P2Y12 Receptor

Tetrasodium Cangeler is the only effective intravenous direct potential adenosine diphosphate (ADP) P2Y12 receptor clamping agent [1]. The hP2Y12 receptor pKb of tetrasodium cangeler is 8.6-9.2 [3].

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Cangrelor is a selective, rapidly reversible P2Y12 platelet receptor inhibitor that directly blocks adenosine diphosphate (ADP)-induced activation and aggregation of platelets and achieves a 90% level of platelet inhibition within five minutes [1].

Cangrelor tetrasodium (10 mg/kg) not only significantly reduces BLM-induced release of inflammatory cytokines (PF4, CD40L, and MPO) but also reduces vasculature, neutrophil damage, and neutrophils in fibrotic lungs and peripheral vessels. Neutrophils damage cell aggregates and increase in BLM-treated blood [2].

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Researchers have reported that cangrelor, a non-sepesific GPR17 antagonist, alleviates pulmonary fibrosis partly by inhibiting macrophage inflammation in mice. Cangrelor is also a well-known anti-platelet agent. To test whether cangrelor mitigated pulmonary fibrosis partly through the inhibition of platelets, bleomycin (BLM) was used to induce pulmonary fibrosis in C57BL/6 J mice. We found that cangrelor (10 mg/kg) not only significantly decreased BLM-induced release of inflammatory cytokines (PF4, CD40 L and MPO), but also decreased the increment of platelets, neutrophils and platelet-neutrophil aggregates in the fibrotic lung and in the peripheral blood of BLM-treated mice. In addition, cangrelor decreased the number of CD40 and MPO double positive neutrophils and the expression level of CD40 in BLM-treated mouse lungs. Based on these results we conclude that cangrelor alleviates BLM-induced lung inflammation and pulmonary fibrosis in mice, partly through inhibition of platelet activation, therefore reducing the infiltration of neutrophils due to the adhesion of platelets and neutrophils mediated by CD40 - CD40 L interaction. Cangrelor could be a potential therapeutic medicine for pulmonary fibrosis. [2]

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Methods Complete Freund's adjuvant (CFA)-induced chronic inflammatory pain was induced in wild-type and P2ry12 gene-deficient (P2ry12-/- ) mice, and the potent, direct-acting and reversible P2Y12 receptor antagonists PSB-0739 and cangrelor were used. Results CFA-induced mechanical hyperalgesia was significantly decreased in P2ry12-/- mice for up to 14 days, and increased neutrophil myeloperoxidase activity and tumor necrosis factor (TNF)-α and CXCL1 (KC) levels in the hind paws were also attenuated in the acute inflammation phase. At day 14, increased interleukin (IL)-1β, IL-6, TNF-α and KC levels were attenuated in P2ry12-/- mice. PSB-0739 and cangrelor reversed hyperalgesia in wild-type mice but had no effect in P2ry12-/- mice, and PSB-0739 was also effective when applied locally. The effects of both local and systemic PSB-0739 were prevented by A-803467, a selective NaV1.8 channel antagonist, suggesting the involvement of NaV1.8 channels in the antihyperalgesic effect. Platelet depletion by anti-mouse CD41 antibody decreased hyperalgesia and attenuated the proinflammatory cytokine response in wild-type but not in P2ry12-/- mice on day 14. Conclusions In conclusion, P2Y12 receptors regulate CFA-induced hyperalgesia and the local inflammatory response, and platelet P2Y12 receptors contribute to these effects in the chronic inflammation phase[3].

Animals and reagents [2]
C57BL/6 J mice (male, 6–8 weeks, 22−25 g) were used. The mice are free access to water and food in air-conditioned rooms (23 °C, relative humidity 50 %) on a 12 h light / dark cycle. Four treatments were performed in these mice: sham-operated control (Con, n = 6), cangrelor (Cang, 10 mg/kg, n = 6), bleomycin + saline (BLM, 3 mg/kg, n = 6) and bleomycin + cangrelor (BLM + Cang 10 mg/kg, n = 6). Cangrelor and bleomycin (Hisun Pharmaceutical Co., Ltd., China) were stored at 4 °C and diluted in saline before use.

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Experimental procedure and cangrelor administration [2]
According to the previous report (Zhan et al., 2018, 2018; Tanaka et al., 2017), pulmonary fibrosis was induced by intratracheal administration of bleomycin (BLM, 3 mg/kg) in C57BL/6 J mice, and cangrelor (10 mg/kg) was administrated via subcutaneous injection. BLM was administrated on day 0, the treatment of cangrelor was started 2 days before BLM administration and lasted for 16 days (once per day). On day 14, the mice were sacrificed by cervical dislocation after the pulmonary resistance was determined. The bronchoalveolar lavage fluid (BALF) was collected from right lung, then the right lung tissues were stored at -80 °C for Western blotting analysis and quantitative reverse-transcription polymerase chain reaction (qRT-PCR). The left lung tissues were fixed in 10 % formaldehyde for histological inspection. The blood and BALF were collected for flow cytometry and ELISA assay.

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Mice were treated with P2Y12R antagonists, or with their vehicle (sterile saline), intraperitoneally ([dichloro‐[[[(2R,3S,4R,5R)‐3,4‐dihydroxy‐5‐[6‐(2‐methylsulfanylethylamino)‐2‐(3,3,3‐trifluoropropylsulfanyl)purin‐9‐yl]oxolan‐2‐yl]methoxy‐hydroxyphosphoryl]‐oxyhydroxyphosphoryl]methyl]phosphonic‐acid, cangrelor , 3 mg kg−1; The Medicines Company, Parsippany, NJ, USA), intraplantarly or intrathecally (1‐amino‐4‐[4‐phenylamino‐3‐sulfophenylamino]‐9,10‐dioxo‐9,10‐dihydroanthracene‐2‐sulfonate, PSB‐0739, 0.3 mg kg−1, selective P2Y12R antagonist synthesized by Y. Baqi and C. E. Müller). on days 3, 4, 7, 10 and 14 after CFA injection. The doses were chosen on the basis of our previous experiments: the pKB values of PSB‐0739 and cangrelor at human P2Y12Rs (hP2Y12Rs) were 9.8 and 8.6, respectively, whereas, in the doses applied in the present study (PSB‐0739, 0.3 mg kg−1 intrathecally; cangrelor , 3 mg kg−1 intraperitoneally), they reversed acute inflammatory pain for up to 96 h. Taking into account that the approximate blood volume of a 25‐mg mice is 1700 μL, these doses correspond to 5 μm and 50 μm, indicating maximal target inhibition. As a reference compound, aspirin, an alternative platelet antagonist, was used at a low dose (2‐acetyloxybenzoic acid, 20 mg kg−1 intraperitoneally). The mechanonociceptive thresholds of hind paws were measured 15 min or 30 min after intrathecal/intraperitoneal or intraplantar injections, with the exception of day 3, when PWT measurements were performed before drug administration. 5‐(4‐chlorophenyl)‐N‐(3,5‐dimethoxyphenyl)‐2‐furancarboxamide (A‐803467, 30 mg kg−1), a potent and selective NaV1.8 sodium channel antagonist or its vehicle (polyethylene glycol and dimethyl sulfoxide [9 : 1]) was administered intraperitoneally 5 min before the respective PSB‐0739/saline injection. The dose of A‐803467 was chosen on the basis of a previous study, and a submaximal dose (30 mg kg−1 intraperitoneally) in the reduction of mechanical allodynia was selected to reveal any additive interactions between PSB‐0739 and A‐803467. In some experiments, paw edema was also volumetrically quantified by plethysmometry (7140; Ugo Basile). [3]

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Assessment of platelet CD62P levels by flow cytometry [3]
To investigate how P2Y12R antagonists and antiplatelet agents administered via different routes altered platelet activation, we measured ADP‐induced changes in platelet CD62P levels ex vivo, in platelet‐rich plasma (PRP) samples. Wild‐type mice were treated with PSB‐0739 (0.3 mg kg−1 intrathecally), cangrelor (3 mg kg−1 intraperitoneally), aspirin (20 mg kg−1 intraperitoneally), or their vehicle. Blood samples were taken directly from the vena cava of anesthetized mice 15 min or 30 min after the treatment. Apyrase (1 U mL−1) was added to the samples to prevent ADP receptor desensitization. After 10 min of centrifugation at 150 × g, PRP was collected. Platelet activation was induced by ADP (500 μm), and changes in platelet CD62P levels were assessed after 60 min of incubation. Platelets were stained with anti‐human/mouse CD62P antibody for 10 min. Samples were acquired with a BD FACSVerse machine, and analyzed with BD facsuite software. Changes in CD62P mean fluorescence intensity values were determined on CD42d‐positive platelets.

Absorption, Distribution and Excretion
Following IV administration of [3H] cangrelor, 58% of radioactivity was recovered in urine. The remaining 35% of radioactivity was in feces, presumably following biliary excretion.
In a study in healthy volunteers administration at a dose of 30 mcg/kg bolus plus 4 mcg/kg/min showed a volume of distribution of 3.9 L.
The mean clearance is about 43.2 L/h.
/MILK/ It is not known whether Kengreal is excreted in human milk.
Following IV administration of 3(H) Kengreal 58% of radioactivity was recovered in urine. The remaining 35% of radioactivity was in feces, presumably following biliary excretion. The average elimination half-life of Kengreal is about 3-6 minutes.
In a study in healthy volunteers, Kengreal administration at a dose of 30 ug/kg bolus plus 4 mcg/kg/min showed a volume of distribution of 3.9 L. Plasma protein binding of Kengreal is about 97-98%.

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Metabolism / Metabolites
Cangrelor is deactivated rapidly in the circulation by dephosphorylation to its primary metabolite, a nucleoside, which has negligible anti-platelet activity. Cangrelor's metabolism is independent of hepatic function and it does not interfere with other drugs metabolized by hepatic enzymes.
Kengreal is deactivated rapidly in the circulation by dephosphorylation to its primary metabolite, a nucleoside, which has negligible anti-platelet activity. Kengreal's metabolism is independent of hepatic function and it does not interfere with other drugs metabolized by hepatic enzymes.

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Biological Half-Life
The average elimination half-life of cangrelor is about 3-6 minutes.
Following IV administration of 3(H) Kengreal, ... elimination half-life of Kengreal is about 3-6 minutes.

Toxicity Summary
IDENTIFICATION AND USE: Cangrelor is a platelet aggregation inhibitor and purinergic P2Y receptor antagonist. HUMAN STUDIES: Cangrelor is a potent intravenous platelet P2Y12 receptor antagonist with rapid onset and offset of action. In patients undergoing percutaneous coronary interventions (PCI), compared with control, cangrelor (30 ug/kg bolus, followed immediately by a 4 ug/kg per minute infusion for 2-4 hr or until the conclusion of the index PCI, whichever was longer) reduces periprocedural thrombotic complications without an increase in major bleeding complications, although minor bleeding is increased. In a large clinical trial program of patients undergoing PCI, cangrelor overdosing was rare and not associated with an increase in bleeding complications, an observation that may be attributed to its very short-half life and rapid offset of action. Platelet P2Y12 receptor expression is significantly increased and the receptor is constitutively activated in patients with type 2 diabetes mellitus, which contributes to platelet hyperactivity and limits antiplatelet drug efficacy in type 2 diabetes mellitus. Cangrelor was non-mutagenic and non-clastogenic in genetic toxicology studies, including chromosome aberration assay in human peripheral lymphocytes. ANIMAL STUDIES: Cangrelor had no significant effect on male or female rats fertility treated for 28 days, or on early embryonic development. In embryo-fetal development studies in rats, cangrelor produced dose-related fetal growth retardation characterized by increased incidences of incomplete ossification and unossified hind limb metatarsals. In rabbits, cangrelor was associated with increased incidences of abortion and intrauterine losses, as well as fetal growth retardation. Cangrelor was non-mutagenic and non-clastogenic in genetic toxicology studies, including in vitro bacterial gene mutation assay, mouse lymphoma thymidine kinase assay, and in vivo bone marrow micronucleus assay in mice.

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Hepatotoxicity
In several large clinical trials, serum ALT elevations were no more frequent with cangrelor therapy than with placebo [9% vs 12%] or with comparator arms [6.6% vs 6.8%] and no cases of clinically apparent liver injury with jaundice were reported. In addition, since marketing and release, there have been no published reports of clinically apparent liver injury or jaundice associated with cangrelor therapy and hepatotoxicity is not mentioned in the product label.
Likelihood score: E (unlikely cause of clinically apparent liver injury).

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Protein Binding
about 97-98%.

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Interactions
BACKGROUND: Agents that act as antagonists at P2Y(12) ADP receptors on platelets are in use (clopidogrel), and in development for use (cangrelor and prasugrel), in patients with cardiovascular disease. Cangrelor is a direct-acting reversible antagonist being developed for short-term infusion; clopidogrel and prasugrel are oral prodrugs that provide irreversible inhibition via transient formation of active metabolites. At the cessation of cangrelor infusion, patients are likely to receive clopidogrel or prasugrel as a means of maintaining antiplatelet therapy. OBJECTIVES: To apply an experimental in vitro approach to investigate the possibility that cangrelor influences the ability of the active metabolites of clopidogrel and prasugrel to inhibit ADP-mediated platelet function. METHODS: The effects of cangrelor and the active metabolites of clopidogrel (C-AM) and prasugrel (P-AM) on platelet function were assessed by ADP-induced platelet P-selectin expression in whole blood. The method involved rapid removal of the antagonists by dilution, and measurement of residual platelet inhibition. RESULTS: Cangrelor, C-AM and P-AM markedly inhibited P-selectin expression. The effect of cangrelor, but not of C-AM and P-AM, was reversible following antagonist removal. Preincubation of blood with cangrelor prior to addition of C-AM or P-AM reduced the ability of metabolites to irreversibly antagonize P2Y(12). Irreversible inhibition was maintained when blood was preincubated with metabolites prior to cangrelor. CONCLUSIONS: Cangrelor influences the ability of the active metabolites of clopidogrel or prasugrel to inhibit platelet function irreversibly. Careful consideration should be given to the timing of administration of an oral P2Y(12) antagonist following cangrelor infusion.
Concomitant administration of cangrelor with the thienopyridine antiplatelet drugs clopidogrel or prasugrel decreases the antiplatelet effect of clopidogrel and prasugrel by blocking P2Y12-receptor binding of the active metabolites of these drugs. Oral maintenance antiplatelet therapy with clopidogrel or prasugrel should not be administered until the cangrelor infusion has been discontinued.

[1]. Intravenous cangrelor as a peri-procedural bridge with applied uses in ischemic events. Ann Transl Med. 2019;7(17):408.

[2]. Cangrelor alleviates bleomycin-induced pulmonary fibrosis by inhibiting platelet activation in mice. Mol Immunol. 2020;120:83-92.

[3]. Contribution of platelet P2Y12 receptors to chronic Complete Freund's adjuvant-induced inflammatory pain. J Thromb Haemost. 2017;15(6):1223-1235.

Cangrelor tetrasodium is an organic sodium salt that is the tetrasodium salt of cangrelor. Used as an intravenous antiplatelet drug that prevents formation of harmful blood clots in the coronary arteries. It has a role as a platelet aggregation inhibitor and a P2Y12 receptor antagonist. It contains a cangrelor(4-).
Cangrelor Tetrasodium is the tetrasodium salt form of cangrelor, an inhibitor of the platelet adenosine diphosphate (ADP) P2Y12 receptor (P2Y12R), with antiplatelet activity. Upon administration, cangrelor selectively and reversibly binds to P2Y12R, and blocks the platelet signaling pathway. This inhibits the activation of the glycoprotein complex GPIIb/IIIa, fibrinogen binding to platelets and platelet adhesion and aggregation.

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Drug Indication
Kengrexal, co-administered with acetylsalicylic acid (ASA), is indicated for the reduction of thrombotic cardiovascular events in adult patients with coronary artery disease undergoing percutaneous coronary intervention (PCI) who have not received an oral P2Y12 inhibitor prior to the PCI procedure and in whom oral therapy with P2Y12 inhibitors is not feasible or desirable.

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Due to the increasing number of patients on antiplatelet therapy for cardiovascular and neurological conditions, it can be challenging to manage these patients peri-operatively. It is critical to prevent ischemia and thrombosis, and at the same time decrease the risk of bleeding, necessitating the need for a successful bridging therapy. Cangrelor looks promising as a bridging therapy with its distinctive pharmacokinetic profile with fast activity and easy reversibility. However, large prospective studies are required to delineate clear guidelines to identify the patient population that would receive maximum benefit from bridging antiplatelet therapy, determine optimal dosing and titration, monitoring therapy, and manage adverse events. Although guidelines recommend IV bridge therapy in these settings, no agent currently has FDA-approval for this indication and positive, randomized controlled data for GPIs in this setting is also lacking. Cangrelor bridging therapy appears to have advantages over the previous standard using GPIs due to its faster offset and non-renal clearance. Future research is warranted for use of cangrelor in special populations, such as those with CAD on DAPT as a bridge to LVAD implantation. Overall, in this complex era of advancing medical technologies, therapies such as cangrelor may mitigate thrombotic and bleeding risks in the peri-operative period. [1]

C17H21CL2F3N5NA4O12P3S2

864.27

862.876

C, 23.63; H, 2.45; Cl, 8.20; F, 6.59; N, 8.10; Na, 10.64; O, 22.21; P, 10.75; S, 7.42

163706-36-3

Cangrelor; 163706-06-7

10260031

White to off-white solid powder

979ºC at 760 mmHg

545.9ºC

0mmHg at 25°C

4.676

3

21

14

48

1110

4

CSCCNC1=C2C(=NC(=N1)SCCC(F)(F)F)N(C=N2)C3C(C(C(O3)COP(=O)([O-])OP(=O)(C(P(=O)([O-])[O-])(Cl)Cl)[O-])O)O.[Na+].[Na+].[Na+].[Na+]

COWWROCHWNGJHQ-OPKBHZIBSA-J

InChI=1S/C17H25Cl2F3N5O12P3S2.4Na/c1-43-5-3-23-12-9-13(26-15(25-12)44-4-2-16(20,21)22)27(7-24-9)14-11(29)10(28)8(38-14)6-37-42(35,36)39-41(33,34)17(18,19)40(30,31)32;;;;/h7-8,10-11,14,28-29H,2-6H2,1H3,(H,33,34)(H,35,36)(H,23,25,26)(H2,30,31,32);;;;/q;4*+1/p-4/t8-,10-,11-,14-;;;;/m1..../s1

tetrasodium;[dichloro(phosphonato)methyl]-[[(2R,3S,4R,5R)-3,4-dihydroxy-5-[6-(2-methylsulfanylethylamino)-2-(3,3,3-trifluoropropylsulfanyl)purin-9-yl]oxolan-2-yl]methoxy-oxidophosphoryl]oxyphosphinate

ARC69931; AR-C69931; AR C69931; Cangrelor; AR-C69931MX; Cangrelor tetrasodium; 163706-36-3; AR-C69931MX; kengrexal; Cangrelor Tetrasodium [USAN]; Cangrelor tetrasodium salt; 5'-Adenylic acid, N-[2-(methylthio)ethyl]-2-[(3,3,3-trifluoropropyl)thio]-, anhydride with P,P'-(dichloromethylene)bis[phosphonic acid], sodium salt (1:1:4); 2144G00Y7W; AR C69931MX; ARC69931MX; Cangrelor tetrasodium salt

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture and light.

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

H2O : ~125 mg/mL (~144.63 mM)
DMSO : ~12.5 mg/mL (~14.46 mM)

Solubility in Formulation 1: 1.25 mg/mL (1.45 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution; with sonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 12.5 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: 1.25 mg/mL (1.45 mM) 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; with ultrasonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 12.5 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 3: 1.25 mg/mL (1.45 mM) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 12.5 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 100 mg/mL (115.70 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.

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