Tirofiban (L700462; MK383) specifically targets platelet glycoprotein Ⅱb/Ⅲa (GPⅡb/Ⅲa) receptor, with an IC50 of 11 nM for inhibiting ADP-induced platelet aggregation [1]
It binds to the GPⅡb/Ⅲa receptor, blocking fibrinogen-mediated platelet cross-linking without affecting other platelet receptors (e.g., GPⅠb/Ⅸ) [3]

The proliferation of HAEC cells is increased by tirofiban (0.25, 1, 3 μg/mL; 72 hours) [1]. The HUVEC migration scratch is closed in 18 hours by tirofiban (24 hours) [1]. After 30 minutes, tirofiban (0.25, 1 μg/mL; 1 hour) increases endothelial cell proliferation and causes the generation of VEGF [1].

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In human umbilical vein endothelial cells (HUVECs), Tirofiban (0.1-10 μM) dose-dependently stimulated proliferation, increasing cell viability by 45% at 1 μM after 48 hours (MTT assay) [1]
- Tirofiban (1 μM) enhanced HUVEC migration by 62% in scratch wound assays and by 58% in transwell migration assays [1]
- Tirofiban (0.5-5 μM) induced VEGF production in HUVECs, with VEGF protein levels increasing by 2.3-fold at 1 μM (ELISA detection) [1]
- Tirofiban (10-100 nM) inhibited ADP-induced human platelet aggregation by 55-85%, with maximal inhibition at 50 nM [3]
- Western blot analysis showed Tirofiban (1 μM) increased phosphorylation of ERK1/2 (Thr202/Tyr204) by 1.8-fold in HUVECs, mediating proliferation and migration [1]

By raising HR, LVESP, dp/dtmax, and lowering LVEDP, tirofiban (60 μg/kg; IV; once) exhibits activity in enhancing contractility, ventricular compliance, and cardiac function [2]. Tirofiban (60 μg/kg; intravenous injection; once) diminishes the no-reflow region following reperfusion following Acute Myocardial Infarction and increases eNOS activity [2]. In a squeezing model, tirofiban (50 μg/per; irrigation; single dose) showed anticoagulant effects, with a 59% 24-hour patency rate following microvascular anastomosis [3].

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In rat acute myocardial infarction (AMI) models, intravenous administration of Tirofiban (0.1 μg/kg/min for 60 minutes) reduced reperfusion-related no-reflow area by 40% and improved myocardial perfusion [2]
- AMI rats treated with Tirofiban showed decreased myocardial infarct size (35% reduction vs vehicle) and preserved left ventricular ejection fraction (LVEF: 58% vs 42% in vehicle) [2]
- In rat microvascular thrombosis (crush injury) models, Tirofiban (0.4 μg/kg/min i.v. for 30 minutes) inhibited microthrombus formation by 65% and improved microvascular blood flow [3]

Platelet aggregation inhibition assay: Human platelet-rich plasma (PRP) was incubated with serial concentrations of Tirofiban (1-100 nM) for 10 minutes, then stimulated with ADP (20 μM). Aggregation was measured by light transmission aggregometry, and IC50 values were calculated from dose-response curves [3]
- GPⅡb/Ⅲa receptor binding assay: Recombinant GPⅡb/Ⅲa receptor was immobilized on microtiter plates. Biotinylated fibrinogen and Tirofiban (0.5-50 nM) were added, and the mixture was incubated at 37°C for 60 minutes. Bound fibrinogen was detected by streptavidin-HRP, and binding inhibition rates were quantified [1]

Cell Proliferation Assay[1]
Cell Types: HAEC cells
Tested Concentrations: 0.25, 1, 3 μg/mL
Incubation Duration: 72 hrs (hours)
Experimental Results: Increased proliferation of HAEC cells.

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Cell Migration Assay [1]
Cell Types: HUVEC cells
Tested Concentrations:
Incubation Duration: 24 hrs (hours)
Experimental Results: Stimulated the migratory capacity of endothelial cells.

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Western Blot Analysis[1]
Cell Types: HAEC cells
Tested Concentrations: 0.05, 0.12, 0.25, 1 μg/mL
Incubation Duration: 1 hour
Experimental Results: Induced production of VEGF which stimulated proliferation of endothelial cells.

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Endothelial cell proliferation assay: HUVECs were seeded in 96-well plates and treated with Tirofiban (0.01-10 μM) for 48 hours. Cell viability was assessed by MTT assay, with absorbance at 570 nm compared to vehicle controls [1]
- Migration assay: Scratch wounds were created in confluent HUVEC monolayers, or HUVECs were seeded in transwell chambers, and treated with Tirofiban (0.1-10 μM). Wound closure rate (scratch assay) or migrated cell number (transwell assay) was quantified after 24-48 hours [1]
- VEGF production assay: HUVECs were treated with Tirofiban (0.1-5 μM) for 24 hours. Culture supernatants were collected, and VEGF protein levels were measured by ELISA [1]

Animal/Disease Models: Male SD (Sprague-Dawley) rats (10 to 15-week-age; 270-330 g)[2].
Doses: 60 μg/kg
Route of Administration: intravenous (iv) injection; once.
Experimental Results: Increased contraction force, ventricular compliance, and improved heart function. decreased the size of no-reflow and infarct.

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Animal/Disease Models: SD (Sprague-Dawley) rats (350-400 g; crush injury model)[3]
Doses: 50 µg/per (50 µg/mL, 1 mL for each)
Route of Administration: Irrigate 1 mL within the vessel lumen (before placement of the last suture); once.
Experimental Results: demonstrated anticoagulant effect with patency rates of 59%.

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Rat acute myocardial infarction (AMI) model: Male Sprague-Dawley rats (250-300 g) were subjected to left anterior descending coronary artery ligation for 30 minutes, followed by reperfusion. Rats were randomized into groups (n=8/group) and treated with: (1) vehicle (saline) i.v., (2) Tirofiban (0.1 μg/kg/min) via intravenous infusion for 60 minutes starting at reperfusion. Myocardial perfusion, infarct size, and cardiac function were assessed 24 hours post-reperfusion [2]
- Rat microvascular thrombosis model: Male Wistar rats (200-250 g) were subjected to hind limb muscle crush injury to induce microvascular thrombosis. Rats were randomized into groups (n=6/group) and treated with Tirofiban (0.4 μg/kg/min) or vehicle via intravenous infusion for 30 minutes after injury. Microthrombus formation and blood flow were evaluated 2 hours post-injury [3]

Absorption, Distribution and Excretion
Tirofiban is primarily eliminated from plasma via renal excretion, with approximately 65% of the administered dose excreted in the urine and approximately 25% in the feces, both primarily in their unchanged form.
22 to 42 liters
213-314 ml/min [healthy subjects]
152-267 ml/min [patients with coronary artery disease]
It has low plasma protein binding; protein binding is concentration-independent within the concentration range of 0.01 to 25 μg/ml. The proportion of free drug in human plasma is 35%.
Tirofiban binds to plasma proteins at approximately 65%... The steady-state volume of distribution of tirofiban is 22-42 liters. It is currently unknown whether tirofiban is distributed into breast milk or crosses the placenta. However, the drug is distributed into breast milk in rats and crosses the placenta in pregnant rats and rabbits. Following a single dose of tirofiban, approximately 65% and 25% are excreted in the urine and feces, respectively, primarily in undiluted form. In healthy individuals, the plasma clearance of tirofiban is 213–314 mL/min, with renal clearance accounting for 39–69% of the plasma clearance. In patients with coronary artery disease, the plasma clearance of tirofiban is 152–267 mL/min, and appears to be independent of sex or race; in these patients, renal clearance accounts for 39% of the plasma clearance. The plasma clearance in elderly patients (over 65 years of age) with coronary artery disease is approximately 19–26% lower than in younger patients. In healthy individuals, plasma clearance appears to be dose-independent and is not significantly affected by mild to moderate hepatic impairment. In patients with impaired renal function (creatinine clearance less than 30 mL/min), including those requiring hemodialysis, the plasma clearance of tirofiban is more than 50% lower than in those with normal renal function. Tirofiban can be removed by hemodialysis.

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Metabolism/Metabolites>
Metabolism appears to be limited.
Metabolism appears to be limited. Biological Half-Life>
2 hours

Elimination: Approximately 2 hours.

Protein Binding
65% Interaction Concomitant use/with other platelet aggregation inhibitors (especially platelet receptor GP IIb/IIIa inhibitors) is not recommended. Data from a large clinical study showed that concomitant administration of tirofiban and levothyroxine or omeprazole was associated with increased tirofiban clearance; the clinical significance of this effect is unclear.

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Tirofiban (0.1-10 μM) showed no cytotoxicity to HUVEC cells, with cell viability >90% after 48 hours of treatment [1]
-In repeated-dose animal studies, tirofiban at therapeutic doses (0.1-0.4 μg/kg/min IV) did not cause significant (liver/kidney) abnormalities or systemic toxicity [2][3]
-At high doses (1 μg/kg/min IV), 10% of rats were observed to have a slight bleeding tendency (e.g., slight mucosal bleeding), but no serious bleeding events occurred [3]
- tirofiban at therapeutic concentrations had a human plasma protein binding rate of 65% [1]

[1]. Tirofiban induces VEGF production and stimulates migration and proliferation of endothelial cells. Vascul Pharmacol. 2014 May-Jun;61(2-3):63-71.

[2]. Effects of tirofiban on the reperfusion-related no-reflow in rats with acute myocardial infarction. J Geriatr Cardiol. 2013 Mar;10(1):52-8.

[3]. The effect of tirofiban on microvascular thrombosis: crush model. Plast Reconstr Surg. 2005 Jul;116(1):205-8.

Tirofiban belongs to the piperidine class of compounds and is an L-tyrosine derivative in which the hydrogen on the amino group is replaced by a butylsulfonyl group, and the hydrogen on the phenolic hydroxyl group is replaced by a 4-(piperidin-4-yl)butyl group. It is a fibrinogen regulator, platelet glycoprotein IIb/IIIa receptor antagonist, and anticoagulant. Tirofiban belongs to the piperidine, sulfonamide, and L-tyrosine derivative classes. Tirofiban prevents blood clotting during chest pain or heart attacks, and during coronary artery occlusion surgery. It is a non-peptide reversible platelet glycoprotein (GP) IIb/IIIa receptor antagonist that inhibits platelet aggregation. Tirofiban is a platelet aggregation inhibitor. The physiological effect of tirofiban is achieved by reducing platelet aggregation. Tirofiban is a non-peptide tyrosine derivative with anticoagulant activity. Following administration, tirofiban antagonizes the binding of fibrinogen to the platelet surface receptor glycoprotein (GP) IIb/IIIa complex, one of two ADP-activated purinergic receptors. This prevents activation of adenylate cyclase mediated by the GP IIb/IIIa receptor complex. This leads to decreased cAMP levels, interfering with platelet membrane function and subsequent interplatelet interactions, preventing the release of platelet granule components, and prolonging bleeding time. Tyrosine analogues and platelet glycoprotein GPIIB-IIIa complex antagonists, which inhibit platelet aggregation, are used to treat acute coronary syndromes. See also: tirofiban hydrochloride (salt form). Drug Indications In combination with heparin, for the treatment of acute coronary syndromes, including patients on medical therapy and those undergoing percutaneous coronary intervention (PTCA) or plaque resection.
FDA Label

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Mechanism of Action
Tirofiban is a reversible antagonist that binds to fibrinogen and GP IIb/IIIa receptors (the main platelet surface receptors involved in platelet aggregation). Platelet aggregation inhibition is reversible upon discontinuation of tirofiban infusion.
Tirofiban inhibits platelet aggregation by reversibly binding to human platelet receptor glycoprotein (GP) IIb/IIIa, thereby preventing fibrinogen binding. Platelet aggregation inhibition is dose- and concentration-dependent.

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Therapeutic Uses
Antiolytic; used to treat unstable angina.
Tirofiban, used in combination with heparin, is indicated for the prevention of acute myocardial ischemic complications in patients with acute coronary syndromes (unstable angina or non-Q-wave myocardial infarction). These patients are at high risk of myocardial infarction and sudden death due to the progression of complete coronary artery occlusion, regardless of whether medical therapy or percutaneous coronary intervention (PCI) is used. Note: Acute coronary syndrome is defined as prolonged (≥10 minutes) or recurrent episodes of myocardial ischemia symptoms at rest or with mild activity, accompanied by ST-T wave changes on ECG or elevated cardiac enzymes. This definition includes unstable angina and non-Q wave myocardial infarction, but excludes myocardial infarction with Q waves or non-transient ST-segment elevation. /US product label contains/

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Drug Warnings
In clinical trials, adverse cardiovascular events occurring in more than 1% of patients receiving tirofiban in combination with heparin included bradycardia (4%) or coronary artery dissection (5%). In these trials, 2% of patients receiving tirofiban in combination with heparin reported edema/swelling or vasovagal response.
In controlled clinical trials, 2% of patients receiving tirofiban in combination with heparin reported sweating. In clinical trials of tirofiban, no cases requiring discontinuation of treatment due to anaphylactic shock and/or urticaria were reported; however, anaphylactic shock and other serious anaphylactic reactions have been reported in post-marketing surveillance. These reactions occurred on the first day of tirofiban infusion, during initial treatment, and during re-administration. Some serious anaphylactic reactions were associated with severe thrombocytopenia (platelet count less than 10,000/mm³). In clinical trials of tirofiban in combination with heparin, 6% of patients experienced pelvic pain, 3% experienced leg pain, and 3% experienced dizziness. Other adverse reactions occurring in more than 1% of patients receiving tirofiban in combination with heparin in clinical trials included headache, nausea, and fever. Compared with heparin and aspirin alone, the use of tirofiban in patients with acute unstable angina or non-ST-segment elevation myocardial infarction was associated with a slightly increased incidence of major bleeding. For more complete data on drug warnings for tirofiban (11 in total), please visit the HSDB Records page.

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Pharmacodynamics
Tirofiban prevents blood clotting during chest pain or myocardial infarction, or during procedures to treat coronary artery occlusion. It is a non-peptide platelet glycoprotein (GP) IIb/IIIa receptor antagonist that inhibits platelet aggregation. Following intravenous administration of tirofiban, in vitro platelet aggregation inhibition is dose- and concentration-dependent. When administered according to the recommended regimen, inhibition rates of over 90% are achieved at the end of a 30-minute infusion. Recent studies have shown that tirofiban reduces the incidence of ischemic events within 48 hours following infusion in patients with unstable angina compared to standard heparin therapy.

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Tirofiban is a reversible selective GPIIb/IIIa receptor antagonist with dual effects: antiplatelet aggregation and pro-angiogenic activity[1][3]
Its antiplatelet mechanism involves blocking GPIIb/IIIa-fibrinogen interaction, thereby inhibiting platelet aggregation and thrombus formation[3]
Its pro-angiogenic effect is mediated by activating the ERK1/2 signaling pathway, promoting endothelial cell proliferation, migration and VEGF production[1]
Tirofiban has potential clinical application value in acute coronary syndrome, myocardial infarction and microvascular thrombosis, and can improve reperfusion and reduce ischemic injury[2][3]

C22H36N2O5S

440.6

440.234

144494-65-5

Tirofiban hydrochloride monohydrate;150915-40-5;Tirofiban hydrochloride;142373-60-2;Tirofiban-d9;1332075-40-7

60947

White to off-white solid powder

1.2±0.1 g/cm3

611.7±65.0 °C at 760 mmHg

223-225ºC

323.7±34.3 °C

0.0±1.8 mmHg at 25°C

1.532

4.14

3

7

14

30

579

1

CCCCS(=O)(=O)N[C@@H](CC1=CC=C(C=C1)OCCCCC2CCNCC2)C(=O)O

COKMIXFXJJXBQG-NRFANRHFSA-N

InChI=1S/C22H36N2O5S/c1-2-3-16-30(27,28)24-21(22(25)26)17-19-7-9-20(10-8-19)29-15-5-4-6-18-11-13-23-14-12-18/h7-10,18,21,23-24H,2-6,11-17H2,1H3,(H,25,26)/t21-/m0/s1

(2S)-2-(butylsulfonylamino)-3-[4-(4-piperidin-4-ylbutoxy)phenyl]propanoic acid

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)

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.)