Factor Xa (FXa) (Ki = 41 nM)

The synthetic compound DX-9065a represents a low molecular weight, direct, competitive inhibitor of factor Xa (FXa) with a high affinity and selectivity for the enzyme. Under experimental conditions DX-9065a exerts strong anticoagulant actions in vitro and is antithrombotically effective in various thrombosis models. It inhibits proliferation of vascular smooth muscle cells in cell culture systems. As a small molecule inhibitor, DX-9065a inactivates both free and fibrin-bound FXa. By this mechanism it effectively affects the clot-associated procoagulant activity which might be responsible for the propagation of intravascular thrombi as well as for recurrent thrombosis and thrombotic reocclusion after lysis. [1]

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The synthetic compound DX-9065a represents a low molecular weight, direct inhibitorof FXa with a high affinity and selectivity for the enzyme. The first kinetic studies re-vealed that DX-9065a inhibited competitively human FXa with an inhibition constant Ki of 41 nM, whereas other serine proteases are not or only weakly affected (Ki values in ìM:thrombin > 2000, trypsin = 0.62, chymotrypsin > 2000, plasmin = 23, tPA = 21, plasmakallikrein = 2.3 and tissue kallikrein = 1000). In following studies slightly different Kivalues for the inhibition of human FXa were found ranging from 3.1 and 7.8 nM (14)to 24 nM. [1]

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A potent and selective FXa inhibitor such as DX-9065a does not influence preformedthrombin, but, based on the amplification mechanisms within the coagulation cascade andthe important role of the prothrombinase complex, it is expected to effectively inhibit thegeneration of thrombin which is probably the most important mechanism for the anti-thrombotic effectiveness of these agents. Thrombin-mediated feedback reactions such asthe activation of the cofactors V and VIII that amplify thrombin formation and the effectof thrombin on platelets and other cellular elements will also be altered. The inhibitoryeffect of DX-9065a on the generation of thrombin was demonstrated in human wholeblood where it caused a time- and concentration-dependent delay in or inhibition ofthrombin generation as well as in a biochemical thrombin generation assay whereDX-9065a delayed and reduced the generation of thrombin resulting in a decreasedamount of thrombin measured in plasma. [1]

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DX-9065a is the first member of a newly developed series of synthetic and selective inhibitors of factor Xa. DX 9065A inhibited in a dose-dependent manner human factor Xa with K iota value of 3.1 +/- 0.5 nM. Steady-state studies revealed that DX 9065A was a competitive inhibitor of factor Xa. DX 9065A inhibited thrombin generation occurring via both the extrinsic and intrinsic pathway in vitro. [2]

Studies on platelet-derived microparticles which are formed following platelet acti-vation showed that both the prothrombinase activity on the surface of these microparticlesand the resulting prothrombotic effect in vivo are inhibited by direct (DX-9065a) and in-direct (pentasaccharide) FXa inhibitors. The inhibitory effect of these anti-FXa agents isbased on the inhibition of FXa present at the surface of microparticles, on the delay of theburst of thrombin and on the inhibition of the coagulation cascade triggered at the surfaceof microparticles. An important aspect for the development of small molecule inhibitors of FXa is theirability to inactivate the clotting enzyme not only in plasma but also when it is bound tofibrin within a clot, an effect which is not seen with antithrombin and antithrombin-de-pendent inhibitors. Because of the presence of FXa and active prothrombinase complexesin intravascular and mural thrombi (10,11,39), the inactivation of FXa and the resulting in-hibition of thrombin formation may be an effective way to affect clot-associated proco-agulant activity which is considered to be responsible for the propagation of intravascularthrombi and for recurrent thrombosis after successful thrombolysis. As a small molecularweight inhibitor with a high affinity for the enzyme DX-9065a inhibits both free and pro-thrombinase-bound FXa and is able to penetrate into the clot and inhibit the clot-boundenzyme [1].

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After i.v. injection to rabbits, DX-9065a displayed prolonged anti-factor Xa activity and inhibition of thrombin generation. Pretreatment of mice with DX-9065a dose-dependently improved the survival rate of mice injected with a lethal dose of tissue factor (ED50 = 1.1 +/- 0.2 mg/kg). After p.o. administration, DX 9065A caused a reduction in tissue factor-induced mortality of mice with ED50 value of 56 +/- 7 mg/kg. When given i.v. to rats, DX 9065A exhibited a dose-dependent antithrombotic effect against factor Xa + stasis-induced venous thrombosis (ED50 = 1.2 +/- 0.7 mg/kg i.v.), but also in an arteriovenous shunt thrombosis model (ED50 = 8.1 +/- 3.5 mg/kg i.v.) without affecting bleeding time significantly. Similar effects were obtained after s.c. or p.o. administration. In rabbits, after i.v., s.c. or p.o. administration, DX 9065A inhibited stasis-induced thrombosis after injection of tissue factor with ED50 values of 0.03 +/- 0.01, 0.3 +/- 0.07 and 50.5 +/- 19 mg/kg, respectively (n = 10). DX 9065A inhibited in a dose-dependent manner endotoxin-induced venous thrombosis in the rabbit (ED50 = 0.25 +/- 0.1 mg/kg i.v.) (n = 5) and reduced the decrease in platelet number and circulating fibrinogen levels in an experimental model of tissue factor-induced disseminated intravascular coagulation. Compared to standard heparin, DX-9065a exhibited a favorable antithrombotic/bleeding ratio, therefore showing that it might be considered as a promising compound in the treatment and prevention of various thrombotic diseases.[2]

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Anticoagulant Action [1]
After parenteral administration there was a predictable quantitative relation betweenthe administered dose of DX-9065a and its anticoagulant and anti-FXa activity. A dose-dependent prolongation of clotting times in the APTT and PT assay was also seen afteroral administration of the inhibitor. The direct comparison between orally and intra-venously effective doses of DX-9065a revealed that for the anticoagulant and/or anti-thrombotic action at least 5 to 10 times higher doses are required by oral than by intrave-nous administration (see Table 1). It was also obvious that experimentalthrombus formation was inhibited with DX-9065a at doses which only modestly increasedthe clotting times APTT and PT measured ex vivo. It has to be mentioned that the anticoagulant and anti-Xa activities of DX-9065a arespecies-dependent. DX-9065a was nearly equally effective in prolonging the PT inhuman and common squirrel monkey plasma. In contrast, in rat plasma the inhibitor was40 times less potent than in human plasma. The anticoagulant response to DX-9065a incynomolgus monkey, dog, rabbit and mouse plasma ranged between those in human andrat plasma.

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Effect on Platelet Function [1]
DX-9065a, at concentrations up to 100 ìM did not inhibit ADP-, collagen-, or throm-bin-induced platelet aggregation. The direct inactivation of factor Xa by DX-9065adid not prevent platelet activation in human whole blood which was induced by agonists such as arachidonic acid, TRAP or ã-thrombin. However, DX-9065a showed a time- andconcentration-dependent inhibitory effect on platelet activation mediated by either tissuefactor or factor Xa. It seems that the antiplatelet effect of DX-9065a is mainly due to theinhibition of generation of thrombin which is known to be the most important physiologicagonist for platelet activation and aggregation.

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Antithrombotic Action [1]
It was clearly demonstrated by various experimental studies that DX-9065a is an effi-cacious antithrombotic drug by either intravenous, subcutaneous or oral administration.DX-9065a inhibits or prevents the development of several types of vascular thrombosissuch as venous and arterial thrombosis, occlusion of vascular shunts and disseminatedintravascular coagulation. Because of the great differences in the experimental models, thethrombogenic stimuli, the kind of drug administration as well as the species used it is dif-ficult to assess the usefulness of DX-9065a for a definite thrombotic disorder. Based onthe mechanism of action of DX-9065a it is expected that the inhibitor might have a greaterinhibitory effect on a thrombotic process where the underlying pathophysiological mech-anism primarily involves the activation of the coagulation cascade with the final con-version of fibrinogen to fibrin such as in venous thrombosis. A summary of experimentalthrombosis studies carried out with DX-9065a is given in Table 1.

Pharmacokinetics [1] Animal Studies The pharmacokinetics and pharmacodynamics of FXa inhibitors are closely related because circulating blood is the primary site of action for these inactivated thrombin compounds. Oral administration is the preferred route for long-term clinical use of anticoagulants/antithrombotic drugs. DX-9065a is a low molecular weight FXa inhibitor designed to overcome the limitations of peptide compounds, particularly the low oral bioavailability. Animal and human studies have confirmed the efficacy of oral DX-9065a. DX-9065a has shown potent anticoagulant and/or antithrombotic effects after parenteral or oral administration (Table 1). However, comparative studies have shown that the oral dose required to achieve antithrombotic effects is much higher (at least 10 times higher) than the intravenous dose (Table 1). This is consistent with the results of pharmacokinetic studies in baboons, which estimated its oral bioavailability to be approximately 5% to 12%. Studies on the time course of action of DX-9065a in rats showed that after intravenous injection, the anti-FXa activity reached its maximum immediately after injection and lasted for approximately 30 minutes. After oral administration, the anti-FXa activity reached its maximum 15 to 30 minutes after administration and lasted for approximately 90 minutes. The same time course was observed for the antithrombotic effect: a short duration of 10 to 20 minutes after intravenous injection, while the effect lasted for more than 3 hours after oral administration. Other researchers found that after oral administration of DX-9065a in rats, the peak activity occurred approximately 1 hour after administration and lasted for 4 hours. In baboons, intravenous injection of DX-9065a revealed an α-phase plasma half-life of 6.3 minutes and a β-phase plasma half-life of 99 minutes. After oral administration, the peak plasma concentration of DX-9065a occurred at 30 minutes, subsequently declining gradually over approximately 6 to 8 hours. Human Studies: The pharmacokinetics of DX-9065a in humans differ from those in animals. This is likely due to species differences, as clearly demonstrated in studies of the inhibitor's anticoagulant/antithrombotic effects (see above). Of particular note is the significantly longer duration of action of DX-9065a in humans compared to rats or baboons. This longer duration of action is especially important given the potential clinical applications of DX-9065a in the prevention or treatment of thromboembolic diseases. Following a single intravenous bolus (0.625–2.5 mg) or a 1-hour intravenous infusion (total dose 5 to 30 mg) in male volunteers, a double- or triple-exponential decrease in DX-9065a plasma concentrations was observed. The terminal-phase half-life was 10.7 hours after a single 2.5 mg intravenous bolus, and 22.8 to 26.1 hours after 60 minutes of intravenous infusion. Plasma protein binding ranged from 64.6% to 83%, and cumulative urinary excretion ranged from 32.3% to 40.9%. The concentration of DX-9065a in feces was below the limit of quantitation. Within the studied dose range, the pharmacokinetics of DX-9065a in human subjects were linear, regardless of whether it was administered as a bolus or continuous intravenous infusion over 1 hour, and there was no statistically significant difference between the two treatment groups. In a healthy white male volunteer, a single intravenous injection of 10 mg [14C]DX-9065a followed by continuous infusion over 1 hour resulted in a biexponential decrease in plasma drug concentration, which fell below the limit of detection 48 hours after administration. The distribution phase half-life was 6.93 hours. The primary route of excretion was urine, accounting for over 77.6% of the administered dose. Renal tubular secretion may contribute to the urinary excretion of DX-9065a. Biotransformation of DX-9065a did not appear to play a significant role in its clearance in humans. In the XaNADU-IB (Xa Neutralization Therapy for Atherosclerotic Disease) trial, patients with stable coronary artery disease received an intravenous bolus of 1 mg DX-9065a, followed by continuous infusion over 72 hours at four different doses (see below). The half-lives of DX-9065a were t1/2a = 0.14 to 0.30 hours, t1/2a = 1.93 to 3.20 hours, and t1/2a = 76.57 to 98.86 hours. Inter-individual variability increased with increasing dose. In a double-blind, placebo-controlled study, researchers administered 2.5, 5, or 10 mg of DX-9065a subcutaneously to healthy male subjects. Peak plasma concentrations were reached 1 hour after injection and decreased below the detection limit 4 to 8 hours after treatment.

Bleeding Effects in Animal Studies [1]
The main side effect of anticoagulant/antithrombotic drugs during treatment is bleeding complications. At effective antithrombotic doses, the risk of bleeding should be as low as possible. Data on the impairment of primary hemostasis by anti-FXa drugs suggest that FXa inhibitors may cause fewer bleeding complications than thrombin inhibitors. As a competitive inhibitor, DX-9065a does not completely inhibit thrombin production, and therefore still produces trace amounts of thrombin. Since thrombin has a much higher affinity for platelets than for fibrinogen, these trace amounts of thrombin are sufficient to form platelet-dependent thrombi, thereby preventing bleeding. In several preclinical studies, DX-9065a has been shown to inhibit experimental thrombosis without causing bleeding complications. In different bleeding time measurement models, such as rat tail transection models, gastrointestinal bleeding models, or rabbit ear bleeding models, DX-9065a, whether administered via parenteral or oral routes, did not prolong bleeding time at effective antithrombotic doses. Direct comparison of the thrombin inhibitor argatroban, low molecular weight heparin flammin, unfractionated heparin, and DX-9065a showed that argatroban and antithrombin III-dependent anticoagulants prolonged bleeding time in rats at doses slightly higher than the effective antithrombotic dose, while DX-9065a had no effect on bleeding time at doses ten times higher. Human Side Effects [1] In human volunteers, DX-9065a did not produce any serious adverse reactions during or after the study, whether administered intravenously (0.625 to 2.5 mg) or via intravenous infusion (5 to 30 mg/60 min). No clinically significant changes were observed in serum chemistry, hematology, bleeding time, or urinalysis. In the XaNADU-IB trial, DX-9065a was well tolerated in patients with stable coronary artery disease. No significant adverse effects on renal function, liver function, platelet count, or hemoglobin were observed. No major bleeding complications occurred. Only in the highest dose group was there a slight, but not statistically significant, dose-related increase in the incidence of minor bleeding compared to the placebo group.

[1]. Brigitte Kaiser. DX-9065a, a direct inhibitor of factor Xa. Cardiovasc Drug Rev. 2003 Summer;21(2):91-104.

[2]. DX 9065A a novel, synthetic, selective and orally active inhibitor of factor Xa: in vitro and in vivo studies. J Pharmacol Exp Ther. 1996 Mar;276(3):1030-8.

In recent years, significant progress has been made in the development of small-molecule direct thrombin inhibitors. These inhibitors can block the coagulation process at different stages of the coagulation cascade. Highly effective and selective inhibitors of activated coagulation factors (such as thrombin and FXa) hold promise for overcoming the limitations of current antithrombotic regimens using heparin or vitamin K antagonists. The synthetic small-molecule FXa inhibitor DX-9065a inactivates the enzyme without any endogenous cofactors, representing a new class of anticoagulant/antithrombotic drugs with broad application prospects. Under experimental conditions, DX-9065a exhibits potent anticoagulant activity both in vitro and in vivo, demonstrating antithrombotic efficacy in various thrombosis models and inhibiting the proliferation of vascular smooth muscle cells in both cell culture systems and in vivo models. As a small-molecule anti-FXa drug, DX-9065a inhibits both free FXa and thrombus-bound FXa. This effect, coupled with its inhibition of thrombin generation, may be an effective method for controlling thrombosis-related procoagulant activity. However, despite the proven efficacy of DX-9065a, a comprehensive assessment of its therapeutic potential requires consideration of many other aspects. Developing an inhibitor targeting the active site of FXa necessitates attention not only to its optimal binding to the target enzyme but also to the physicochemical properties that determine the compound's pharmacokinetic and pharmacodynamic behavior. One of the main goals of synthetic FXa inhibitor development is to find an intravenous drug for treating acute thrombotic diseases, followed by an oral formulation for long-term outpatient treatment. However, many small-molecule FXa inhibitors are highly basic, resulting in poor pharmacokinetic properties, particularly limited oral bioavailability. Synthetic anti-Xa drugs are primarily absorbed in the intestine via passive diffusion, and more lipophilic molecules can improve this absorption. DPC423 is a good example; it is a highly effective, selective, and orally bioavailable Xa factor inhibitor. In a series of 3-trifluoromethylpyrazole derivatives, the strongly basic benzylamine group was replaced with a weaker basic group, and the molecular structure was further optimized, ultimately yielding a compound with favorable pharmacokinetic and pharmacodynamic characteristics. DPC423 has an oral bioavailability of 57% in dogs, a plasma half-life of 7.5 hours, and has shown antithrombotic activity in a rabbit arteriovenous shunt thrombosis model. DX-9065a, containing a naphthamidin group, is one of the few FXa inhibitors that has been shown to be orally effective in experimental studies. However, its oral bioavailability is relatively low and may be insufficient for long-term treatment needs. Nevertheless, preliminary clinical trials in healthy volunteers and patients with cardiovascular disease have shown that DX-9065a has at least predictable pharmacodynamic and pharmacokinetic characteristics via intravenous bolus and continuous infusion. Intravenously administered DX-9065a has proven to be an effective anticoagulant/antithrombotic agent with a higher safety profile compared to other drugs such as heparin or warfarin. Furthermore, specific FXa inhibitors are expected to be superior to direct thrombin inhibitors (such as hirudin) in selectively inhibiting thrombus formation without impairing platelet hemostasis. Preclinical and preliminary clinical studies suggest that DX-9065a may be a promising alternative for the prevention or treatment of thrombotic events without bleeding side effects. Therefore, it can be used as adjunctive therapy to thrombolytic therapy or in combination with antiplatelet drugs without increasing the risk of bleeding. Another important issue with the use of specific FXa inhibitors is whether and how their efficacy can be monitored in clinical practice, i.e., whether there are simple and reproducible assays available. A dose-dependent increase in DX-9065a plasma concentrations is associated with prolonged clotting times measured in overall coagulation tests such as prothrombin time (PT) and activated partial thromboplastin time (APTT). It remains unclear whether DX-9065a prolongs coagulation parameters at therapeutic doses and plasma concentrations, which biomarkers are most sensitive to the drug, and whether it should be routinely used to monitor the efficacy of DX-9065a. Furthermore, it is unclear how to neutralize the effects of this compound in the event of overdose or adverse reactions. Experimental results indicate that FXa plays a role in the complex pathogenesis of restenosis and atherosclerosis. Thrombin and FXa both appear to affect the proliferation of vascular smooth muscle cells in vivo. However, the exact role of serine proteases, particularly the significance of their mitogenic activity in restenosis and atherosclerosis, and the practical significance of specific inhibitors inactivating these enzymes, remains to be elucidated. Platelet activation and aggregation, as well as thrombin generation, are crucial in arterial thrombosis. Therefore, direct inhibition of FXa by DX-9065a appears to be a safe and effective novel approach for preventing atherosclerotic thrombotic complications. A comprehensive evaluation of the therapeutic potential of DX-9065a requires consideration of several other aspects. In addition to pharmacokinetic characteristics such as oral bioavailability, biological half-life, metabolic transformation, and excretion pathways, its interactions with other drugs or endogenous factors need to be investigated. The most promising clinical indications for DX-9065a and the benefits of combining it with drugs with different mechanisms of action or sites of action also need further clarification. Such combinations may have a synergistic effect on therapeutic efficacy but may also exacerbate adverse side effects such as bleeding complications. In summary, the FXa inhibitor DX-9065a is a promising drug for the prevention and/or treatment of various thromboembolic diseases. Further experimental studies, particularly comprehensive clinical trials, are expected to confirm the inhibitory properties, efficacy, and superiority of this compound over other drug regimens for cardiovascular diseases. [1]

C26H39CLN4O8

571.068

570.246

C, 54.68; H, 6.88; Cl, 6.21; N, 9.81; O, 22.41

155204-81-2

150612-55-8;150611-74-8 (HCl);155204-81-2 (HCl hydrate);201933-30-4 (racemic); 155204-80-1 (R-isomer);

122128

Typically exists as solid at room temperature

632.9ºC at 760mmHg

336.6ºC

6.9E-17mmHg at 25°C

5.303

10

10

8

39

720

2

CC(=N)N1CC[C@@H](C1)OC2=CC=C(C=C2)[C@H](CC3=CC4=C(C=C3)C=CC(=C4)C(=N)N)C(=O)O.Cl.O.O.O.O.O

LJCBAPRMNYSDOP-LVCYMWGESA-N

InChI=1S/C26H28N4O3.ClH.5H2O/c1-16(27)30-11-10-23(15-30)33-22-8-6-19(7-9-22)24(26(31)32)13-17-2-3-18-4-5-20(25(28)29)14-21(18)12-17;;;;;;/h2-9,12,14,23-24,27H,10-11,13,15H2,1H3,(H3,28,29)(H,31,32);1H;5*1H2/t23-,24-;;;;;;/m0....../s1

(2S)-3-(7-carbamimidoylnaphthalen-2-yl)-2-[4-[(3S)-1-ethanimidoylpyrrolidin-3-yl]oxyphenyl]propanoic acid;pentahydrate;hydrochloride

DX9065A; DX 9065A; dx-9065a; 155204-81-2; DX 9065-a; DX-9065-A HCl hydrate; QXQVEPEVI2; 155204-81-2 (HCl, hydrate); (2S)-2-(4-(((3S)-1-acetimidoyl-3-pyrrolidinyl)oxy)phenyl)-3-(7-amidino-2-naphtyl)propanoic acid; (2S)-3-(7-Carbamimidoylnaphthalen-2-yl)-2-[4-[(3S)-1-ethanimidoylpyrrolidin-3-yl]oxyphenyl]propanoic acid;pentahydrate;hydrochloride; DX-9065A

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.)