The in-vitro and in-vivo metabolism of the oral anticoagulant phenprocoumon as influenced by genetic polymorphisms of cytochrome P4502C9

Oral anticoagulants are widely used for the prevention of thromboembolic disorders. Warfarin (WA) is most commonly used world-wide, while phenprocoumon (PPC) is the first-line anticoagulant in some European countries including Germany. Each anticoagulant exists in two different enantiomeric forms and is administered orally as a racemate. Due to a narrow therapeutic index the anticoagulant response needs to be monitored throughout treatment. Despite such effect monitoring, differences of the dose-response relationship often give rise to bleeding complications or insufficient anticoagulation. These variations are mainly due to genetic and environmental factors that influence the pharmacokinetics of oral anticoagulants.

Previous knowledge in this regard principally referred to WA and cytochrome P450 (CYP) 2C9 has been established as main catalyst responsible for the metabolism of its more potent S-enantiomer. The significant role of CYP2C9 polymorphisms in the response to WA treatment is increasingly appreciated. The general aims of this thesis were to identify those CYP enzymes catalysing S- and RPPC hydroxylation and to evaluate the impact of genetic polymorphims of the identified isoforms on the biotransformation of PPC in vitro and in vivo.

In Study 1, CYP2C9 and CYP3A4 were identified as major catalysts of S- and R-PPC hydroxylation in vitro by kinetic, correlation and inhibition studies using CYP-specific chemical and immunological inhibitors. Overall, CYP2C9 was little more important than CYP3A4 in catalysing PPC hydroxylation with minor contribution of CYP2C8.

In Study 2 and 3, analytical techniques for the quantification of PPC, WA and their monohydroxylated metabolites in human plasma and urine were developed. High selectivity and sensitivity is a prerequisite to study PPC biotransformation due to its slow metabolite formation rate and was achieved by HPLC-MS or HPLC-MS/MS.

In Study 4, the pharmacokinetics of S- and R-PPC were studied in healthy volunteers expressing all six allele combinations of CYP2C9*1, 2C9*2 and 2C9*3. Overall, the plasma clearance of S-PPC was moderately reduced in CYP2C9 variant allele carriers, while RPPC clearance was essentially unaffected. However, the metabolite formation clearances were impaired in a gene-dose dependent manner.

In Study 5, the impact of CYP2C9 polymorphisms on the hydroxylation of PPC was stereoselectively studied in vitro and in vivo. The S-7-hydroxylation -being the major metabolic pathway- was significantly compromised in a gene-dose dependent manner, while other reactions were much less influenced by CYP2C9 genotype.

In conclusion, CYP2C9 appears markedly less important for the clearance of PPC than of WA due to the role of CYP3A4 as additional catalyst of S- and R-PPC hydroxylation and a significant excretion of unchanged drug. Consequently, CYP2C9 polymorphisms only moderately influence the pharmacokinetics and the anticoagulant response of PPC that seems preferable over WA for therapeutic anticoagulation in poor metabolisers of CYP2C9.

List of scientific papers

I. Ufer M, Svensson JO, Krausz KW, Gelboin HV, Rane A, Tybring G (2004). Identification of cytochromes P450 2C9 and 3A4 as the major catalysts of phenprocoumon hydroxylation in vitro. Eur J Clin Pharmacol. 60(3): 173-82. Epub 2004 Mar 31
https://pubmed.ncbi.nlm.nih.gov/15054565

II. Kammerer B, Kahlich R, Ufer M, Laufer S, Gleiter CH (2004). Determination of (R)- and (S)-phenprocoumon in human plasma by enantioselective liquid chromatography/electrospray ionisation tandem mass spectrometry. Rapid Commun Mass Spectrom. 18(4): 458-64.
https://pubmed.ncbi.nlm.nih.gov/14966853

III. Ufer M, Kammerer B, Kirchheiner J, Rane A, Svensson JO (2004). Determination of phenprocoumon, warfarin and their monohydroxylated metabolites in human plasma and urine by liquid chromatography-mass spectrometry after solid-phase extraction. J Chromatogr B Analyt Technol Biomed Life Sci. 809(2): 217-26.
https://pubmed.ncbi.nlm.nih.gov/15315768

IV. Kirchheiner J, Ufer M, Walter EC, Kammerer B, Kahlich R, Meisel C, Schwab M, Gleiter CH, Rane A, Roots I, Brockmoller J (2004). Effects of CYP2C9 polymorphisms on the pharmacokinetics of R- and S-phenprocoumon in healthy volunteers. Pharmacogenetics. 14(1): 19-26.
https://pubmed.ncbi.nlm.nih.gov/15128047

V. Ufer M, Kammerer B, Kahlich R, Kirchheiner J, Yasar U, Brockmoller J, Rane A (2004). Genetic polymorphisms of cytochrome P450 2C9 causing reduced phenprocoumon (S)-7-hydroxylation in vitro and in vivo. Xenobiotica. [Accepted]