liu.seSearch for publications in DiVA
Change search
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf
Studies on warfarin treatment with emphasis on inter-individual variations and drug monitoring
Linköping University, Department of Clinical and Experimental Medicine, Clinical Chemistry. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Centre for Laboratory Medicine, Department of Clinical Chemistry.
2007 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [sv]

Waran används sedan 60 år som blodförtunnande läkemedel för att förebygga eller förhindra progress av blodproppssjukdom. I Sverige behandlas årligen cirka 1 % av befolkningen med waran. I Östergötland uppskattas antal waranpatienter till cirka 3000. Waran hämmar enzymet VKORC1 som ansvarar för vitamin K omsättningen i kroppen. Vitamin K behövs som kofaktor för flera koagulationsfaktorer.

Behandling med waran är förenad med en svår balansgång och kräver en noggrann dosering. Stora skillnader i dosbehov mellan olika individer, beroende på ärftliga och miljöfaktorer, gör waran till ett svårhanterligt läkemedel. För låg dos medför otillräcklig effekt och därmed risk för minskat skydd mot blodproppssjukdom. För hög dos leder till allvarliga blödningskomplikationer. Uppskattningsvis 1 – 3 % livshotande blödningsfall registreras årligen efter waranbehandling. Därför måste behandlingen kontrolleras noga med analys av protrombinkomplex (PK) och dosjusteringar göras med ledning av resultaten. Två olika metoder finns att använda för mätning av PK. I Norden och i Japan används Owrens metod (utvecklat i Norge under 40- och 50-talet av Paul Owren). I de flesta andra länder används Quickmetoden (utvecklat i USA under 30-talet av Armand Quick). Den senare metoden är förenad med stora variationer mellan olika analyslaboratorier. I Norden, däremot, där Owrens metod används finns det ofta bra överensstämmelse mellan olika laboratorier i PK-resultat. Beroende på vilken PK-metod som används, kan samma patient få olika warandoser vilket ökar risker för under- eller överbehandling.

Vi har i samarbete med flera sjukhus och antikoagulationsmottagningar (AK-mottagningar) i sydöstra Sverige studerat dels mekanismerna bakom skillnader i warandos mellan olika patienter, och dels tittat varför de olika PK-metoder skiljer sig så mycket som de gör.

I studien har vi identifierat genetiska varianter av enzymet VKORC1. Av de undersökta patienter som gick på waran under längre tid, har vi identifierat en grupp som markant skiljde sig från de övriga. Denna grupp hade warandoser som var betydligt lägre än de övriga. När vi kartlade deras arvsmassa, fann vi att lågdospatienterna hade genvarianten VKORC1*2. Dessutom hade patienter med denna variant svårigheter att få stabila PK-värden. De gjorde också fler besök på AK-mottagningar än andra patienter. Vi har därför konstaterat att en del av de problem som är förenade med waranbehnadlingen kan förklaras av VKORC1*2 varianten. Vetskap om denna variant skulle troligen underlätta behandlingen framför allt under inledningsfasen då patienter med VKORC1*2 riskerar blödningar på grund av överdosering.

Vi har identifierat att provförspädning enligt Owrens metod är nödvändig för harmonisering av PK-resultatet mellan olika länder. Quickmetoden använder inte förspädning av patientprov till skillnad från Owrens metod. När vi modifierade en Quickmetod genom att förspäda prover enligt Owrens metod noterade vi en bra överensstämmelse mellan de två olika metoderna. Däremot var resultatet sämre utan provförspädning. Vi anser att Quickmetoder kan uppnå lika bra resultat som Owrens metod om prover förspäds som i Owrens metod. Det skulle gynna patienter som reser mellan olika regioner eller länder och leda till en bättre övervakning av waranbehandling internationellt.

I studien har dessutom en metod för mätning av waran i blodet utvecklats. Metoden som är den enda i sitt slag i Norden ger möjlighet att studera hur läkemedlet beter sig i kroppen. Vi har med denna metod kunnat upptäcka patienter som har onormala nedbrytningar av waran.

Abstract [en]

Warfarin was introduced more than 60 years ago and is used worldwide for the prophylaxis of arterial and venous thromboembolism in primary and secondary prevention. The drug is orally administered as a racemic mixture of (R)- and (S)-enantiomers. The (S)-form is mainly responsible for the anticoagulant effect and is metabolised by CYP2C9 enzyme in the liver microsomes. Warfarin exerts its pharmacological action by inhibiting the key enzyme (VKORC1) that regenerates vitamin K from an oxidised state to a reduced form. The latter is a cofactor for the post-translational modification of a number of proteins including coagulation factors II, VII, IX and X. The vitamin K-dependent modification provides these factors with the calcium-binding ability they require for the interaction with cell membranes of their target cells such as platelets.

Warfarin is monitored by measuring prothrombin time (PT) expressed as INR. Two main methods exist for PT analysis. The Owren method is used mainly in the Nordic and Baltic countries, in Japan, whereas the Quick method is employed in most other countries. Warfarin management is associated with some complications. Unlike many other drugs the dose for a given patient cannot be estimated beforehand, dose-response relationship is not predictable, and the prevention of thrombosis must be balanced against the risk of bleeding. Furthermore, the different PT methods used to monitor the drug are sometimes not in agreement and show significant discrepancies in results.

In an attempt to clarify the mechanisms influencing the inter-individual variations in warfarin therapy and to detect the factors that contribute to differences between PT methods, studies were conducted in collaboration with hospitals and anticoagulation clinics in the south-eastern region of Sweden. First, a stereo-specific HPLC method for measurement of warfarin enantiomers was developed and validated. With this method, the levels of plasma warfarin following its oral administration can be studied and evaluated. Abnormal clearance in some patients can be detected, and patient compliance can be verified. Furthermore, differing ratios of (S)- and (R)-isomers can be identified.

The impact of common VKORC1 polymorphisms on warfarin therapy was investigated. This study has shown that the VKORC1*2 haplotype is an important genetic determinant for warfarin dosage and is associated with difficulties in attaining and retaining therapeutic PT-INR. Further, significant differences in warfarin S/R-ratio was detected between patients with VKORC1*2 and VKORC1*3 or VKORC1*4 variants. This difference was not coupled with CYP2C9 genotype.

The effects of predilution of patient plasma samples, sources of thromboplastin and deficient plasma on between PT methods agreement were studied. This study has revealed that sample predilution according to the Owren method is to be preferred for the harmonisation of PT results. Undiluted samples, in contrast, according to the Quick method have shown reduced correlation between two different thromboplastin reagents. Sources of thromboplastin and deficient plasma were only of minor importance.

Place, publisher, year, edition, pages
Institutionen för biomedicin och kirurgi , 2007. , 68 p.
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 1000
Keyword [en]
Warfarin, VKORC1, Haplotype, Prothrombin time, INR
National Category
Other Clinical Medicine
Identifiers
URN: urn:nbn:se:liu:diva-8874ISBN: 978-91-85715-45-9 (print)OAI: oai:DiVA.org:liu-8874DiVA: diva2:23586
Public defence
2007-06-01, Berzeliussalen, Campus HU, Universitetssjukhuset, Linköping, 09:00 (English)
Opponent
Supervisors
Available from: 2007-05-21 Created: 2007-05-21 Last updated: 2009-08-22
List of papers
1. A new high-performance liquid chromatographic method for determination of warfarin enantiomers.
Open this publication in new window or tab >>A new high-performance liquid chromatographic method for determination of warfarin enantiomers.
2005 (English)In: Journal of Chromatography B, ISSN 1570-0232, Vol. 826, no 1-2, 75-80 p.Article in journal (Refereed) Published
Abstract [en]

Warfarin is the most common agent used for control and prevention of venous as well as arterial thromboembolism. Although warfarin is administered as a racemic mixture of two stereoisomers (S and R), the S-form is mainly responsible for the anticoagulant effect. The anticoagulant effect of the drug is monitored by analysis of prothrombin complex (International Normalised Ratio,INR). In some cases, however, the measurements of plasma warfarin concentration are needed. Here, we present a new, rapid, sensitive and cost-effective HPLC-method for the determination of warfarin enantiomers in plasma. The chromatographic system consisted of Waters 616 gradient pump, Waters 996 photo diode array detector, Gilson 230 autoinjector and Pirkle (R,R) Whelk-O1 column (25 cm × 4.6 mm I.D., 5 μm). An isocratic mobile phase of methanol/acetonitrile/water (50/10/40, v/v) with 0.1% glacial acetic acid was used. The follow rate was 1 mL/min. Data analysis was carried out with Waters Millennium32. The absorbance at 305 nm was measured with a total run-time of 15 min. Method linearity was studied by establishing regression data containing eight points over the range 0.08–10 μg/mL. In this range, warfarin showed to be linear (r2 = 0.9997 for S-warfarin and r2 = 0.9998 for R-warfarin). The limit of detection in plasma was 16 ng/mL for S-warfarin and 18 ng/mL for R-warfarin. Limit of quatitation was defined as 10 × LOD. The extraction recovery was approximately 80%. Also the relation between INR and warfarin concentration was investigated. As expected, there was a low correlation between these two variables (r = 0.23, y = 0.3044x + 0.9712). This method offers a rapid and cost-effective determination of warfarin enantiomers in human plasma.

Keyword
S- and R-warfarin; Oxybenzone; INR
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-14487 (URN)10.1016/j.jchromb.2005.08.011 (DOI)
Available from: 2007-05-21 Created: 2007-05-21 Last updated: 2013-09-03
2. Main haplotypes and mutational analysis of vitamin K epoxide reductase (VKORC1) in a Swedish population: A retrospective analysis of case records
Open this publication in new window or tab >>Main haplotypes and mutational analysis of vitamin K epoxide reductase (VKORC1) in a Swedish population: A retrospective analysis of case records
Show others...
2006 (English)In: Journal of Thrombosis and Haemostasis, ISSN 1538-7933, Vol. 4, no 8, 1723-1729 p.Article in journal (Refereed) Published
Abstract [en]

Background: Vitamin K epoxide reductase (VKORC1) is the site of inhibition by coumarins. Several reports have shown that mutations in the gene encoding VKORC1 affect the sensitivity of the enzyme for warfarin. Recently, three main haplotypes of VKORC1; *2, *3 and *4 have been observed, that explain most of the genetic variability in warfarin dose among Caucasians.

Objectives: We have investigated the main haplotypes of the VKORC1 gene in a Swedish population. Additional objective was to screen the studied population for mutations in the coding region of VKORC1 gene.

Patients/methods: Warfarin doses and plasma S- and R-warfarin of 98 patients [with a target International Normalized Ratio (INR) of 2.0–3.0] have been correlated to VKORC1 haplotypes. Controls of 180 healthy individuals have also been haplotyped. Furthermore, a retrospective analysis of case records was performed to find any evidence indicating influence of VKORC1 haplotypes on warfarin response in the first 4 weeks (initiation phase) and the latest 12 months of warfarin treatment.

Results and conclusions: Our result shows that VKORC1*2 is the most important haplotype for warfarin dosage. Patients with VKORC1*2 haplotype had more frequent visits than patients with VKORC1*3 or *4 haplotypes, higher coefficient of variation (CV) of prothrombin time-INR and higher percentage of INR values outside the therapeutic interval (i.e. 2.0–3.0) than patients with VKORC1*3 or *4 haplotypes. Also, there was a statistically significant difference in warfarin dose (P < 0.001) and R-warfarin plasma levels (P < 0.01) between VKORC1*2 and VKORC1*3 or 4 haplotypes. Patients with VKORC1*2 haplotype seem to require much lower warfarin doses than other patients.

Keyword
INR, VKORC1, warfarin
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-14488 (URN)10.1111/j.1538-7836.2006.02039.x (DOI)
Available from: 2007-05-21 Created: 2007-05-21 Last updated: 2013-09-03
3. Plasma S/R ratio of warfarin co-varies with VKORC1 haplotype
Open this publication in new window or tab >>Plasma S/R ratio of warfarin co-varies with VKORC1 haplotype
2007 (English)In: Blood Coagulation and Fibrinolysis, ISSN 0957-5235, E-ISSN 1473-5733, Vol. 18, no 3, 293-296 p.Article in journal (Refereed) Published
Abstract [en]

We recently reported that the low-dose VKORC1*2 haplotype is an important genetic determinant for warfarin dose requirement and is associated with difficulties to attain stable therapeutic prothrombin time-International Normalized Ratio in patients undergoing anticoagulation therapy. The aim of this study was to investigate whether patients with VKORC1*2 compared with patients carrying high-dose haplotypes VKORC1*3 or VKORC1*4 had different warfarin S/R ratios in their plasma, and whether that was related to CYP2C9 variants CYP2C9*2 and CYP2C9*3 or other factors. Samples from patients previously haplotyped for VKORC1 and measured for plasma warfarin concentration were genotyped for the CYP2C9 variants CYP2C9*2 and CYP2C9*3. Nonparametric statistical analysis was performed to elucidate whether there was any significant difference in the warfarin S/R ratio between the two patient groups. Our result shows that there is a significant difference (P < 0.01) in warfarin S/R ratios between VKORC1*2 and VKORC1*3 or VKORC1*4 patients. This difference did not originate from CYP2C9 variants CYP2C9*2 and CYP2C9*3. We speculate that VKORC1 haplotypes possibly are linked to some unidentified factors involved in the metabolic clearance of warfarin enantiomers. Dose-dependent variations in (S)-warfarin and (R)-warfarin clearance in these patients can also be a probable explanation for the difference in warfarin S/R ratios.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-14489 (URN)10.1097/MBC.0b013e3280444bfd (DOI)
Available from: 2007-05-21 Created: 2007-05-21 Last updated: 2017-04-05Bibliographically approved
4. Plasma sample dilution improves the correlation: between reagents for PT methods
Open this publication in new window or tab >>Plasma sample dilution improves the correlation: between reagents for PT methods
2007 (English)In: XXIst ISTH Congress, 2007Conference paper, Published paper (Refereed)
Abstract [en]

Introduction: The Quick (plain thromboplastins) and Owren (combined thromboplastins) PT methods are used worldwide for the monitoring of anticoagulation therapy with vitamin K antagonists. Although both methods measure the activity of vitamin K dependent coagulation factors, the Quick PT-result in addition is dependent on the activity of factor V and of fibrinogen. The Quick PT-methods are also associated with larger inter-laboratory variations than Owren PT-methods. We have investigated whether the dilution of the sample, the source of depleted plasma or the source of thromboplastin have an impact on the correlation between the different PT-reagents.

Methods: Patient and quality control samples were analysed undiluted and prediluted 2x, 7x, 10x, and 12x in Owren's buffer. One part of sample was then mixed with two parts of reagent and calcium. Bovine vs. human depleted plasmas and rabbit brain vs. human placenta thromboplastins were compared in combinations. The assays were run on an ACL Top from Instrumentation Laboratory (Italy).

Results: Our results show that the dilution of the sample is important for the correlation between different PT-reagents. We found the best correlation (r = 0.95) when the sample was 7-fold prediluted (1 part + 6 parts). The lowest correlation was found when the sample was undiluted (r = 0.67). The source of thromboplastin had a small, if any, impact on the PT-results provided the sample was prediluted.The source of depleted plasma had no significance for the relationship between the PT-reagents. The depleted plasma is necessary to in order enable clotting of prediluted plasma.

Conclusions: We conclude that the Owren style sample dilution is to prefer for the harmonization of PT-results and to overcome the large inter-laboratory variations that are associated with Quick PT-methods.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-14490 (URN)
Note
This paper is published in volume 5, supplement 1 of Journal of Thrombosis and Haemostasis (2007).Available from: 2007-05-21 Created: 2007-05-21 Last updated: 2009-12-18

Open Access in DiVA

cover(262 kB)34 downloads
File information
File name COVER01.pdfFile size 262 kBChecksum MD5
9a5b3d6c226f930129700ef7538e0bdb67703b9b5dbc4bbe01adabfa52a0fa2cf0bde468
Type coverMimetype application/pdf
fulltext(517 kB)4083 downloads
File information
File name FULLTEXT01.pdfFile size 517 kBChecksum MD5
1f93047be440b73afb4fcc76a46d458cece9db7cbeab348539a4e9ef1f2efc922104a36f
Type fulltextMimetype application/pdf

Search in DiVA

By author/editor
Osman, Abdimajid
By organisation
Clinical ChemistryFaculty of Health SciencesDepartment of Clinical Chemistry
Other Clinical Medicine

Search outside of DiVA

GoogleGoogle Scholar
Total: 4083 downloads
The number of downloads is the sum of all downloads of full texts. It may include eg previous versions that are now no longer available

Total: 2128 hits
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf