vitamin K1 recycling
Warfarin targets the enzyme vitamin K epoxide reductase (VKORC1). By inhibiting VKORC1, it prevents the regeneration of the active, reduced form of vitamin K. This is a critical cofactor for the γ-carboxylation of glutamate residues on several clotting factors, including factors II (prothrombin), VII, IX, and X, as well as the anticoagulant proteins C and S. This results in the production of inactive clotting factors and a subsequent reduction in blood coagulation.

In vitro, Warfarin is a potent inhibitor of VKORC1 activity in liver microsomal preparations. Its effect is to inhibit the vitamin K-dependent carboxylation of clotting factors. The S-enantiomer of warfarin is approximately 3-5 times more potent than the R-enantiomer in its anticoagulant effect. It has no direct effect on existing clotting factors, which is why its therapeutic effect takes several days to manifest.

In vivo, Warfarin is a highly effective oral anticoagulant used to prevent thrombosis and thromboembolism. It is indicated for the treatment and prevention of deep vein thrombosis (DVT), pulmonary embolism (PE), and for preventing stroke in patients with atrial fibrillation or prosthetic heart valves. Its therapeutic effect is monitored by the international normalized ratio (INR), and its dosage must be carefully individualized to maintain a therapeutic INR range.

In vitro enzyme assays for Warfarin typically use liver microsomes or recombinant VKORC1 to measure its ability to inhibit the reduction of vitamin K 2,3-epoxide to vitamin K. The enzyme is incubated with its substrate and varying concentrations of warfarin, and the production of vitamin K is measured, typically by HPLC. The IC₅₀ is then calculated to determine the compound's potency.

In vitro cell-based assays for Warfarin are not commonly performed, as its primary pharmacological effect is not on isolated cells but on the complex coagulation cascade. However, the compound's effect on cellular uptake of vitamin K can be studied in liver cell lines like HepG2. Cells are treated with warfarin, and the intracellular levels of vitamin K and its epoxide are measured.

In vivo animal studies for Warfarin are conducted in rodent models to assess its anticoagulant effect. The compound is typically administered orally, and its efficacy is measured by the prothrombin time (PT) or INR. In toxicity studies, it has been used as a rodenticide, where it causes lethal hemorrhage.

Absorption, Distribution and Excretion
Warfarin is completely absorbed from the gastrointestinal tract. The average time to peak concentration (Tmax) of warfarin sodium tablets is 4 hours.
Warfarin is almost completely eliminated through metabolism, with a small amount excreted unchanged. 80% of the total dose is excreted in the urine, and the remaining 20% in the feces.
The volume of distribution (Vd) is 0.14 L/kg. The distribution period of warfarin lasts 6–12 hours. Warfarin is known to cross the placenta and reach concentrations in fetal serum similar to those in the mother.
Warfarin clearance depends on the CYP2C9 genotype. The 2 and 3 alleles, with frequencies of 11% and 7% respectively in Caucasian populations, are known to reduce warfarin clearance. Other genotypes that reduce clearance include the 5, 6, 9, and 11 alleles. Genotypes with estimated population clearance rates are as follows: 1/1 = 0.065 mL/min/kg; 1/2, 1/3 = 0.041 mL/min/kg; 2/2, 2/3, 3/3 = 0.020 mg/min/kg. Warfarin sodium is rapidly and extensively absorbed from the gastrointestinal tract, but absorption rates vary significantly between individuals. Oral absorption of warfarin sodium is controlled by the dissolution rate, and the absorption rate and extent may differ between different commercially available tablets. Studies using warfarin sodium have shown that the presence of food in the gastrointestinal tract reduces the rate of drug absorption but does not affect the extent of absorption. Warfarin sodium crosses the placenta, and fetal plasma drug concentrations may be comparable to maternal plasma concentrations. Limited data indicate that warfarin is not excreted into human breast milk. A study found that warfarin was undetectable in the breast milk and plasma of breastfed infants of 13 lactating women who received an initial dose of 30 or 40 mg warfarin and a maintenance dose of 2–12 mg daily. Researchers investigated salivary excretion in rabbits after intravenous and oral administration of warfarin. After intravenous administration (50 mg/kg), the salivary decay curve conformed to a two-compartment open model. …After oral administration (100 mg/kg), the distribution of warfarin conformed to a one-compartment model. A good linear relationship was observed between salivary and plasma warfarin concentrations. The salivary/plasma (S/P) ratio was approximately 0.07. A good correlation was also observed between salivary warfarin concentrations and plasma free warfarin concentrations. The ratio of salivary to plasma free warfarin concentrations (S/PF) was approximately 0.92. … For more complete data on the absorption, distribution, and excretion of warfarin (18 types), please visit the HSDB records page.

---

Metabolism/Metabolites
Warfarin metabolism exhibits stereoselectivity and regioselectivity. The primary metabolic pathway is oxidation to various hydroxywarfarin derivatives, accounting for 80-85% of total metabolites. CYP2C9 is the main enzyme catalyzing the 6- and 7-hydroxylation of S-warfarin, while 4'-hydroxylation is catalyzed by CYP2C18, with CYP2C19 contributing less. R-warfarin is metabolized to 4'-hydroxywarfarin via CYP2C8, with CYP2C19 also participating in some of the metabolism; 6- and 8-hydroxywarfarin are metabolized by CYP1A2 and CYP2C19; 7-hydroxywarfarin is metabolized by CYP1A2 and CYP2C8; and finally, it is metabolized to 10-hydroxywarfarin by CYP3A4. The 10-hydroxywarfarin metabolite and a benzyl alcohol metabolite undergo elimination steps to generate dehydrowarfarin. A minor metabolic pathway is the reduction of the ketone group to warfarinol, accounting for approximately 20% of the metabolites. Binding to sulfate and uronic acid groups was limited, but these metabolites were only confirmed in R-hydroxywarfarin. Of all the recovered metabolites, only 4'-hydroxywarfarin and DHG (2,3-dihydro-2-methyl-4-phenyl-5-oxo-γ-pyranol (3,2-c)(1)benzopyran) showed anticoagulant activity. The oxidative biotransformation of (R)-warfarin and (S)-warfarin was investigated in human liver microsomes. The quantitative pattern of warfarin oxidation products in vitro changed significantly with substrate concentration. The apparent Km values of 4', 6, 7, and 8-hydroxywarfarin production indicated the presence of two easily distinguishable human hepatic cytochrome P450 subsets: a high-affinity subset with a Km value of 15 μM; and a low-affinity subset of isoenzymes with Km values greater than 200 μM. The high-affinity subset primarily responsible for the metabolism of the more bioactive (S)-warfarin in vivo, while the low-affinity subset primarily responsible for the metabolism of (R)-warfarin. The apparent maximum reaction rate (Vmax) alone cannot reflect the relative proportions of phenolic metabolites produced by the two compounds in vivo, as the low-affinity, high-capacity fraction masks the metabolic profile of (S)-warfarin. The intrinsic clearance rates (Vmax/Km) of each metabolite are well-aligned with the regioselective and stereoselective metabolic results in vivo. In humans, the (S) isomer primarily undergoes 7-hydroxylation, while the (R) isomer is reduced to (R,S)-alcohols. In rats, the (S) isomer primarily undergoes 4'-hydroxylation, while the (R) enantiomer undergoes 7-hydroxylation. The involvement of different cytochrome P-450 forms was used to explain these results.
The contributions of human p450 2A6 and mouse p450 2a-5 isoenzymes (both highly active in the 7-hydroxylation of coumarin) to warfarin metabolism were investigated in several in vitro systems using human and mouse liver preparations. Recombinant p450 2a-5 purified from DBA/2 mouse liver could not metabolize warfarin. While anti-p450 2a-5 antibody inhibited over 90% of the 7-hydroxylation of coumarin, it did not persistently inhibit any warfarin biotransformation catalyzed by human or mouse liver microsomes. In some human liver microsomal samples, anti-p450 2a-5 antibody inhibited the 4- and 8-hydroxylation of warfarin to some extent. Warfarin (at concentrations below 1 μM) did not inhibit the 7-hydroxylation of coumarin by human or mouse liver microsomes in vitro. These results indicate that mouse and human coumarin 7-hydroxylases do not oxidize warfarin. For more complete data on the metabolism/metabolites of warfarin (9 metabolites in total), please visit the HSDB record page. Warfarin is stereoselectively and regioselectively metabolized by hepatic microsomal enzymes. S-warfarin is primarily metabolized by cytochrome P450 (CYP) 2C9, producing 6- and 7-hydroxylated metabolites. R-warfarin is metabolized by CYP1A1, 1A2, and 3A4, producing 6-, 8-, and 10-hydroxylated metabolites. Hydroxylated metabolites may undergo further binding before excretion in bile and urine. UGT1A1 appears to be responsible for the formation of warfarin's 6-O-glucuronide, and UGT1A10 may also contribute. Five UGT1A molecules may be involved in the formation of warfarin's 7-O-glucuronide. S-warfarin is more potent than R-warfarin, and CYP2C9 gene polymorphisms may significantly reduce drug clearance and increase its toxicity. In the human body, the dextrorotatory enantiomer of warfarin is metabolized to secondary alcohols via side-chain reduction, while levorotatory warfarin is metabolized via epoxidation, primarily producing 7-hydroxywarfarin. These inactive metabolites are bound to glucuronic acid to some extent, undergo enterohepatic circulation, and are ultimately excreted in urine and feces. (A613)
Elimination pathway: Warfarin is eliminated almost entirely through metabolism. Very little warfarin is excreted unchanged in urine. Metabolites are mainly excreted in urine, with a small amount excreted in bile.
Half-life: R-warfarin t_1/2 = 37-89 hours; S-warfarin t_1/2 = 21-43 hours.
The clearance rate of R-warfarin is slower than that of S-warfarin, approximately half that of the latter. The t_1/2 of R-warfarin is 37-89 hours. The t_1/2 of S-warfarin is 21–43 hours. After a single dose, the terminal half-life of warfarin is approximately one week; however, the effective half-life is 20–60 hours, with an average of approximately 40 hours. The clearance rate of R-warfarin is typically half that of S-warfarin; therefore, due to their similar volumes of distribution, R-warfarin has a longer half-life than S-warfarin. The half-life of R-warfarin is 37–89 hours, while that of S-warfarin is 21–43 hours.
Warfarin is rapidly and completely absorbed from the gastrointestinal tract. It is highly protein-bound (approximately 99%) and has a volume of distribution of about 0.14 L/kg. It is metabolized in the liver by cytochrome P450 enzymes, primarily CYP2C9 (for the S-enantiomer) and CYP1A2 and CYP3A4 (for the R-enantiomer). The half-life of S-warfarin is about 40 hours, while R-warfarin is about 45 hours. It is excreted in urine as inactive metabolites.

Toxicity Summary
Warfarin inhibits vitamin K reductase, leading to depletion of reduced vitamin K (vitamin KH2). Since vitamin K is a cofactor for the carboxylation of N-terminal glutamate residues in vitamin K-dependent proteins, it restricts the γ-carboxylation and subsequent activation of vitamin K-dependent clotting proteins. The synthesis of vitamin K-dependent clotting factors II, VII, IX, and X, as well as anticoagulants C and S, is inhibited. Inhibition of three of the four vitamin K-dependent clotting factors (factors II, VII, and X) results in decreased prothrombin levels and reduced thrombin production and fibrin binding. This reduces the thrombotic potential of thrombi.

---

Toxicity Data
LD50: 374 mg/kg (oral, mouse) (A308)Interactions
After a single intravenous co-administration of warfarin (1.2 mg/kg) and furosemide (1.67 mg/kg), the pharmacokinetic complex activity was not significantly different from that of the warfarin-only group; however, when co-administered with 5 mg/kg furosemide, the elimination rate constant was significantly increased, and the pharmacokinetic complex activity was significantly enhanced more than 60 hours after administration. These results indicate that an interaction occurs between warfarin and furosemide when high doses are administered concurrently, for example, the binding of warfarin at the albumin binding site is displaced.

A review of cimetidine interactions with clinically important drugs. Cimetidine reduces the metabolism of warfarin (and other coumarin anticoagulants).
...Metronidazole has no effect on the serum protein binding rate of racemic warfarin over a wide concentration range in vitro, but it reduces the protein binding rate of R-(+)-warfarin and S-(-)-warfarin... (In rats) Intraperitoneal injection of 100 mg/kg metronidazole every 6 hours reduces the plasma clearance of free warfarin. ...Metronidazole does not affect the activity of plasma prothrombin complex in vitro, but reduces its activity in vivo. Chloramphenicol has no significant effect on the serum protein binding rate of R-(+)-warfarin or S-(-)-warfarin in vitro or in vivo (in rats). Intraperitoneal injection of chloramphenicol at 50 mg/kg every 4 hours or 30 mg/kg every 6 hours reduces the plasma clearance of free warfarin by half or more, without significant stereoselectivity. Volume of distribution is not significantly affected; chloramphenicol significantly prolongs the serum half-life of each warfarin enantiomer. The significant enhancement of warfarin's anticoagulant effect by chloramphenicol appears to be solely due to the inhibition of warfarin metabolism, and this effect is not stereoselective. For more complete data on warfarin interactions (out of 85), please visit the HSDB record page. Non-human toxicity values: Rat intravenous LD50: 25 mg/kg /warfarin sodium/
Rat oral LD50: 8700 μg/kg /warfarin sodium/
Mouse oral LD50: 374 mg/kg /warfarin sodium/
Mouse intravenous LD50: 160 mg/kg /warfarin sodium/
For more complete data on warfarin non-human toxicity values (out of 20), please visit the HSDB record page.
Warfarin has a narrow therapeutic index and can cause serious bleeding, which is its most significant toxicity. The risk of bleeding is increased by factors such as drug interactions, genetic polymorphisms in CYP2C9 and VKORC1, and co-morbidities. Other side effects include skin necrosis, purple toe syndrome, and osteoporosis with long-term use. It is contraindicated in pregnancy due to its teratogenic effects.

[1]. Pharmacology and management of the vitamin K antagonists: American College of Chest Physicians Evidence-Based Clinical Practice Guidelines (8th Edition). Chest, 2008. 133(6 Suppl): p. 160S-198S.

Therapeutic Uses
Anticoagulant
While warfarin treatment can present various challenges in children (e.g., dietary differences, adherence issues, monitoring difficulties, lack of commercially available liquid formulations), it has been used in some pediatric patients with venous thromboembolism. /Not included in US product label/
The American College of Chest Physicians (ACCP) recommends routine thromboprophylaxis (using pharmacological and/or mechanical methods, such as intermittent pneumatic compression) in all patients undergoing major orthopedic surgery (including total hip replacement, total knee replacement, and hip fracture surgery) due to the high risk of postoperative venous thromboembolism; thromboprophylaxis should continue for at least 10–14 days and may continue up to 35 days postoperatively. The ACCP recommends several antithrombotic agents (e.g., low molecular weight heparin, fondaparinux, low-dose unfractionated heparin, warfarin, aspirin) for pharmacological thromboprophylaxis in patients undergoing major orthopedic surgery. Although the ACCP recommends that low molecular weight heparin (LMWH) be the first-line treatment due to its relative effectiveness, safety, and extensive clinical experience, alternative drugs such as warfarin may be a reasonable option in cases where LMWH is unavailable or cannot be used (e.g., patients with heparin-induced thrombocytopenic purpura or those who refuse or are uncooperative with subcutaneous injections). /US Product Label Contains/
Warfarin and/or aspirin are used to prevent thromboembolism associated with various valvular heart diseases; the choice of antithrombotic therapy regimen depends on the balance between the risk of thromboembolism and the risk of bleeding complications from antithrombotic therapy. /US Product Label Contains/
For more complete data on the therapeutic uses of warfarin (17 types), please visit the HSDB record page.

---

Drug Warning
/Black Box Warning/ Warning: Bleeding Risk Warfarin may cause serious or fatal bleeding. INR values should be monitored regularly in all patients receiving treatment. Medications, dietary changes, and other factors can affect the INR levels achieved after warfarin treatment. Instruct patients to take preventative measures to minimize the risk of bleeding and report any signs and symptoms of bleeding. Rarely, potentially fatal skin or other tissue gangrene and/or subcutaneous infarction, vasculitis, and localized thrombosis have occurred in patients receiving coumarin derivatives (including warfarin). This reaction can occur at first exposure to these drugs or during subsequent treatments, usually early after the start of treatment (e.g., 1–10 days); tissue damage primarily occurs in adipose tissue sites such as the abdomen, breast, buttocks, and thighs. Most cases of warfarin-induced necrosis are reported in women. Necrotic lesions typically begin as painful erythema on the skin and rapidly develop into dark red hemorrhagic areas. Necrosis may involve the skin, soft tissues, and muscles; gangrene often follows, often with infection. In severe cases, surgical debridement, skin grafting, or amputation of the affected tissue may be necessary. Patients with congestive heart failure receiving oral anticoagulants… may experience an enhanced response to hypoprothrombinemia; this response usually lessens as myocardial function improves. ...High metabolic states, such as fever and hyperthyroidism, increase responsiveness to oral anticoagulants, while patients with myxedema require higher doses...Patient age is positively correlated with the degree of responsiveness to oral anticoagulants; this effect is independent of body weight, and the pharmacokinetics of warfarin are unaffected. ...During pregnancy, increased activity of clotting factors VII, VIII, IX, and X leads to decreased responsiveness to oral anticoagulants. However, this condition only affects the mother; the fetus is highly sensitive to oral anticoagulants because...they can freely cross the placenta, and the fetus has a limited capacity to synthesize clotting factors. Uremia significantly increases the proportion of free drug in plasma and accelerates the clearance of warfarin from circulation.
Warfarin use in early pregnancy (especially weeks 6-9) is associated with embryological disorders...Of infants exposed to warfarin during this period, only about one-third are born normal and live. Other abnormalities, including central nervous system and eye defects (e.g., blindness), are thought to be caused by longer exposures, possibly in the second and third trimesters. For more complete data on warfarin warnings (38 in total), please visit the HSDB records page.

---

Pharmacodynamics
Warfarin is an anticoagulant, therefore it interferes with the coagulation cascade, thus reducing the frequency and severity of thrombosis. In patients with deep vein thrombosis or atrial fibrillation, the risk of thrombosis is increased due to reduced blood flow. In patients with valvular heart disease or who have undergone valve replacement, this increased coagulation activity is due to tissue damage. Thrombi from venous thrombosis can travel to the lungs, forming a pulmonary embolism, which obstructs blood supply to parts of the lung tissue. Thrombi from the heart can travel to the brain, causing ischemic stroke. Preventing these events is a primary goal of warfarin treatment. However, limiting thrombosis is also a source of adverse reactions. In patients with atherosclerotic plaques, plaque rupture often leads to thrombosis. When these patients receive anticoagulation therapy, plaque rupture can cause cholesterol to be released from the lipid core, forming atherosclerotic emboli or cholesterol microemboli. These emboli are smaller than blood clots and can block smaller blood vessels, typically less than 200 micrometers in diameter. The consequences vary depending on the location of the blockage. Effects include visual disturbances, acute kidney injury or exacerbation of chronic kidney disease, central nervous system ischemia, and violet-toe or blue-toe syndrome. Blue-toe syndrome is reversible if it hasn't progressed to tissue necrosis, but other effects of microembolism are often permanent. Anticoagulation appears to be a mediating factor in warfarin-related nephropathy (a seemingly spontaneous kidney injury or exacerbation of chronic kidney disease associated with warfarin treatment). In this case, the nephropathy appears to result from increased permeability of red blood cells through the glomeruli, followed by red blood cell casts obstructing the renal tubules. Pre-existing kidney injury can exacerbate or potentially induce this condition. The risk of warfarin-related nephropathy increases when the INR exceeds 3.0, but beyond this value, the risk no longer increases with further increases in INR. Warfarin is associated with the development of calcifying uremia. This is thought to be because warfarin inhibits the reuse of vitamin K (VKA), which is essential for the carboxylation of matrix Gla protein. This protein is an anti-calcification factor, and inhibiting the carboxylation step in its synthesis shifts the calcification balance toward calcifying uremia. Tissue necrosis may occur early in warfarin treatment. This is attributed to the influence of vitamin K reuse inhibition on the half-lives of clotting factors. Protein C and protein S are anticoagulant factors with half-lives of 8 hours and 24 hours, respectively. The half-lives of clotting factors IX, X, VII, and thrombin (factor II) are 24 hours, 36 hours, 6 hours, and 50 hours, respectively. This means that, except for factor VII, protein C and protein S are inactivated faster than procoagulant proteins, resulting in a prothrombotic state during the first few days of treatment. Thrombi formed during this period can obstruct small arteries in different locations, impeding blood flow and leading to tissue necrosis due to ischemia.
Warfarin is a vitamin K antagonist oral anticoagulant. It is used for the prevention of stroke and systemic embolism in patients with atrial fibrillation, and for the treatment and prevention of venous thromboembolism. It requires careful monitoring of the INR to ensure a safe and effective therapeutic range. This product is for human therapeutic use.

308.104

C, 74.01; H, 5.23; O, 20.76

81-81-2

Warfarin-d5;75472-93-4

54678486

White to off-white solid powder

1.3±0.1 g/cm3

515.2±50.0 °C at 760 mmHg

162-164 °C(lit.)

188.8±23.6 °C

0.0±1.4 mmHg at 25°C

1.635

3.42

1

4

4

23

502

0

PJVWKTKQMONHTI-UHFFFAOYSA-N

InChI=1S/C19H16O4/c1-12(20)11-15(13-7-3-2-4-8-13)17-18(21)14-9-5-6-10-16(14)23-19(17)22/h2-10,15,21H,11H2,1H3

4-hydroxy-3-(3-oxo-1-phenylbutyl)chromen-2-one

WARF42; Athrombine-K; NSC 59813; NSC-59813; NSC59813; d-Con; Rat-ola; Tox-Hid; Warfarin Q

2934.99.03.00

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)

DMSO: ≥ 50 mg/mL (~162.2 mM)
H2O: < 0.1 mg/mL (Insoluble)

Solubility in Formulation 1: ≥ 2.5 mg/mL (8.11 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% 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 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.

---

Solubility in Formulation 2: ≥ 2.5 mg/mL (8.11 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.

---

View More

Solubility in Formulation 3: ≥ 2.5 mg/mL (8.11 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.

---

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