe renal impairment, and metabolic acidosis. For research applications, standard safety precautions should be followed. The compound is for research and clinical use under medical supervision.

[1]. Whole-blood pharmacokinetics and metabolic effects of the topical carbonic anhydrase inhibitor dorzolamide. Eur J Clin Pharmacol. 1995;47(5):455-60.

[2]. Inhibition of carbonic anhydrase activity in cultured bovine corneal endothelial cells by dorzolamide. Invest Ophthalmol Vis Sci. 2002 Oct;43(10):3273-8.

[3]. Dorzolamide synergizes the antitumor activity of mitomycin C against Ehrlich's carcinoma grown in mice: role of thioredoxin-interacting protein. Naunyn Schmiedebergs Arch Pharmacol. 2015 Dec;388(12):1271-82.

Dorzolamide is a 5,6-dihydro-4H-thieno[2,3-b]thienopyran-2-sulfonamide 7,7-dioxide, in which the hydrogen atoms at positions 4 and 6 are replaced by ethylamino and methyl groups, respectively (4S, trans configuration). It is a carbonic anhydrase inhibitor, used in ophthalmic solutions as a hydrochloride salt to lower intraocular pressure and treat open-angle glaucoma and ocular hypertension. It has EC 4.2.1.1 (carbonic anhydrase) inhibitor, antihypertensive, and antiglaucoma-preventive effects. It is a sulfonamide compound belonging to the thiophene class of compounds. Dorzolamide is a non-bacterial sulfonamide derivative and a topical carbonic anhydrase (CA) inhibitor used to treat elevated intraocular pressure associated with open-angle glaucoma and ocular hypertension. Dorzolamide works by blocking an enzyme in the ciliary process that regulates ion balance and fluid pressure within the eye. Unlike oral carbonic anhydrase inhibitors, dorzodamine has minimal effects on acid-base balance or electrolyte disturbances and other systemic adverse reactions. Dorzodamine was first marketed in 1995 as a single-drug eye drop (brand name Trusopt) or in combination with timolol (brand name Cosopt PF). Dorzodamine is a carbonic anhydrase inhibitor. Its mechanism of action is as a carbonic anhydrase inhibitor. Dorzodamine is an inhibitor of carbonic anhydrase, a zinc-containing enzyme that catalyzes the rapid conversion of carbon dioxide and water into carbonic acid, protons, and bicarbonate ions. Various carbonic anhydrases are distributed in many cells and tissues and play important roles in mineral and metabolic homeostasis. (NCI04) See also: Dorzodamine hydrochloride (salt form).

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Drug Indications
Dolzodamine is indicated for the treatment of elevated intraocular pressure in patients with ocular hypertension or open-angle glaucoma. For patients with an inadequate response to ocular beta-blockers, dazolamide can also be used in combination with timolol for the same indication. Furthermore, the efficacy of preoperative dazolamide in preventing increased intraocular pressure (IOP) after Nd:YAG laser-assisted posterior capsulotomy has been investigated.
FDA Label

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Mechanism of Action
Elevated IOP is a characteristic manifestation of high intraocular pressure or open-angle glaucoma. IOP levels depend on the balance between aqueous humor production (generated by the ciliary processes) and outflow of aqueous humor from the anterior segment via the trabecular meshwork (conventional pathway) or the uveal-scleral meshwork (non-conventional pathway). IOP increases when resistance to aqueous humor outflow via the trabecular meshwork increases. Subsequently, optic nerve damage can occur due to restricted blood flow and mechanical deformation of ocular structures. Optic nerve damage can further lead to optic disc cupping and progressive visual field defects (and in some cases, even blindness). Carbonic anhydrase (CA) is a ubiquitous enzyme that catalyzes the reversible hydration of carbon dioxide to bicarbonate ions and the dehydration of carbonic acid to carbonic acid. In the ciliary process, bicarbonate ions produced locally by carbonic anhydrase promote the transport of sodium ions and fluids. Carbonic anhydrase II (CA-II) is a key isoenzyme mainly found in erythrocytes (RBCs) and is responsible for regulating aqueous humor production. Dozolem is a highly specific CA-II inhibitor with an affinity for carbonic anhydrase II that is 4000 times higher than for carbonic anhydrase I. Inhibition of CA-II in the ciliary process disrupts bicarbonate ion production and reduces sodium ion and fluid transport, leading to decreased aqueous humor secretion and lower intraocular pressure.

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Pharmacodynamics
Dozolem is a carbonic anhydrase inhibitor that lowers intraocular pressure in patients with open-angle glaucoma or high intraocular pressure. When used in combination with topical β-adrenergic antagonists, dozolem has an additive intraocular pressure-lowering effect. The peak intraocular pressure-lowering effect of dozolem is observed approximately 2 hours after ocular administration.
Dorzolamide (also known as L671152, MK507) is a potent carbonic anhydrase II inhibitor with CAS number 120279-96-1. Its molecular formula is C10H16N2O4S3 and molecular weight is 324.44. The compound is FDA-approved for the treatment of elevated intraocular pressure in patients with ocular hypertension and open-angle glaucoma. It is administered as a 2% ophthalmic solution three times daily. The mechanism of action involves inhibition of carbonic anhydrase II in the ciliary body, reducing aqueous humor production and lowering intraocular pressure. The compound is also being investigated for anti-tumor activity.

C10H16N2O4S3

324.44

324.027

120279-96-1

Dorzolamide hydrochloride;130693-82-2;Dorzolamide-d5;1227097-70-2

5284549

Typically exists as solid at room temperature

1.53 g/cm3

575.8ºC at 760 mmHg

302ºC

1.626

4.666

2

7

3

19

534

2

O=S(C(S1)=CC2=C1S([C@@H](C)C[C@@H]2NCC)(=O)=O)(N)=O

IAVUPMFITXYVAF-XPUUQOCRSA-N

InChI=1S/C10H16N2O4S3/c1-3-12-8-4-6(2)18(13,14)10-7(8)5-9(17-10)19(11,15)16/h5-6,8,12H,3-4H2,1-2H3,(H2,11,15,16)/t6-,8-/m0/s1

(4S,6S)-4-(ethylamino)-6-methyl-7,7-dioxo-5,6-dihydro-4H-thieno[2,3-b]thiopyran-2-sulfonamide

L671152MK507 L671152 MK507 Trusopt UNII-9JDX055TW1 UNII9JDX055TW1 UNII 9JDX055TW1

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)