P2 receptor; CYP2; CYP3; adenosine diphosphate/ADP receptor

Ticlopidine displays activity at apparent Ki,app values of 14 µM against human CD39[1]. Ticlopidine has a Ki value of 127±12 µM, which inhibits recombinant human CD39 expressed in COS-7-cells[1]. Ticlopidine (30 and 150 µM) has an impact on growth rate in the first few days of culture, but its effects diminish in the days that follow[4].

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The effect of Ticlopidine at various concentrations (150, 30, 6 microM), has been studied on cultured endothelial cells derived from human umbilical cord vein. Ticlopidine affects the initial attachment of endothelial cells to artificial substrata and has an inhibitory effect on endothelial cell growth rate which correlates to the concentration of the chemical in the culture medium. These effects are related to a marked reduction of intra- and extracellular fibronectin as evidentiated by immunofluorescence. The drug seems to interfere with the formation of fibronectin filaments from intracellular granules. The reduction of fibronectin availability could affect platelet adhesion to subendothelium as well as endothelial cell repair, and subsequently influence the bleeding time. The inhibition of cell proliferation and its possible effect on the thickness of the vessel wall should be considered as additional mechanisms of action for this substance [4].

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Inhibition of CYP2C9 and 3A4 [3]
The inhibitory effects of ticlopidine on CYP2C9 and 3A4 activities are shown in Figure 1. Ticlopidine inhibited CYP2C9 and 3A4 with IC50 values of 26.0 and 32.3 μmol/L, respectively.

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Rhodamine-123 retention assay [3]
The effects of ticlopidine on the cellular accumulation of rhodamine-123 in MCF-7 and MCF-7/ADR cells are shown in Figure 2. Accumulation of rhodamine-123 was reduced in MCF-7/ADR cells overexpressing P-gp compared to MCF-7 cells lacking P-gp. The relative cellular uptake of rhodamine-123 was comparable between the two conditions at the concentration range of 1–30 μmol/L ticlopidine.

Ticlopidine can effectively prevent losartan metabolism in the intestine and/or liver when given orally in combination with Losartan at a dose of 10 mg/kg. This is indicated by the considerable rise in the AUC (by 65.0%) observed by this combination[3].

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Ticlopidine (10 mg/kg) significantly increased the areas under the plasma concentration-time curves (AUCs) and peak plasma concentration (C(max)) of oral losartan (9 mg/kg), as well as the AUCs of the active metabolite EXP-3174. Ticlopidine (10 mg/kg) did not significantly change the pharmacokinetics of intravenous losartan (3 mg/kg). Ticlopidine inhibited CYP2C9 and 3A4 with IC₅₀ values of 26.0 and 32.3 μmol/L, respectively. The relative cellular uptake of rhodamine-123 was unchanged. Conclusion: The significant increase in the AUC of losartan (9 mg/kg) by ticlopidine (10 mg/kg) could be attributed to the inhibition of CYP2C9- and 3A4-mediated losartan metabolism in small intestine and/or in liver. The inhibition of P-gp in small intestine and reduction of renal elimination of losartan by ticlopidine are unlikely to be causal factors. [3]

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Effect of ticlopidine on the pharmacokinetics of oral losartan [3]
Rats were given oral administration of losartan (9 mg/kg) with and without oral administration of ticlopidine at doses of 4 and 10 mg/kg. The mean arterial plasma concentration-time profiles of losartan are shown in Figure 3. The relevant pharmacokinetic parameters of losartan are listed in Table 1. Absorption of losartan was rapid; losartan was detected in plasma at the first blood sampling time point (5 min) with a rapid tmax (1–2 h) for all rats studied (Figure 3). The AUC and Cmax of losartan after oral administration with losartan and 10 mg/kg ticlopidine were significantly greater (by 65.0% and 49.4%, respectively) than those of control rats. The F of losartan was 64.7%.

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Effect of ticlopidine on the pharmacokinetics of active metabolite EXP-3174 [3]
Rats were given oral administration of losartan (9 mg/kg) with and without oral ticlopidine at doses of 4 and 10 mg/kg. The mean arterial plasma concentration-time profiles of EXP-3174 are shown in Figure 4. The relevant pharmacokinetic parameters of losartan are listed in Table 2. The AUC and Cmax of EXP-3174 after oral administration of losartan with 10 mg/kg ticlopidine were significantly greater than those of control rats (by 41.8% and 36.8%, respectively). The AUC ratio (AUCEXP-3174/AUClosartan) was not significantly decreased (P>0.05) compared to control rats.

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Effect of ticlopidine on the pharmacokinetics of intravenous losartan [3]
Mean arterial plasma concentration-time profiles of losartan following intravenous administration of losartan (3 mg/kg) to rats in the presence or absence of ticlopidine (4 and 10 mg/kg) are shown in Figure 5. The corresponding pharmacokinetic parameters are shown in Table 3. The AUC of losartan was increased by ticlopidine treatment, but was not statistically significant compared to the control. The t1/2 of losartan was also prolonged, but this increase was not significant. The pharmacokinetics of intravenous losartan was not affected by the concurrent use of ticlopidine, in contrast to those of oral losartan. Accordingly, there was enhanced oral bioavailability in the presence of ticlopidine, while there was no significant change in the pharmacokinetics of intravenous losartan. This finding may be due to the inhibition of CYP3A-mediated metabolism of losartan in the small intestine and/or in the liver rather than the reduction of renal elimination of losartan by ticlopidine.

Ticlopidine is an effective inhibitor of platelet aggregation. It is a prodrug that is metabolised to an active form, which blocks the ADP receptor that is involved in GPIIb/IIIa receptor activation leading to platelet aggregation. Ticlopidine is marketed under the brand name Ticlid and is indicated for patients who cannot take aspirin or in whom aspirin has not worked to prevent a thrombotic stroke. The FDA label includes a black-box warning of neutropenia, aplastic anemia, thrombotic thrombocytopenia purpura, and agranulocytosis, so it is necessary to monitor patients' WBC and platelets when they are taking ticlopidine.

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Ticlopidine Hydrochloride is the hydrochloride salt form of ticlopidine, a thienopyridine derivative with anticoagulant property. Ticlopidine hydrochloride irreversibly inhibits adenosine-diphosphate (ADP)-induced platelet-fibrinogen binding by binding to the glycoprotein (GP) IIb/IIIA complex, one of the two purinergic receptors activated by ADP. Inhibition of the receptor activation causes the inhibition of adenylyl cyclase, results in decreased levels of cyclic adenosine monophosphate and thereby interferes with platelet membrane function and subsequent, platelet-platelet interaction, release of platelet granule constituents and prolongation of bleeding time.

Cell Proliferation Assay[4]
Cell Types: Human endothelial cells
Tested Concentrations: 30 and 150 µM
Incubation Duration: 2, 6; 10 days
Experimental Results: Treated cells grow slower if compared with controls and this effect correlates with the concentration of Ticlopidine in the culture medium.

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Rhodamine-123 retention assay [3]
MCF-7/ADR, a doxorubicin-resistant human breast cancer cell line, was used and seeded on 24-well plates at a density of 105 cells. At 80% confluence, the cells were incubated in FBS-free DMEM for 18 h. The culture medium was then changed to Hanks' balanced salt solution, and the cells were incubated at 37 °C for 30 min. After incubating the cells with 20 μmol/L rhodamine-123 in the presence of Ticlopidine (1, 3, 10, and 30 μmol/L) for 90 min, the medium was completely removed. The cells were then washed three times with ice-cold phosphate buffer (pH 7.0) and lysed in lysis buffer. Rhodamine-123 fluorescence was measured in the cell lysates using excitation and emission wavelengths of 480 and 540 nm, respectively. Fluorescence values were normalized to the total protein content of each sample and presented as a ratio compared with control values. Verapamil (100 μmol/L) was used as a positive control.

Animal/Disease Models: Male SD (Sprague-Dawley) rats ( 7-8 weeks old, weighing 270-300 g)[3]
Doses: 4 or 10 mg/kg
Route of Administration: Orally administered 30 min before oral administration of losartan.
Experimental Results: The AUC and Cmax of Losartan after oral administration with Losartan and 10 mg/kg Ticlopidine were Dramatically greater (by 65.0% and 49.4%, respectively) than those of control rats.

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Oral and intravenous administrations of losartan and Ticlopidine [3]
Losartan was orally or intravenously administrated, whereas ticlopidine was administrated orally. Oral losartan and ticlopidine were dissolved in distilled water (5 and 4 mL, respectively). Intravenous losartan was dissolved in 0.9% NaCl solution (4 mL). The rats were randomly divided into six groups (n=6 each): oral losartan (9 mg/kg) without and with oral ticlopidine (4 or 10 mg/kg); intravenous losartan (3 mg/kg) without and with oral ticlopidine (4 or 10 mg/kg). The rats were fasted for at least 24 h prior to the beginning of the experiments. Each animal was anaesthetized with light ether and the right femoral artery (for blood sampling) or vein (for iv administration of losartan) was cannulated with a polyethylene tube (SP45, ID 0.58 mm, OD 0.96 mm). Ticlopidine was orally administered 30 min before oral administration of losartan. Oral losartan and ticlopidine were administered using a gastric gavage tube. A blood sample (approximately 0.5 mL) was collected into a heparinized tube at 0 (control), 0.017 (end of the infusion), 0.1, 0.25, 0.5, 1, 2, 4, 8, and 12 h after iv administration, and 0, 0.1, 0.25, 0.5, 1, 2, 4, 6, 8, and 12 h after oral administration. Approximately 1 mL of whole blood collected from untreated rats was infused via the femoral artery at 0.25, 1, and 4 h to replace the blood loss due to blood sampling. Each blood sample was centrifuged at 16 810×g for 5 min, and 200 μL of each plasma sample was stored at -40 °C until HPLC analysis of losartan and EXP-3174 was performed.

Absorption, Distribution and Excretion
Absorption rate is greater than 80%. Food can increase absorption by approximately 20%. Ticlopidine is primarily excreted in the urine (60%), with a small amount excreted in the feces (23%). Volume of distribution was not quantified. Ticlopidine clearance was not quantified, but clearance decreases with age. Metabolisms/Metabolites Ticlopidine is primarily metabolized in the liver, with only trace amounts of intact drug detected. At least 20 metabolites have been identified. Known metabolites of ticlopidine include ticlopidine S-oxide and thienodihydropyridinium. Biological Half-Life Following a single 250 mg dose, the half-life is approximately 7.9 hours in subjects aged 20 to 43 years and approximately 12.6 hours in subjects aged 65 to 76 years. After repeated administration (250 mg twice daily), the half-life was approximately 4 days in subjects aged 20 to 43 years and approximately 5 days in subjects aged 65 to 76 years.

Hepatotoxicity
During ticlopidine treatment, approximately 4% of patients experience elevated serum enzymes. These elevations are usually mild, asymptomatic, and rarely require dose adjustment or discontinuation. Ticlopidine has also been associated with clinically significant acute liver injury. Although these reactions are rare, more than 50 cases have been reported in the literature, some of which are severe. Symptoms usually appear within 6 weeks (range 1 to 24 weeks), with fatigue, jaundice, and pruritus as the main manifestations. The most common pattern of elevated liver enzymes is cholestatic (approximately 75%), but mixed or hepatocellular liver enzyme elevations have also been reported. Immune allergic reactions, such as fever, rash, and eosinophilia, may occur but are uncommon and, if they do occur, are usually mild. Autoantibody formation is rare. Liver biopsy typically shows cholestatic hepatitis with mixed cellular infiltration. Most cases are self-limiting and resolve within 1 to 3 months, but some cases present with persistent jaundice or abnormal liver function, including at least one suspected case of vanishing bile duct syndrome, ultimately requiring liver transplantation. Ticlopidine treatment is also associated with aplastic anemia and thrombotic thrombocytopenic purpura (TTP), which can be severe and even fatal; these patients may also have cholestatic liver injury.

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Protein Binding
Ticlopidine binds reversibly (98%) to plasma proteins, primarily serum albumin and lipoproteins. Over a wide concentration range, the binding of ticlopidine to albumin and lipoproteins is unsaturated. Ticlopidine also binds to α1-acid glycoprotein (with a binding rate of approximately 15% or less).

[1]. 2-Substituted thienotetrahydropyridine derivatives: Allosteric ectonucleotidase inhibitors. Arch Pharm (Weinheim). 2021 Dec;354(12):e2100300.

[2]. Ticlopidine-Induced Cholestatic Inflammatory Hepatitis: New Insights into Pathogenetic Mechanisms of Drug-Related Hepatotoxicity.

[3]. Effects of ticlopidine on pharmacokinetics of losartan and its main metabolite EXP-3174 in rats. Acta Pharmacol Sin. 2011 Jul;32(7):967-72.

[4]. The effect of Ticlopidine on human endothelial cells in culture. Thromb Res. 1984 Feb 1;33(3):323-32.

Ticlopidine is a thienopyridine drug, chemically named 4,5,6,7-tetrahydrothieno[3,2-c]pyridine, in which the hydrogen atom bonded to the nitrogen atom is replaced by an o-chlorobenzyl group. It has various pharmacological effects, including fibrinogen regulation, hematologic action, anticoagulant, platelet aggregation inhibitor, and P2Y12 receptor antagonist. Ticlopidine belongs to the thienopyridine class of drugs and is also a monochlorobenzene compound. Ticlopidine is a potent platelet aggregation inhibitor. It is a prodrug that, after being metabolized to its active form, blocks ADP receptors involved in GPIIb/IIIa receptor activation, thereby inhibiting platelet aggregation. Ticlopidine is marketed under the brand name Ticlid and is indicated for patients who cannot take aspirin or whose aspirin treatment is ineffective, for the prevention of thrombotic stroke. The FDA label contains boxed warnings for neutropenia, aplastic anemia, thrombotic thrombocytopenic purpura, and agranulocytosis, therefore patients must have their white blood cell and platelet counts monitored while taking ticlopidine. Ticlopidine is a platelet aggregation inhibitor. Its physiological action is achieved by reducing platelet aggregation. Ticlopidine is a platelet aggregation inhibitor used to reduce the risk of stroke in patients known to have atherosclerosis. Elevated serum enzymes during ticlopidine treatment occur less frequently but are associated with rare, specific, clinically significant cases of acute liver injury. Ticlopidine is a thienopyridine derivative with anticoagulant activity. Ticlopidine inhibits the binding of adenosine diphosphate (ADP) to its platelet receptors. This prevents ADP activation, inhibits the expression of platelet glycoprotein (GP) IIb/IIIa receptors, the binding of fibrinogen to platelet glycoprotein GP IIb-IIIa, and platelet-to-platelet interactions. This can lead to prolonged bleeding time.
A potent platelet aggregation inhibitor commonly used in coronary artery stenting.
See also: Ticlopidine hydrochloride (salt form).
Indications

For the prevention of recurrent thrombotic stroke in patients who have experienced a stroke or stroke precursors and cannot take aspirin or are unresponsive to aspirin.
FDA Label
Mechanism of Action

The active metabolite of ticlopidine prevents adenosine diphosphate (ADP) from binding to its platelet receptors, thereby inhibiting ADP-mediated activation of the glycoprotein GPIIb/IIIa complex. Studies have shown that this inhibition is associated with defects in the mobilization of platelet particles from their storage sites to the adventitia. The GPIIb/IIIa receptor is not directly interfered with. Since the glycoprotein GPIIb/IIIa complex is the main receptor for fibrinogen, impaired activation of this complex prevents fibrinogen from binding to platelets, thereby inhibiting platelet aggregation. The active metabolite of ticlopidine inhibits platelet aggregation induced by other agonists besides ADP by blocking the amplifying effect of released ADP on platelet activation.

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Pharmacodynamics
Ticlopidine is a prodrug that metabolizes to an unidentified metabolite that can act as a platelet aggregation inhibitor. Inhibition of platelet aggregation prolongs bleeding time. At the concentrations reached in vivo, the prodrug form of ticlopidine has no significant activity in vitro.

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Objective: Losartan and the antiplatelet drug ticlopidine can be used in combination for the prevention or treatment of cardiovascular disease. Therefore, this study evaluated the effect of ticlopidine on the pharmacokinetics of losartan and its active metabolite EXP-3174 in rats. Methods: Ticlopidine (4 or 10 mg/kg) was administered orally 30 minutes before losartan (9 mg/kg orally or 3 mg/kg intravenously). The activities of human CYP2C9 and 3A4 were determined using a CYP inhibition assay kit. The activity of P-gp was assessed in MCF-7/ADR cells using the rhodamine-123 retention assay. [3]

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After oral administration of losartan and ticlopidine (10 mg/kg), the AUC of losartan was significantly increased, which is likely due to the inhibition of losartan metabolism in the small intestine and/or liver mediated by CYP2C and 3A subfamilies. The inhibition of P-gp in the small intestine by ticlopidine and the decrease in renal clearance of losartan are unlikely to be pathogenic factors. [3]

C14H14CLNS

263.79

263.053

55142-85-3

Ticlopidine hydrochloride;53885-35-1;Ticlopidine-d4;1246817-49-1

5472

Light yellow to yellow oil

1.3±0.1 g/cm3

367.3±37.0 °C at 760 mmHg

approx. 1 189°C

175.9±26.5 °C

0.0±0.8 mmHg at 25°C

1.638

3.77

0

2

2

17

261

0

PHWBOXQYWZNQIN-UHFFFAOYSA-N

InChI=1S/C14H14ClNS/c15-13-4-2-1-3-11(13)9-16-7-5-14-12(10-16)6-8-17-14/h1-4,6,8H,5,7,9-10H2

5-[(2-chlorophenyl)methyl]-6,7-dihydro-4H-thieno[3,2-c]pyridine

5-(2-Chlorobenzyl)-4,5,6,7-tetrahydrothieno[3,2-c]pyridine; Ticlopidina; Ticlopidinum; Ticlopidin-Puren; PCR 5332; Ticlopidinum [INN-Latin];

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product requires protection from light (avoid light exposure) during transportation and storage.

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

DMSO: ≥ 100 mg/mL (379.09 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (9.48 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
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.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (9.48 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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