Timolol can significantly decrease the increase in myocardial lipid peroxidation levels in diabetic rats. Timolol can create a well-balanced ratio between oxidative stress and antioxidant defense systems in diabetic mice, and has an essential cardioprotective effect on diabetes-induced ERS and associated apoptosis [3].

Timolol inhibits the ERS response in diabetic rats, which has cardioprotective effects [3].

Animal/Disease Models: Experimental diabetes model: 3-month-old male Wistar rat [3].
Doses: 5 mg/kg
Route of Administration: Timolol (5 mg/kg daily for 12 weeks)
Experimental Results: demonstrated cardioprotective effects.

Absorption, Distribution and Excretion
A study in healthy volunteers showed an absorption rate of 78.0 ± 24.5% for ophthalmic eye drops, indicating that caution must be exercised when using this drug as it may be absorbed in large quantities and produce various systemic effects. Another study determined the bioavailability of timolol eye drops in healthy volunteers to be 60%. In most subjects, the peak plasma concentration (Cmax) of timolol was approximately 1.14 ng/ml within 15 minutes after ophthalmic administration. The mean area under the curve (AUC) after intravenous injection was approximately 6.46 ng/ml/h, and after ophthalmic administration, it was approximately 4.78 ng/ml/h. Timolol and its metabolites are primarily excreted in the urine. The distribution dose of timolol is 1.3–1.7 L/kg, and it is distributed in the following tissues: conjunctiva, cornea, iris, sclera, aqueous humor, kidneys, liver, and lungs. A pharmacokinetic study in healthy volunteers yielded a total plasma clearance of 557 ± 61 ml/min for timolol. Another study measured a total clearance of 751.5 ± 90.6 ml/min and a renal clearance of 97.2 ± 10.1 ml/min in healthy volunteers. Systemic absorption of timolol after topical application is not fully elucidated. However, drug absorption appears to still exist, as adverse systemic reactions have been observed after ocular instillation. In a small number of subjects, the mean peak plasma concentrations after twice-daily topical instillation of 0.5% timolol solution were 0.46 ng/ml in the morning and 0.35 ng/ml in the afternoon. In subjects receiving once-daily topical instillation of 0.5% timolol gel ocular solution in the morning, the mean peak plasma concentration was 0.28 ng/ml. After topical application of a 0.25% or 0.5% solution, intraocular pressure typically decreases within 15-30 minutes, peaks within 1-5 hours, and remains elevated for approximately 24 hours. Approximately 90% of oral timolol maleate is rapidly absorbed from the gastrointestinal tract. Food does not impair absorption. Due to extensive metabolism following first-pass metabolism in the liver, only about 50% of the oral dose enters systemic circulation unchanged. Peak plasma concentrations are typically reached within 1-2 hours after oral administration. Significant individual differences in plasma concentrations have been reported even with the same oral dose of timolol. The binding rate of timolol to plasma proteins is 10-60%, depending on the detection method used. The drug is excreted into breast milk. The plasma half-life of timolol is 3-4 hours; this remains largely unchanged in patients with moderate renal impairment. Approximately 80% of timolol is metabolized in the liver to inactive metabolites. Both the unchanged drug and its metabolites are excreted in the urine. Hemodialysis can only remove small amounts of the drug. In six healthy volunteers, the β-receptor blocking and binding activity of timolol was investigated after intravenous administration of 0.25 mg. Plasmokinetics were determined using radioreceptor assay (RRA). Blocking activity was determined by comparing the dose ratio (DR) (I25) of the isoproterenol infusion rate required to increase heart rate by 25 bpm. Binding activity was determined by measuring the extent to which timolol occupied rabbit lung β1 receptors and mouse reticulocyte β2 receptors in undiluted plasma samples. Timolol was eliminated from plasma, with a mean half-life of 2.6 hours during the elimination phase. Timolol effectively antagonized isoproterenol-induced tachycardia for at least 4 hours. The activity correlated very well with the estimated β2 receptor binding activity of timolol in circulating plasma. In conclusion, the plasma clearance of low-dose intravenous timolol was very similar to that of 80-fold higher-dose oral timolol previously reported in the literature. A 0.25 mg dose exhibits considerable systemic β-receptor blocking and binding activity, which may help explain the reported side effects following ocular administration. The binding of circulating β-receptor blockers to rabbit lung β1 receptors and mouse reticulocyte β2 receptors appears to predict the strength and selectivity of their β-receptor blocking effect in healthy volunteers. Metabolism/Metabolites Timolol is primarily metabolized in the liver by the cytochrome P450 2D6 enzyme, with a smaller contribution from CYP2C19. 15–20% of the dose undergoes first-pass metabolism. Although the first-pass metabolism rate of timolol is relatively low, its overall metabolism rate is still as high as 90%. Four metabolites of timolol have been identified, with the hydroxyl metabolite being the most prevalent. Timolol is primarily metabolized in the liver, with only trace amounts of the parent drug detected in the urine. One study investigated the metabolism of timolol maleate in 108 patients with essential hypertension who received a single oral dose of 10 mg timolol maleate. Studies have found that the metabolism of timolol maleate is partially controlled by a debrominated monogene. Individuals with weaker debrominated metabolism had average plasma timolol maleate concentrations twice that of those with stronger metabolism. Timolol maleate/
Timolol's known metabolites include 4-[4-[3-(tert-butylamino)-2-hydroxypropoxy]-1,2,5-thiadiazol-3-yl]morpholin-2-ol.
Biological half-life

In a clinical study of healthy volunteers, the half-life of timolol was 2.9 ± 0.3 hours.
The plasma half-life is 3–5 hours.

Toxicity Summary
Identification: Timolol is a beta-adrenergic receptor blocker, belonging to class II antiarrhythmic drugs. It is a white, odorless powder. It is soluble in water, ethanol, and chloroform; soluble in methanol; practically insoluble in ether. Human Exposure: Major Risks and Target Organs: Beta-blockers exert their effects by competing with endogenous and/or exogenous beta-adrenergic agonists. Timolol is a non-cardiac-selective beta-blocker (with similar affinity for both β1 and β2 receptors) and has no intrinsic sympathomimetic or membrane-stabilizing effects. The major risks are likely atrioventricular block and negative inotropic effects. Clinical Presentation Overview: Only one case of acute poisoning has been reported in a 24-year-old male. The patient presented with moderate poisoning symptoms: drowsiness, dizziness, headache, and first-degree atrioventricular block. He recovered without sequelae after treatment with atropine and isoproterenol. Systemic adverse reactions have been reported in patients using timolol eye drops. Indications: Oral: Timolol has been used to treat hypertension, angina pectoris, arrhythmias, migraines, and to reduce mortality after myocardial infarction. Ophthalmic: Timolol eye drops are used to treat glaucoma to lower intraocular pressure. Contraindications: Timolol is contraindicated in patients with asthma, second- and third-degree atrioventricular block, and cardiogenic shock. Caution should be exercised when using timolol in patients with chronic obstructive pulmonary disease, sinus bradycardia, heart failure, myasthenia gravis, or Raynaud's syndrome. Timolol should not be used concomitantly with other beta-blockers. Route of Administration: Oral: Poisoning may occur after taking timolol tablets, but only one case has been reported so far. Ocular: Systemic toxicity symptoms may occur after using timolol eye drops. Absorption: Oral: Timolol is almost completely (90%) absorbed from the gastrointestinal tract. Peak plasma concentrations occur 0.5–3 hours after administration. Timolol exhibits a moderate first-pass effect. Ocularly: Its intraocular pressure-lowering effect begins 10-20 minutes and lasts for at least 24 hours. Timolol is systemically absorbed. Distribution: Oral: Bioavailability is approximately 60%. Apparent volume of distribution is 1.3-1.7 L/kg. Plasma protein binding is approximately 10%. Timolol crosses the placental barrier. Ocular administration: Timolol is distributed in the conjunctiva, cornea, sclera, iris, aqueous humor, liver, kidneys, and lungs. Transdermal administration: After application to the skin, 50%–60% of timolol ointment is systemically absorbed. Biological half-life of different routes of administration: Oral: The half-life after oral administration is 2.5–5 hours. The half-life varies due to genetic differences in hepatic metabolism: It has been reported that individuals with high metabolic capacity have a half-life of 3.7 hours, while those with low metabolic capacity have a half-life of 7.5 hours. Metabolism: Oral: Timolol is extensively metabolized in the liver via hydrolytic cleavage of the morpholine ring followed by oxidation. After oral administration, 80% is metabolized, and 20% is excreted unchanged in the urine. Metabolism depends on genetic polymorphism. Clearance: Oral: Renal: Approximately 20% of the dose is excreted unchanged in the urine, and 40% to 60% is excreted as metabolites. Breast Milk: Timolol is present in breast milk. The milk/plasma concentration ratio is 0.80 after oral administration to the mother. Ocular: Breast Milk: After ocular instillation, the drug concentration in breast milk is approximately 6 times that in serum. Pharmacology and Toxicology: Mechanism of Action: Toxicological Effects: At toxic doses, timolol may produce significant negative chronotropic and negative inotropic cardiac effects. Pharmacodynamics: The exact mechanism by which timolol lowers intraocular pressure is unknown. The most likely mechanism of action is through reducing aqueous humor secretion. At therapeutic doses, timolol can slightly reduce heart rate, supraventricular conduction, and cardiac output. Adults: Only one case of acute timolol poisoning has been reported; the patient presented with moderate to severe symptoms. Children: An 18-month-old girl developed bradycardia, respiratory depression, and cyanosis 30 minutes after using timolol eye drops. Teratogenicity: No epidemiological studies have been reported on congenital abnormalities in infants born to women treated with timolol during pregnancy. Drug Interactions: Sinus bradycardia has been reported when timolol eye drops are used in combination with quinidine. Clinical Manifestations: Acute Poisoning: Eye Contact: Systemic adverse reactions have been reported after treatment with timolol ophthalmic solutions. Chronic Poisoning: Eye Contact: Dry eye symptoms have been reported in a man treated with 75 mg of timolol daily. Corneal numbness has been reported in a patient treated with timolol eye drops. Systemic description of clinical manifestations: Cardiovascular system: Acute: There have been reports of first-degree atrioventricular block, blood pressure of 120/80 mmHg, and heart rate of 58 bpm after timolol administration. Bradycardia, hypotension, atrioventricular block, and congestive heart failure may occur after timolol use. Respiratory system: Acute: A 62-year-old woman experienced reversible respiratory arrest after using timolol eye drops; respiratory arrest may also occur after oral administration of timolol. Bronchospasm may occur in susceptible patients after timolol use. Nervous system: Central nervous system: Acute: There has been one report of somnolence, dizziness, and headache. Fatigue, confusion, depression, and hallucinations have been reported after timolol use. Peripheral nervous system: Acute: Timolol use may exacerbate myasthenia gravis. Autonomic nervous system: Acute: Effects of beta-blockers. Gastrointestinal tract: Acute: Abdominal pain, nausea, vomiting, and diarrhea may occur after oral or intravenous administration of timolol. Skin: Acute: Urticaria may occur. Eyes, ears, nose, throat: Local effects: Acute: Eyelid erythema and edema have been reported after ocular application. Metabolism: Acid-base imbalance. Fluid and electrolyte disturbances: Hyperkalemia has been reported. Other clinical effects: Sexual dysfunction has been reported after topical application of commonly used doses of timolol eye drops, and may also occur after oral administration. Special risks: Timolol can be excreted in breast milk. There are no epidemiological studies reported on congenital abnormalities in infants born to women who took timolol during pregnancy.

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Hepatotoxicity
Less than 2% of timolol patients experience mild to moderate elevations in serum transaminase levels, which are usually transient and asymptomatic and resolve with continued treatment. Despite the widespread use of timolol, there is no conclusive evidence that it is associated with clinically significant cases of liver injury. Other beta-blockers have been associated with rare cases of acute liver injury, with an incubation period of 2 to 24 weeks, hepatocellular elevation of serum enzymes, mild and spontaneous course, and no evidence of hypersensitivity or autoimmune reactions.
Probability Score: E (Unlikely a cause of clinically apparent acute liver injury).
Pregnancy and Lactation Effects
◉ Overview of Lactation Use
Due to individual variability in timolol secretion in breast milk and limited experience with its use during lactation, alternative medications should be preferred, especially in breastfeeding newborns or premature infants.
The risk to breastfed infants is low when mothers use timolol eye drops, but some guidelines indicate that gel formulations are preferred over solution formulations. To significantly reduce the amount of medication entering breast milk after using eye drops, press the tear duct at the corner of the eye for at least 1 minute, then blot away excess medication with absorbent tissue.
◉ Effects on Breastfed Infants
Currently, there are no relevant reports, but β-adrenergic blockers with similar excretory properties to breast milk have caused adverse reactions in breastfed newborns.
One case report shows that a mother of a 9-week-old breastfed infant used 0.5% timolol eye drops twice daily in one eye without experiencing side effects.
Another mother used 2 drops of 0.5% timolol eye drops daily, along with pilocarpine eye drops twice daily and 250 mg of acetazolamide orally twice daily, and was born prematurely at 36 weeks of gestation. The infant was exclusively breastfed 6 hours after birth and continued for 5 months. On the second day after birth, the infant developed electrolyte disturbances, manifested as hypocalcemia, hypomagnesemia, and metabolic acidosis. The infant received oral calcium gluconate and a single intramuscular injection of magnesium sulfate. Despite continued breastfeeding and ongoing maternal medication, the infant's mild metabolic acidosis resolved by day four postnatally, and weight gain was normal at 1, 3, and 8 months, although mild hypotonia persisted. The authors suggest the metabolic disturbances were caused by transplacental transport of acetazolamide, and these disturbances eventually resolved despite continued breastfeeding. The infant gained weight well during breastfeeding but still had mild residual hypertonia in the lower extremities, requiring physical therapy. A newborn was breastfed while the mother received multiple ophthalmic treatments with timolol, dipivirine, dazolamide, brimonidine, and multiple acetazolamide combinations. Ultimately, the mother received 0.5% timolol gel solution and 2% dazolamide eye drops. The medications were administered immediately after breastfeeding, and punctal occlusion was performed; the infant did not experience apnea or bradycardia.
◉ Effects on Lactation and Breast Milk
As of the revision date, no published information was found regarding the effects of beta-blockers or timolol during normal lactation. A study of 6 patients with hyperprolactinemia and galactorrhea found no change in serum prolactin levels after beta-adrenergic blockade with propranolol.

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Protein Binding
Timolol has low plasma protein binding, estimated at approximately 10%.

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Drug Interactions
The intraocular pressure-lowering effect of timolol maleate may have an additive effect when used in combination with topical miotics, topical dipiformin, topical epinephrine, and/or systemically administered carbonic anhydrase inhibitors. This effect can be used to treat glaucoma. However, the long-term efficacy of combination therapy with beta-adrenergic blockers using adrenergic agonists (e.g., dipiformin, epinephrine) remains to be determined. While topical timolol alone has little effect on pupil size, there are occasional reports of topical timolol combined with epinephrine causing mydriasis. /Timolol maleate/
For patients receiving both systemic β-adrenergic blockers and topical timolol, the additive effect of both on intraocular pressure and/or systemic β-adrenergic blockade should be considered.
When topical timolol is used in combination with catecholamine-depleting drugs (e.g., reserpine), patients should be closely monitored for potential additive effects, as well as hypotension and/or significant bradycardia, which may lead to dizziness, syncope, and/or orthostatic hypotension.
Because reserpine has a catecholamine-depleting effect, the combination of timolol and reserpine may increase the incidence of hypotension and bradycardia compared to timolol alone. Timolol has an additive effect with other antihypertensive drugs (e.g., hydralazine, methyldopa) and may enhance their antihypertensive effects. This effect is often used for treatment, but dosage should be carefully adjusted when these drugs are used concurrently. For more complete data on interactions of timolol (8 types), please visit the HSDB record page. Non-human toxicity values: Female mice: 1190 mg/kg LD50 /timolol maleate/ Female rats: 900 mg/kg LD50 /timolol maleate/

[1]. James Barnes; Majid Moshirfar. Timolol.

[2]. Treatment of open-angle glaucoma and ocular hypertension with preservative-free tafuprost/timolol fxed-dose combination therapy: 6 case reports and clinical outcomes. BMC Ophthalmol. 2022 Apr 2;22(1):152.

[3]. Beta-blocker timolol alleviates hyperglycemia-induced cardiac damage via inhibition of endoplasmic reticulum stress. J Bioenerg Biomembr. 2014 Oct;46(5):377-87.

Therapeutic Uses
Adrenergic beta-blockers; antiarrhythmics; antihypertensives; sympathomimetic drugs. Antihypertensives, antiarrhythmics, antianginals, and antiglaucoma medications. In ophthalmology, topical application of timolol maleate can reduce intraocular pressure elevation caused by various diseases, including open-angle glaucoma, aphakic glaucoma, ocular hypertension, and certain secondary glaucomas. It can reduce or prevent glaucomatous visual field defects or optic nerve damage, and avoid surgery. It lowers intraocular pressure. /Timolol Maleate/ Used to treat hypertension. Timolol can be used alone or in combination with other classes of antihypertensive drugs. In the treatment of hypertension, the efficacy of timolol is similar to other beta-adrenergic blockers. For more complete data on the therapeutic uses of timolol (13 types), please visit the HSDB record page.

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Drug Warning
Patients using topical timolol and systemic β-adrenergic blockers concurrently should be closely monitored for potential additive effects on intraocular pressure and/or the effects of systemic β-adrenergic blockers.
Patients with a history of atopic dermatitis or severe anaphylactic reactions to multiple allergens have been reported to have a more intense reaction to repeated accidental exposure, diagnostic or therapeutic exposure to such allergens while taking β-adrenergic blockers, and may not respond to the usual dose of epinephrine used to treat anaphylactic reactions.
The above situation has been reported when using multi-dose packages of topical ophthalmic timolol. Bacterial keratitis preparations. These containers may be unintentionally contaminated by patients, most of whom also have corneal disease or ocular surface epithelial damage. Patients should be informed that improper handling of ophthalmic solutions can lead to contamination with common bacteria known to cause eye infections, and should be instructed to avoid contact between the tip of the dispensing container and the eye or surrounding tissues. Using contaminated ophthalmic solutions may cause serious eye damage and subsequent vision loss. Furthermore, patients should be advised to consult their doctor immediately regarding continued use of their current multidose container if complications such as eye disease (e.g., trauma, eye surgery, or infection) occur. Because timolol has little effect on pupil size, it should not be used alone in patients with angle-closure glaucoma but should be used in combination with a miotic. It should not be used concurrently with other ophthalmic beta-adrenergic blockers; patients switching from other beta-blockers to timolol should discontinue the other beta-blocker before starting timolol eye drops. For more complete data on timolol (26 total), please visit the HSDB records page.
Pharmacodynamics
Timolol eye drops rapidly lower intraocular pressure. When administered in tablet form, it lowers blood pressure, heart rate, and cardiac output, and reduces sympathetic nerve activity. The drug has a rapid onset of action, usually within 20 minutes of eye drop administration. Timolol maleate at concentrations of 0.5% or 0.25% can exert its pharmacological effects for up to 24 hours.

C13H24N4O3S

316.41966

316.157

26839-75-8

(S)-Timolol Maleate;26921-17-5;(Rac)-Timolol-d5 maleate;1217260-21-3;Timolol hemihydrate;91524-16-2

33624

Typically exists as solid at room temperature

1.224 g/cm3

487.2ºC at 760 mmHg

71.5 - 72.5ºC

248.5ºC

2.62E-10mmHg at 25°C

1.548

0.958

2

8

7

21

310

1

CC(C)(C)NC[C@@H](COC1=NSN=C1N2CCOCC2)O

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.)