Warfarin: Metabolism and mode of action

doi:10.1016/0006-2952(88)90750-2

Biochemical Pharmacology

Volume 37, Issue 1, 1 January 1988, Pages 19-27

https://doi.org/10.1016/0006-2952(88)90750-2 Get rights and content

Abstract

The various stages involved in the transport, pharmacological action and elimination of warfarin involve the specific binding of warfarin to a chiral macromolecular complex. However, it seems that the degree of stereoselectivity is variable, which presumably reflects the importance of the side-chain in binding to each type of macromolecule. It would appear that there is greater stereoselective control in the interaction of warfarin with cytochrome P-450 enzymes than that observed for interaction with the receptor, vitamin K₁ epoxide reductase. Indeed, warfarin has been developed as a powerful stereochemical probe for in vitro studies of the terminal enzyme in the mixed-function oxidase system, cytochrome P-450. Warfarin undergoes hydroxylation in the 6, 7 and 8-positions of the aromatic ring which must interact with the active (haemoprotein) portion of the molecule, leaving the side-chain, which contains the chiral centre, free for recognition by the substrate binding site. In vitro studies indicate that the interaction of warfarin at its receptor, vitamin K₁ epoxide reductase, is completely non-stereoselective. This suggests that only the 4-hydroxycoumarin ring portion of the drug binds to the enzyme. Consistent with this hypothesis, salicylate, which can mimic part of the 4-hydroxycoumarin ring system, produces hypothrombinaemia by inhibition of vitamin K₁ epoxide reductase. These findings suggest that the coumarin ring system is largely responsible for the pharmacodynamic properties of warfarin, whereas the side-chain dictates the disposition and metabolism of the drug.

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We have performed the structural and spectroscopic analysis of a warfarin derivative (3-(1-(4-fluorophenyl)-3-(2-hydroxyphenyl)-3-oxopropyl)-4-hydroxy-2H-chromen-2-one). One-dimensional potential energy scanning (PES) of a warfarin derivative has been performed with the aim of finding low-lying energy conformations of this compound. During PES and other computations, we adopted DFT/B3LYP level along with the prevailing basis set 6-31++G(d,p). The conformational analysis through PES revealed five conformers and the global minima for conformer-4. The structures, IR and Raman spectra of conformers were deducted and the IR spectrum of the most stable conformer was correlated with the experimental FTIR spectrum of the target compound. The potential energy distributions (PEDs) for the most stable conformer was computed by employing the standard normal coordinate analysis (NCA) method in the input of Molvib program. Additionally, the HOMO/LUMO analysis and hyper-conjugative interaction energies of donor-acceptor interactions were calculated using NBO calculations. This conformational and vibrational spectroscopic investigation of the target molecule can be a crucial prerequisite for similar investigations on the target and its closely similar molecules.
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Overall, the spheroids captured the clinically relevant metabolites for all drugs tested, both primary and secondary metabolites (Table 2). Explicitly, the major human metabolites were identified for all test compounds in all three donors, i.e., carvedilol O-glucuronide and hydroxylations in the carbazolyl group followed by glucuronidation40; imipramine-demethylation to the active metabolite desipramine41,42; major metabolite N,N-deethylated sildenafil43,44; the two major human metabolites for diazepam, N-desmethyl-diazepam (nordiazepam) and 3-hydroxydiazepam (temazepam)45,46; disopyramide's CYP3A4 product N-desisopropyldisopyramide47; and finally, warfarin hydroxylations on the aromatic ring in positions 6′, 7′, and 8′, replicating some enantioselective preference between R- and S-warfarin.48–50 It is technically appealing that all these biotransformation reactions can be observed at incubation concentrations of 1 μM, minimising the risk that inhibitory effects from elevated substrate concentrations may influence the metabolite profiles.51
3D cultures of primary human hepatocytes (PHH) are emerging as a more in vivo-like culture system than previously available hepatic models. This work describes the characterisation of drug metabolism in 3D PHH spheroids. Spheroids were formed from three different donors of PHH and the expression and activities of important cytochrome P450 enzymes (CYP1A2, 2B6, 2C9, 2D6, and 3A4) were maintained for up to 21 days after seeding. The activity of CYP2B6 and 3A4 decreased, while the activity of CYP2C9 and 2D6 increased over time (P < 0.05). For six test compounds, that are metabolised by multiple enzymes, intrinsic clearance (CL_int) values were comparable to standard in vitro hepatic models and successfully predicted in vivo CL_int within 3-fold from observed values for low clearance compounds. Remarkably, the metabolic turnover of these low clearance compounds was reproducibly measured using only 1–3 spheroids, each composed of 2000 cells. Importantly, metabolites identified in the spheroid cultures reproduced the major metabolites observed in vivo, both primary and secondary metabolites were captured. In summary, the 3D PHH spheroid model shows promise to be used in drug discovery projects to study drug metabolism, including unknown mechanisms, over an extended period of time.
Binding of warfarin differently affects the thermal behavior and chain packing of anionic, zwitterionic and cationic lipid membranes
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It is administered orally to fluidize the blood in patients who require the prevention of venous thrombosis, or need treatment for pulmonary embolism, stroke, and heart diseases. Warfarin acts by lowering the amount of vitamin K that performs the action of blood clotting [9]. It is transported in the human body by proteins present in the biological fluid such as human serum albumin [10], and binds to the active site of the cytochrome P450 2C9 enzyme [11].
Warfarin is a coumarin derivative drug widely used for its anticoagulant properties. The interaction of warfarin with fully hydrated lipid bilayers has been studied by combining differential scanning calorimetry, spectrophotometry, electron spin resonance of chain-labelled lipids and molecular docking. Bilayers formed by lipids with different chemico-physical properties were considered, namely dimyristoyl-phosphatidylcholine (DMPC), dimyristoyl-phosphatidylglycerol (DMPG), and dioleoyltrimethyl-ammoniumpropane (DOTAP). We observed in all cases the binding of warfarin in proximity of the surface of the bilayers, leading to a variety of distinct effects on key molecular properties of the membranes. The drug associates with the lipid bilayers in the deprotonated open chain form, with an association constant similar for DMPC and DMPG (1.27·10⁴ and 2.82·10⁴ M⁻¹, respectively) and lower for DOTAP (0.46·10⁴ M⁻¹). In DMPC bilayers, which are zwitterionic and with saturated symmetrical chains, warfarin at 10 mol% suppresses the pre-transition, slightly stabilizes the fluid state and reduces the cooperativity of the main transition. Moreover, it alters the lateral packing density of the chain segments close to the polar/apolar interface at any temperature through the gel phase. In anionic DMPG bilayers, the drug slightly perturbs the thermotropic phase behavior, and at 10 mol% markedly loosens the compact gel phase packing of the first chain segments. In cationic DOTAP bilayers, possessing unsaturated acyl chains, the drug induces a slightly higher degree of order and motional restriction in the outer hydrocarbon region in the frozen state. In all cases, as a surface adsorbed molecule, warfarin does not affect the segmental chain order and dynamics for temperatures in the fluid phase. The overall results provide an outline of the action of warfarin on membranes formed by lipids of different types.
Heavy rainfall provokes anticoagulant rodenticides' release from baited sewer systems and outdoor surfaces into receiving streams
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As warfarin is the only anticoagulant that is concurrently authorized for biocidal and pharmaceutical use, its presence in wastewater and receiving surface waters can be linked to rodent control measures (Regnery et al., 2019b, 2020) as well as consumption of blood-thinning medication by resident population (Ajo et al., 2018; Regnery et al., 2019a; Santos et al., 2013). Like phenprocoumon, warfarin is extensively metabolized in humans and only about 2% of the typical daily prescription dose is excreted as unchanged active ingredient (Crouse et al., 2012; Park, 1988). According to IQVIA MIDAS, the annual domestic pharmaceutical use of these blood-thinning agents accounted for 801 kg of phenprocoumon and 26 kg of warfarin in Germany in 2018 (IQVIA, 2019).
Prevalent findings of anticoagulant rodenticide (AR) residues in liver tissue of freshwater fish recently emphasized the existence of aquatic exposure pathways. Thus, a comprehensive wastewater treatment plant and surface water monitoring campaign was conducted at two urban catchments in Germany in 2018 and 2019 to investigate potential emission sources of ARs into the aquatic environment. Over several months, the occurrence and fate of all eight ARs authorized in the European Union as well as two pharmaceutical anticoagulants was monitored in a variety of aqueous, solid, and biological environmental matrices during and after widespread sewer baiting with AR-containing bait. As a result, sewer baiting in combined sewer systems, besides outdoor rodent control at the surface, was identified as a substantial contributor of these biocidal active ingredients in the aquatic environment. In conjunction with heavy or prolonged precipitation during bait application in combined sewer systems, a direct link between sewer baiting and AR residues in wastewater treatment plant influent, effluent, and the liver of freshwater fish was established. Moreover, study results confirmed insufficient removal of anticoagulants during conventional wastewater treatment and thus indirect exposure of aquatic organisms in receiving streams via tertiary treated effluents and combined sewer overflows. Nevertheless, further research is required to determine the ecological implications and risks for aquatic organisms as well as fish-eating predators from chronic AR exposure at environmentally relevant concentrations.
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Protein-drug interaction is of prominent interest in determining the pharmacokinetic and pharmacodynamic consequences on drug delivery. Warfarin is a widely used anticoagulant drug in the treatment of venous thrombosis and pulmonary embolism and is carried in the blood almost exclusively by human serum albumin. The effects of the binding of warfarin to the native state of albumin were characterized by UV–vis absorption, conventional and synchronous fluorescence, isothermal titration calorimetry, differential scanning calorimetry and molecular dynamics simulation.
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^∗: Wellcome Senior Lecturer.

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Warfarin: Metabolism and mode of action

Abstract

J Biol Chem

Biochem Pharmacol

J Biol Chem

Biochem Pharmacol

Biochem Pharmacol

Life Sci

Eur J Cancer

Lancet

Biochem Pharmacol

J Biol Chem

Biochem Pharmacol

J Biol Chem

Anticoagulants and Myocardial Infarction: A Reappraisal

Studies of the haemorrhagic agent 3',3' methylenebis (4-hydroxycoumarin)

Am J Med Sci

Novel 4-hydroxycoumarin anticoagulants active against resistant rats

Nature, Lond

Warfarin and the inhibition of vitamin K activity by an oxide metabolite

Nature, Lond

Interindividual differences in the response to oral anticoagulants

Drugs

Studies on the 4-hydroxycoumarins XVII. The resolution and absolute configuration of warfarin

J Am Chem Soc

Absolute configuration of acenocoumarin

J Med Chem

Structure of warfarin in solution

J Med Chem

Conformations of selected 3-substituted 4-hydroxycoumarins in solution by nuclear magnetic resonance. Warfarin and Phenprocoumon

J Med Chem

Inhibition of prothrombin synthesis and epoxidation of vitamin K1 by anticoagulants in vitro

Biochem Biophys Res Commun

The preferred solution conformation of warfarin at the active site of cytochrome P-450 based on the CD Spectra in octanol/water model system

J Med Chem

A study of the relationship between the pharmacokinetics and pharmacodynamics of the 4-hydroxycoumarin anticoagulants warfarin, difenacoum and brodifacoum

Br J Pharmacol

Pharmacokinetics and pharmacodynamics of the enantiomers of warfarin in man

Clin Pharmacol Ther

Studies on the optical enantiomorphs of warfarin in man

Clin Pharmacol Ther

Comparative pharmacokinetics of coumarin anticoagulants. Pharmacokinetics of warfarin enantiomers

Clin Pharmacol Ther

Clinical pharmacokinetics and pharmacodynamics of warfarin—understanding the dose-effect relationship

Clin Pharmacokin

Pharmacokinetics of the enantiomers of acenocoumarol in man

Br J Clin Pharmacol

Stereoselective aspects in the pharmacokinetics and pharmacodynamics of acenocoumarol and its amino and acetamido derivatives in the rat

Drug Metab Dispos

Inhibition of prothrombin synthesis and epoxidation of vitamin K₁ by anticoagulants in vitro