Daria Pašalić
Department of Medical Chemistry, Biochemistry and Clinical Chemistry
Zagreb University School of Medicine
Šalata ul 2.
10 000 Zagreb, Croatia
Phone +385 (1) 4590 205; +385 (1) 4566 940
E-mail: dariapasalic [at] gmail [dot] com

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Plebani M.S02-1: Pharmacogenetic approach in optimizing therapy. Biochemia Medica 2009;19(Suppl 1):S25-S26.
Department of Laboratory Medicine, University Hospital of Padova, Padova, Italy
Corresponding author:mario [dot] plebani [at] unipd [dot] it
Pharmacogenetics is the study of the role of inheritance in inter-individual variation in drug response. The ultimate promise of pharmacogenetics is the possibility that knowledge of a patient’s DNA sequence might be used to enhance drug therapy to maximize efficacy, to target drugs only to those patients that are likely to respond or to avoid adverse drug reactions. In the past 10 to 15 years pharmacogenetics (PGx) has generated attention as a discipline with potential application to patient care. While the literature is still evolving as to clinical impact, the knowledge needed to define and interpret studies to assess clinical utility has drawn much debate. The application of PGx to enhancing the concept of personalized medicine hinges on the ability of clinical laboratories to provide accurate, timely and useful information, thus enabling the clinicians to administer the right drug (efficacy), at the right dose (dosing), for the right patients (safety efficacy) and at the right time.
In particular, cytochrome P450s (CYPs) are an important family of enzymes in the metabolism of many therapeutic agents and endogenous metabolic activities. This presentation will provide a review of the literature and of our personal experience in determining genotypes of alleles of selected CYP genes by using several technologies, including the Roche AmpliChip P450 Array.
A body of evidence demonstrates the importance of CYP genotyping, particularly regarding CYP2D6 and CYP2C19. CYP2D6 genotyping resulted to be very interesting in tamoxifen treated breast-cancer patients, as well as for prodrugs such as codeine opiates and antiarrythmics. CYP2C and its polymorphic isoform CYP2C19 is involved in metabolization of several important drugs including most tricyclic antidepressants, and the proton pump inhibitors omeprazole and lansoprazole. Moreover, being the Warfarin therapy a leading cause of hospitalization among adverse drug reactions, with medical consequences and costs, I will review our data on the application of pharmacogenetic information to predict dosing amounts and to eliminate what has been termed “oversteer” in using warfarin for anticoagulation therapy.
Translation of pharmacogenetic research into clinical practice requires explicit analytical and clinical evaluation, and a careful analysis of the balance of benefits and associated risks, including costs. The barriers to using PGx will be discussed as well as the ways to overcame these barriers.
Štefanović M. S02-2: From pharmacogenetic genotype to drug dose prediction. Biochemia Medica 2009;19(Suppl 1):S26-S27.
University Department of Chemistry, Sestre Milosrdnice University Hospital, Zagreb, Croatia
Corresponding author: mstefan6 [at] gmail [dot] com
Despite quick development of pharmacogenetics in the latest 20 years, there are only few genes with pharmacogenetic polymorphisms that are routinely determined, and their clinical usage is clearly defined. Many questions affect the process that start with a new genetic polymorphism discovery and ends with a clinical usage of its determination. Most of these questions are gathered in Guidelines and Recommendations for Laboratory Analysis and Application of Pharmacogenetics to Clinical Practice (2007, 3rd draft) issued by National Academy of Clinical Biochemistry (NACB). This lecture brings a short overview of those guidelines.
Problems in Pharmacogenetic testing. There are many examples of specific problems in pharmacogenetics. Because of great differences in allele frequencies among individual ethnic groups, every laboratory should perform polymorphism frequencies testing on its own, as well as it should follow their trend statistics. Some methods (e.g. sequencing) are not capable of determining genetic changes like big genomic insertions or deletions. Control material should be derived from the renewable independent source, and while performing the analysis, it should take into account inhibitors or interferences which could lead to misinterpretation of the testing results, when compared to the clinical presentation.
Laboratory results. Recommendation is that laboratory results should unambiguously resemble genetic structure and quote only genetic characteristics that were confirmed, in order not to mislead the physician. Direct “genotype to phenotype” prediction could be dangerous, because phenotype classification does not depend on genotype only, but also on researched drug/substrate. Laboratory results should clarify the clinician a complex picture of metabolic pathways and enzymes affected, evaluate the risk to interactions and analyze the current and previous mode of therapy. Alternatively, the laboratory results should at least cite warning interaction examples.
Laboratory should also provide the possibility of interpretation of the findings, but is not recommended for the laboratory to predict drug dose. Methodology and limitations of pharmacogenetic testing should be clearly indicated on the findings, along with the analytical sensitivity/specificity.
Selection for polymorphism test. Polymorphisms included in testing should have their clinically impact on function, pharmacokinetics, pharmacodynamics, and/or drug toxicity defined, but there is still no consensus on all the criteria that this choice should include.
Economic calculation. Concerning the financial viability of pharmacogenetic testing, recommendation is in favor of developing tests that lead to the mitigation of side effects, improving the effectiveness of therapy, or ultimately reduce the overall cost of treatment. Wider population screening is not recommended.
Applying the models. There are several examples of clinical applications of pharmacogenetic testing. Determination of enzymes CYP2C9 and vitamin K-dependent reductase (VKORC) is a successful approach derived from multivariate regression models, in estimating the dose in warfarin anticoagulant therapy. The next example is determining thiopurine methyltransferase (TPMT) in the dosing strategy of oncology patients on azathioprin therapy. Pharmacogenetic algorithms of CYP2D6 enzyme are successfully applied in the treatment of certain psychiatric disorders with atomoxetine and the treatment of breast cancer with tamoxifen. Testing UGT1A1 enzyme prevents irinotecan toxicity in oncology.
In conclusion, therapy drug monitoring is complementary with pharmacogenetic testing and it enables selection of drug and dose for those medicines with clear correlation between genotype and dosing.
Božina N1, Makar-Aušperger K2.S02-3: Pharmacogenetics of coumarin anticoagulants. Biochemia Medica 2009;19(Suppl 1):S28-S29.
1Clinical Institute of Laboratory Diagnosis, Zagreb University School of Medicine and Clinical Hospital Center, Zagreb, Croatia
2Department of Internal Medicine, Division of Clinical Pharmacology, Zagreb University Hospital, Zagreb, Croatia
Corresponding author:nbozina [at] kbc-zagreb [dot] hr
Introduction: Dosing of the coumarin type anticoagulants presents a challenging task due to their narrow therapeutic range and a large variability in dose-response relationship. This variability can partly be explained by polymorphisms of the CYP2C9 gene which encodes the main metabolizing enzyme of coumarins, and by polymorphisms of vitamin K epoxide reductase (VKOR). Coumarins act by inhibiting VKOR activity, their target having been identified as the protein vitamin K reductase complex subunit 1 (VKORC1) which is encoded by the homonymous gene VKORC1. Carriers of a combination of CYP2C9 polymorphism and VKORC1 polymorphism had an increased risk of severe overanticoagulation compared to subjects with no polymorphism or only one polymorphism. Patients with VKORC1 polymorphism required significantly lower doses than VKORC1 wild-type patients. Empirical dose tables based on pharmacogenetics of these enzymes have been proposed, but none of them has yet been widely accepted. The aim of this study was to assess the frequencies of CYP2C9 (CYP2C9*2 and CYP2C9*3 alleles) and the VKORC1 C1173T genotype and to determine the relationship between genetic variations of CYP2C9 and VKORC1 and warfarin dose.
Patients and methods: Patients with an indication for warfarin hospitalized at the Department of Internal Medicine, University Hospital Center Zagreb (N = 157) were genotyped for CYP2C9 and VKORC1 polymorphisms. Prescribed dose on discharge was retrospectively linked to the enzyme variants. Concomitant medications and age were also taken into account when adjusting the required warfarin dose. The genotyping of CYP2C9 (alleles *2 and *3) was performed by Real time PCR method, using TIB MOLBIOL LightMix in Roche LightCycler instrument. The genotyping of VKORC1 C1173T was performed by Real time PCR method in LightCycler Fast Start DNA Master plus HybProbe master mix, and by PCR-RFLP method.
Results: Slow, intermediate and fast metabolizers accounted for 6%, 61% and 33% regarding CYP2C9, and for 13%, 49% and 38 % regarding VKORC1 variants, respectively. Our findings show that č76% of hospitalized patients on warfarin treatment have at least one “sensitive” (CYP2C9*2,*3, VKORC1 1173T) allele. Warfarin dosage on discharge showed correlation with VKORC1 (P < 0.001) and CYP2C9 allele variants (P = 0.041). Patients with VKORC1 and/or CYP2C9 high activity alleles required statistically higher warfarin doses (6.5 ± 1.38 mg) compared to patients with intermediate (2.48 ± 1.62 mg; P < 0.001) and low activity alleles (0.87 ± 0.88 mg; P < 0.03).
Conclusion: VKORC1 and CYP2C9 variants have significant influence on the definition of warfarin dose and could serve clinicians as important genetic markers for coumarin type drug response.
Nikolac N.S02-4:Sulfonylurea receptor-1 (SUR-1) polymorphisms in regulation of type 2 diabetes. Biochemia Medica 2009;19(Suppl 1):S29-S30.
University Department of Chemistry, Sestre Milosrdnice University Hospital, Zagreb, Croatia
Corresponding author:nora [dot] nikolac [at] gmail [dot] com
Type 2 diabetes has become one of the leading health issues worldwide. In the beginning, therapeutic approach includes only lifestyle modifications. If this approach doesn’t yield desired metabolic control, oral hypoglycaemic agents are introduced and sometimes even insulin therapy is required.
Sulfonylureas are hypoglycaemic agents used for diabetes treatment since the 1950’s. Mechanism of action includes increased insulin release from beta cells in pancreas. They bind to sulfonylurea receptor-1 (SUR-1), which is a functional subunit of the ATP-sensitive potassium channel (KATP) located in pancreatic beta cells in islets of Langerhans. The other component of potassium channel is Kir6.2, inwardly rectifying ion channel forming a pore, encoded by gene KCNJ11. Sulfonylureas bind to SUR-1 subunit of potassium channel causing its closure, opening of voltage-gated Ca2+ channels, increase of intracellular Ca2+ concentration and stimulation of insulin release from secretory granules by exocytosis. Closure of KATP is not initiated only by sulfonylurea action, but also by ATP production in glucose metabolism, indicated significant role of SUR-1 and Kir6.2, not only in diabetics on sulfonylurea therapy, but in diabetes in general.
In therapy of type 2 diabetes, combined approach is very common. In most of the patients sulfonylurea therapy accomplishes desired metabolic control, however in a number of patients that effect is very limited. In that case, sulfonylurea is combined with insulin, and in more severe cases only insulin therapy is indicated. According to the results of large 20-years prospective study (UK Prospective Diabetes Study - UKPDS), each year there is a failure of sulfonylurea therapy and introduction of insulin in 5-7% of type 2 diabetics.
The genes encoding SUR-1 and Kir6.2 are located on the chromosome 11p15.1. Gene for the SUR-1 (ABCC8, ATP-binding cassette, sub-family C (CFTR/MRP), member 8) consists of 39 exons, and spans 100 kbp in length. Gene for the other subunit Kir6.2 (KCNJ11, potassium inwardly-rectifying channel, subfamily J, member 11) consists of only one exon.
Polymorphisms of these genes can lead to increased probability of opening of potassium channel, decreased sensitivity on ATP inhibition and increased cut-off for insulin release. These mechanisms can lead to modulated response to sulfonylurea therapy and poor metabolic control of type 2 diabetes. This can be quantified by measuring biochemical and anthropometric parameters that are in correlation with degree of metabolic control.
This lecture will present results of our research on association of SUR-1 and KCNJ11 polymorphisms with diabetes associated phenotypes in patients on sulfonylurea therapy.
Lovrić M1, Božina N1, Sporiš D2, Hajnšek S2.S02-5: Impact of pharmacogenetic variations on antiepileptic serum level. Biochemia Medica 2009;19(Suppl 1):S30-S31.
1Clinical Institute of Laboratory Diagnosis, Zagreb University School of Medicine and Clinical Hospital Center, Zagreb, Croatia
2Department of Neurology, Zagreb University Hospital, Zagreb, Croatia
Corresponding author:lovricm [at] kbc-zagreb [dot] hr
Introduction: A large number of antiepileptics is used today to treat epilepsy. Therapeutic monitoring of this disorder is frequent and necessary due to considerable interindividual differences in drug concentrations and efficacy. Along with clinical and exogenous factors, genetic predispositions have been recognized as an important factor in therapy individualization and they take a prominent place in developing algorithms for selection of the most appropriate drug and dose for each patient. Among pharmacogenetic markers, polymorphic transport proteins, which are responsible for drug transport across various barriers, offered themselves as significant factors of variability and bioavailability of different antiepileptics. Transporter proteins that play important role in pharmacokinetics are located in intestinal, renal and hepatic epithelial membranes. ABC transporters such as P-glycoprotein (ABCB1) and multidrug resistance-associated protein 2 (ABCC2) affect bioavailability of their substrate drugs.
Aims of the study were to evaluate the impact of polymorphisms in the ABCB1 (C3435T, C1236T, G2677T/A) and ABCC2 (C24T, G1249A) on antiepileptic drug disposition. We therefore correlated plasma levels of lamotrigine in mono- and polytherapy (carbamazepine, oxcarbazepine, levetiracetame, phenytoin, phenobarbiton, topiramate, valproate) with gene variants. As covariates, age, gender, height, weight, liver and renal biochemical parameters were included.
Patients and methods: 122 epileptic patients, from 18-70 years old, were stratified into lamotrigine monotherapy group (N = 25), a group receiving lamotrigine plus inductors (N = 60), inhibitors (N = 19) or both (N = 18). ABCB1 genotyping (C1236T, C3435T, G2677T/A) was performed by Real-time PCR and PCR-RFLP; ABCC2 (G1249A, C24T) by PCR-RFLP methods. Therapeutic drug monitoring was performed by HPLC with diode array detector and immunoassay.
Results: We found significant differences in lamotrigine concentrations depending on: add-on therapy (P < 0.001); ALT, age and weight (P < 0.01). Statistical analysis showed borderline correlation between lamotrigine concentrations and C24T variant ABCC2 gene (P = 0.074; CC+CT: TT, 12.2: 22.8 μmol/L). 1249GG and 1249AA carriers of the ABCC2 gene had mean lamotrigine concentration of 11.6 and 18.9 μmol/L, respectively. Mutation on ABCB1 gene also revealed tendency toward correlation of C1236T genotypes with lamotrigine concentrations (CC: TT, 14.8: 11.1 μmol/L) and for C3435T (CC: TT, 13.3: 10.4 μmol/L), but the number of TT genotype carriers was a limiting factor for statistical significance.
Conclusions: Preliminary data are suggestive and point that ABCB1 and ABCC2 polymorphisms may influence AED disposition and may account for interindividual pharmacokinetic variability. Due to controversial results reported in literature and still indefinite role of ABC transport proteins in the pharmacokinetics of antiepileptics, we believe that further investigation is necessary on a larger number of patients in order to include these pharmacogenetic indicators in dosing algorithms for antiepileptic drugs.