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  • Pasanen, Marja (Helsingfors universitet, 2008)
    Wide interindividual and interethnic variability exists in the plasma concentrations of the cholesterol-lowering drugs HMG-CoA reductase inhibitors (statins) in their efficacy and risk of adverse effects. The risk of muscle toxicity as an adverse effect of statin therapy is known to increase along with elevated plasma statin concentrations. Organic anion-transporting polypeptide 1B1 (OATP1B1) is an uptake transporter located on the basolateral (sinusoidal) membrane of human hepatocytes, encoded by the gene SLCO1B1. OATP1B1 facilitates the hepatic uptake of many endogenous and foreign compounds, such as oestrogen conjugates, bile acids and statins. Taking into consideration the known interindividual and interethnic differences in the disposition of many OATP1B1 substrates, particularly statins, and its functional role in the pharmacokinetics of many drugs, it is important to characterize the diversity of the SLCO1B1 gene in various ethnic groups and to investigate the effects of SLCO1B1 polymorphism on statin disposition and response. The frequencies of SLCO1B1 sequence variations were studied in a population of 468 healthy Finnish volunteers and globally in various ethnic populations. DNA samples from the participants were genotyped for the presence of single nucleotide polymorphisms (SNPs) in SLCO1B1 and the results were analysed using population genetic methods. Secondly, the effects of SLCO1B1 genotypes on the pharmacokinetics and pharmacodynamics of fluvastatin, pravastatin, simvastatin, rosuvastatin and atorvastatin, and on cholesterol homeostasis, were studied in a prospective genotype panel study with 32 healthy volunteers. The subjects ingested a single dose of each investigated statin in five different phases. Blood samples were collected before statin administration and up to 48 hours thereafter to determine the concentrations of plasma statins, statin metabolites, cholesterol and noncholesterol sterols. Functionally significant variants in the SLCO1B1 gene were detected at varying frequencies in different populations. Genetic variation in SLCO1B1 was generally similar to that observed for other autosomal markers, although selective pressure may have acted on SLCO1B1, favouring low-activity variants in the north. The frequency of the low-activity c.521T>C variant allele was 24% (95% CI, 18–32%) in Native American populations, 20% (95% CI, 15–25%) in the Middle East, 18% (95% CI, 14–23%) in Europe, 12% (95% CI, 9.5–15%) in East Asia, 9.4% (95% CI, 6.9–13%) in Central/South Asia and 1.9% (95% CI, 0.7–4.8%) in Sub-Saharan Africa. No carriers of the c.521T>C SNP were found in Oceania. The frequency of the homozygous variant c.521CC genotype was around 2% in Caucasians and around 4% in the Finnish population. The greatest genetic diversity was seen in African populations and SLCO1B1 diversity was generally far greater within than between populations. The SLCO1B1 genotype significantly affected the pharmacokinetics of most of the statins investigated. The mean area under the plasma concentration-time curve (AUC) of simvastatin acid, atorvastatin, pravastatin and rosuvastatin was 3.2-, 2.4-, 1.9- and 1.7-fold, respectively, in subjects with the SLCO1B1 c.521CC variant genotype compared with subjects with the c.521TT control genotype (P < 0.05). The SLCO1B1 genotype had no significant effect on the plasma concentrations of fluvastatin. Despite the considerable effect on the pharmacokinetics of statins, the response to a single dose of any of the statins studied was not affected by the SLCO1B1 genotype. Interestingly, the SLCO1B1 variant genotype was associated with an increased baseline cholesterol synthesis rate, as indicated by a 40% higher desmosterol to cholesterol ratio in subjects with the c.521CC genotype than in those with the c.521TT genotype (P = 0.043). In agreement, there was a tendency toward higher plasma concentrations of absolute desmosterol and lathosterol, as well as lathosterol to cholesterol ratios, and lower plasma concentrations of cholesterol absorption markers in subjects with the variant genotype. In conclusion, the low-activity SLCO1B1 c.521T>C variant occurs at varying frequencies in different ethnic groups and is relatively common in non-African populations. Genetically impaired activity of the hepatic influx transporter OATP1B1, due to the presence of the c.521T>C SNP, leads to elevated plasma concentrations of many but not all statins, thus increasing the risk for muscle toxicity. The SLCO1B1 genotype may partially explain why individual patients respond differently to various statins and may help to identify subjects who are at higher risk of developing statin-induced myopathy.
  • Sistonen, Johanna (Helsingin yliopisto, 2008)
    Pharmacogenetics deals with genetically determined variation in drug response. In this context, three phase I drug-metabolizing enzymes, CYP2D6, CYP2C9, and CYP2C19, have a central role, affecting the metabolism of about 20-30% of clinically used drugs. Since genes coding for these enzymes in human populations exhibit high genetic polymorphism, they are of major pharmacogenetic importance. The aims of this study were to develop new genotyping methods for CYP2D6, CYP2C9, and CYP2C19 that would cover the most important genetic variants altering the enzyme activity, and, for the first time, to describe the distribution of genetic variation at these loci on global and microgeographic scales. In addition, pharmacogenetics was applied to a postmortem forensic setting to elucidate the role of genetic variation in drug intoxications, focusing mainly on cases related to tricyclic antidepressants, which are commonly involved in fatal drug poisonings in Finland. Genetic variability data were obtained by genotyping new population samples by the methods developed based on PCR and multiplex single-nucleotide primer extension reaction, as well as by collecting data from the literature. Data consisted of 138, 129, and 146 population samples for CYP2D6, CYP2C9, and CYP2C19, respectively. In addition, over 200 postmortem forensic cases were examined with respect to drug and metabolite concentrations and genotypic variation at CYP2D6 and CYP2C19. The distribution of genetic variation within and among human populations was analyzed by descriptive statistics and variance analysis and by correlating the genetic and geographic distances using Mantel tests and spatial autocorrelation. The correlation between phenotypic and genotypic variation in drug metabolism observed in postmortem cases was also analyzed statistically. The genotyping methods developed proved to be informative, technically feasible, and cost-effective. Detailed molecular analysis of CYP2D6 genetic variation in a global survey of human populations revealed that the pattern of variation was similar to those of neutral genomic markers. Most of the CYP2D6 diversity was observed within populations, and the spatial pattern of variation was best described as clinal. On the other hand, genetic variants of CYP2D6, CYP2C9, and CYP2C19 associated with altered enzymatic activity could reach extremely high frequencies in certain geographic regions. Pharmacogenetic variation may also be significantly affected by population-specific demographic histories, as seen within the Finnish population. When pharmacogenetics was applied to a postmortem forensic setting, a correlation between amitriptyline metabolic ratios and genetic variation at CYP2D6 and CYP2C19 was observed in the sample material, even in the presence of confounding factors typical for these cases. In addition, a case of doxepin-related fatal poisoning was shown to be associated with a genetic defect at CYP2D6. Each of the genes studied showed a distinct variation pattern in human populations and high frequencies of altered activity variants, which may reflect the neutral evolution and/or selective pressures caused by dietary or environmental exposure. The results are relevant also from the clinical point of view since the genetic variation at CYP2D6, CYP2C9, and CYP2C19 already has a range of clinical applications, e.g. in cancer treatment and oral anticoagulation therapy. This study revealed that pharmacogenetics may also contribute valuable information to the medicolegal investigation of sudden, unexpected deaths.
  • Kajosaari, Lauri (Helsingin yliopisto, 2006)
    Introduction Repaglinide is a short-acting drug, used to reduce postprandial hyperglycaemia in type 2 diabetic patients. Repaglinide is extensively metabolised, and its oral bioavailability is about 60%; its metabolites are mainly excreted into bile. In previous studies, the cytochrome P450 (CYP) 3A4 inhibitors itraconazole and clarithromycin have moderately increased the area under the concentration-time curve (AUC) of repaglinide. Gemfibrozil, a CYP2C8 inhibitor, has greatly increased repaglinide AUC, enhancing and prolonging its blood glucose-lowering effect. Rifampicin has decreased the AUC and effects of repaglinide. Aims The aims of this work were to investigate the contribution of CYP2C8 and CYP3A4 to the metabolism of repaglinide, and to study other potential drug interactions affecting the pharmacokinetics of repaglinide, and the mechanisms of observed interactions. Methods The metabolism of repaglinide was studied in vitro using recombinant human CYP enzymes and pooled human liver microsomes (HLM). The effect of trimethoprim, cyclosporine, bezafibrate, fenofibrate, gemfibrozil, and rifampicin on the metabolism of repaglinide, and the effect of fibrates and rifampicin on the activity of CYP2C8 and CYP3A4 were investigated in vitro. Randomised, placebo-controlled cross-over studies were carried out in healthy human volunteers to investigate the effect of bezafibrate, fenofibrate, trimethoprim, cyclosporine, telithromycin, montelukast and pioglitazone on the pharmacokinetics and pharmacodynamics of repaglinide. Pretreatment with clinically relevant doses of the study drug or placebo was followed by a single dose of repaglinide, after which blood and urine samples were collected to determine pharmacokinetic and pharmacodynamic parameters. Results In vitro, the contribution of CYP2C8 was similar to that of CYP3A4 in the metabolism of repaglinide (< 2 μM). Bezafibrate, fenofibrate, gemfibrozil, and rifampicin moderately inhibited CYP2C8 and repaglinide metabolism, but only rifampicin inhibited CYP3A4 in vitro. Bezafibrate, fenofibrate, montelukast, and pioglitazone had no effect on the pharmacokinetics and pharmacodynamics of repaglinide in vivo. The CYP2C8 inhibitor trimethoprim inhibited repaglinide metabolism by HLM in vitro and increased repaglinide AUC by 61% in vivo (P < .001). The CYP3A4 inhibitor telithromycin increased repaglinide AUC 1.8-fold (P < .001) and enhanced its blood glucose-lowering effect in vivo. Cyclosporine inhibited the CYP3A4-mediated (but not CYP2C8-mediated) metabolism of repaglinide in vitro and increased repaglinide AUC 2.4-fold in vivo (P < .001). The effect of cyclosporine on repaglinide AUC in vivo correlated with the SLCO1B1 (encoding organic anion transporting polypeptide 1, OATP1B1) genotype. Conclusions The relative contributions of CYP2C8 and CYP3A4 to the metabolism of repaglinide are similar in vitro, when therapeutic repaglinide concentrations are used. In vivo, repaglinide AUC was considerably increased by inhibition of both CYP2C8 (by trimethoprim) and CYP3A4 (by telithromycin). Cyclosporine raised repaglinide AUC even higher, probably by inhibiting the CYP3A4-mediated biotransformation and OATP1B1-mediated hepatic uptake of repaglinide. Bezafibrate, fenofibrate, montelukast, and pioglitazone had no effect on the pharmacokinetics of repaglinide, suggesting that they do not significantly inhibit CYP2C8 or CYP3A4 in vivo. Coadministration of drugs that inhibit CYP2C8, CYP3A4 or OATP1B1 may increase the plasma concentrations and blood glucose-lowering effect of repaglinide, requiring closer monitoring of blood glucose concentrations to avoid hypoglycaemia, and adjustment of repaglinide dosage as necessary.
  • Tapaninen, Tuija (Helsingin yliopisto, 2012)
    Aliskiren is an antihypertensive drug approved for clinical use in 2007. It acts by inhibiting renin, the first enzyme in the renin-angiotensin-aldosterone system. Marked interindividual variability exists in the pharmacokinetics of aliskiren. Interestingly, the pharmacokinetic properties of aliskiren suggest an important role for drug transporters in its pharmacokinetics. Aliskiren is poorly absorbed, and therefore, its oral bioavailability is only 2-3%. The elimination of aliskiren occurs mainly as an unchanged drug by biliary and renal excretion, and only a small proportion is metabolized by cytochrome P450 (CYP) 3A4. Organic anion-transporting polypeptide 2B1 (OATP2B1) influx transporter is thought to facilitate the intestinal absorption and hepatic uptake of aliskiren. Based on a more recent finding, OATP1A2 may also contribute to aliskiren absorption. Moreover, aliskiren is a substrate of P-glycoprotein (P-gp) efflux transporter, which can reduce the intestinal absorption of its substrates and enhance their elimination into bile, urine, and intestine. Furthermore, P-gp limits the passage of its substrates across many blood-tissue barriers such as the blood-brain barrier. In previous studies, cyclosporine (an inhibitor of P-gp, OATP2B1, and CYP3A4) as well as ketoconazole and atorvastatin (inhibitors of P-gp and CYP3A4) have raised the area under the plasma aliskiren concentration-time curve (AUC) 5-fold, 1.8-fold, and 1.5-fold, respectively. Considering the interindividual differences in aliskiren pharmacokinetics, information on related pharmacokinetic interactions and genetic variations may improve the safety of aliskiren therapy. This thesis comprises four randomized, placebo-controlled, cross-over pharmacokinetic interaction studies and two prospective genotype panel studies in healthy volunteers to assess the potential pharmacokinetic interactions and genetic variations affecting the pharmacokinetics and pharmacodynamics of aliskiren. The effects of induction and inhibition of P-gp and CYP3A4 were investigated by using rifampicin and itraconazole as a model inducer and inhibitor, respectively. Furthermore, the effects of grapefruit juice, orange juice, and apple juice, all of which have inhibited OATP1A2 and OATP2B1 in vitro, were also examined. Genetic variations of P-gp and OATP2B1 for the pharmacogenetic studies were selected on the basis of previous studies reporting their associations with altered plasma concentrations of the substrates of respective drug transporters, and on the basis of their frequencies in the Finnish population. Therefore, the effects of common haplotypes of the ABCB1 gene encoding P-gp, c.1236C-c.2677G-c.3435C and c.1236T-c.2677T-c.3435T, as well as the effects of c.935G>A single-nucleotide polymorphism (SNP) in the SLCO2B1 gene encoding OATP2B1 were evaluated. In all studies, aliskiren was administered as a single dose. Furthermore, in pharmacokinetic interaction studies, the potentially interacting substances were administered according to relevant dosing schemes. Blood and urine samples were collected for the determination of drug concentrations and plasma renin activity, in addition to which blood pressure was measured. Rifampicin, grapefruit juice, orange juice, and apple juice markedly reduced the plasma concentrations of aliskiren, and the reductions in the AUC values of aliskiren were 56%, 61%, 62%, and 63%, respectively (P < 0.001). In addition, the reduced exposure to aliskiren by rifampicin, orange juice, and apple juice led to the attenuation of the renin-inhibiting effect of aliskiren. During the rifampicin, orange juice, and apple juice phases plasma renin activity 24 hours after aliskiren ingestion was 61% (P = 0.008), 87% (P = 0.037), and 67% (P = 0.036) higher, respectively, than during the placebo or water phases. Itraconazole raised the AUC of aliskiren considerably, 6.5-fold (P < 0.001), and also enhanced the renin-inhibiting effect of aliskiren. Plasma renin activity 24 hours after aliskiren ingestion was 68% lower during the itraconazole phase, than during the placebo phase (P = 0.011). The ABCB1 c.1236C-c.2677G-c.3435C and c.1236T-c.2677T-c.3435T haplotypes and the SLCO2B1 c.935G>A SNP were not significantly associated with the pharmacokinetics or pharmacodynamics of aliskiren. In conclusion, aliskiren was found to be susceptible to transporter-mediated pharmacokinetic interactions of clinical significance. The interactions of rifampicin and itraconazole with aliskiren probably resulted from induction and inhibition of P-gp in the small intestine, respectively, with a minor contribution from a parallel effect on CYP3A4. Grapefruit, orange, and apple juices reduced the absorption of aliskiren from the gastrointestinal tract, possibly by inhibiting intestinal OATP transporters. The genetic variations of P-gp and OATP2B1 examined did not explain the large interindividual differences in aliskiren pharmacokinetics. Clinicians should be aware of the possibility that rifampicin may reduce the antihypertensive efficacy of aliskiren. Itraconazole can markedly raise the plasma concentrations of aliskiren and enhance its renin-inhibiting efficacy, and thus, should not be used with aliskiren. In addition, the inhibition of P-gp by itraconazole may alter the tissue distribution of aliskiren and potentially produce adverse reactions not observed with higher doses of aliskiren alone. Moreover, the concomitant use of aliskiren with grapefruit, orange, or apple juices is best avoided because of the risk of therapeutic failure due to reduced aliskiren exposure.
  • Raaska, Kari (Helsingin yliopisto, 2003)
  • Jaakkola, Tiina (Helsingin yliopisto, 2007)
    Pioglitazone is a thiazolidinedione compound used in the treatment of type 2 diabetes. It has been reported to be metabolised by multiple cytochrome P450 (CYP) enzymes, including CYP2C8, CYP2C9 and CYP3A4 in vitro. The aims of this work were to identify the CYP enzymes mainly responsible for the elimination of pioglitazone in order to evaluate its potential for in vivo drug interactions, and to investigate the effects of CYP2C8- and CYP3A4-inhibiting drugs (gemfibrozil, montelukast, zafirlukast and itraconazole) on the pharmacokinetics of pioglitazone in healthy volunteers. In addition, the effect of induction of CYP enzymes on the pharmacokinetics of pioglitazone in healthy volunteers was investigated, with rifampicin as a model inducer. Finally, the effect of pioglitazone on CYP2C8 and CYP3A enzyme activity was examined in healthy volunteers using repaglinide as a model substrate. Study I was conducted in vitro using pooled human liver microsomes (HLM) and human recombinant CYP isoforms. Studies II to V were randomised, placebo-controlled cross-over studies with 2-4 phases each. A total of 10-12 healthy volunteers participated in each study. Pretreatment with clinically relevant doses with the inhibitor or inducer was followed by a single dose of pioglitazone or repaglinide, whereafter blood and urine samples were collected for the determination of drug concentrations. In vitro, the elimination of pioglitazone (1 µM) by HLM was markedly inhibited, in particular by CYP2C8 inhibitors, but also by CYP3A4 inhibitors. Of the recombinant CYP isoforms, CYP2C8 metabolised pioglitazone markedly, and CYP3A4 also had a significant effect. All of the tested CYP2C8 inhibitors (montelukast, zafirlukast, trimethoprim and gemfibrozil) concentration-dependently inhibited pioglitazone metabolism in HLM. In humans, gemfibrozil raised the area under the plasma concentration-time curve (AUC) of pioglitazone 3.2-fold (P < 0.001) and prolonged its elimination half-life (t½) from 8.3 to 22.7 hours (P < 0.001), but had no significant effect on its peak concentration (Cmax) compared with placebo. Gemfibrozil also increased the excretion of pioglitazone into urine and reduced the ratios of the active metabolites M-IV and M-III to pioglitazone in plasma and urine. Itraconazole had no significant effect on the pharmacokinetics of pioglitazone and did not alter the effect of gemfibrozil on pioglitazone pharmacokinetics. Rifampicin decreased the AUC of pioglitazone by 54% (P < 0.001) and shortened its dominant t½ from 4.9 to 2.3 hours (P < 0.001). No significant effect on Cmax was observed. Rifampicin also decreased the AUC of the metabolites M-IV and M-III, shortened their t½ and increased the ratios of the metabolite to pioglitazone in plasma and urine. Montelukast and zafirlukast did not affect the pharmacokinetics of pioglitazone. The pharmacokinetics of repaglinide remained unaffected by pioglitazone. These studies demonstrate the principal role of CYP2C8 in the metabolism of pioglitazone in humans. Gemfibrozil, an inhibitor of CYP2C8, increases and rifampicin, an inducer of CYP2C8 and other CYP enzymes, decreases the plasma concentrations of pioglitazone, which can necessitate blood glucose monitoring and adjustment of pioglitazone dosage. Montelukast and zafirlukast had no effects on the pharmacokinetics of pioglitazone, indicating that their inhibitory effect on CYP2C8 is negligible in vivo. Pioglitazone did not increase the plasma concentrations of repaglinide, indicating that its inhibitory effect on CYP2C8 and CYP3A4 is very weak in vivo.
  • Koskinen, Mia (Helsingin yliopisto, 2006)
    Background. Hyperlipidemia is a common concern in patients with heterozygous familial hypercholesterolemia (HeFH) and in cardiac transplant recipients. In both groups, an elevated serum LDL cholesterol level accelerates the development of atherosclerotic vascular disease and increases the rates of cardiovascular morbidity and mortality. The purpose of this study is to assess the pharmacokinetics, efficacy, and safety of cholesterol-lowering pravastatin in children with HeFH and in pediatric cardiac transplant recipients receiving immunosuppressive medication. Patients and Methods. The pharmacokinetics of pravastatin was studied in 20 HeFH children and in 19 pediatric cardiac transplant recipients receiving triple immunosuppression. The patients ingested a single 10-mg dose of pravastatin, and plasma pravastatin concentrations were measured up to 10/24 hours. The efficacy and safety of pravastatin (maximum dose 10 to 60 mg/day and 10 mg/day) up to one to two years were studied in 30 patients with HeFH and in 19 cardiac transplant recipients, respectively. In a subgroup of 16 HeFH children, serum non-cholesterol sterol ratios (102 x mmol/mol of cholesterol), surrogate estimates of cholesterol absorption (cholestanol, campesterol, sitosterol), and synthesis (desmosterol and lathosterol) were studied at study baseline (on plant stanol esters) and during combination with pravastatin and plant stanol esters. In the transplant recipients, the lipoprotein levels and their mass compositions were analyzed before and after one year of pravastatin use, and then compared to values measured from 21 healthy pediatric controls. The transplant recipients were grouped into patients with transplant coronary artery disease (TxCAD) and patients without TxCAD, based on annual angiography evaluations before pravastatin. Results. In the cardiac transplant recipients, the mean area under the plasma concentration-time curve of pravastatin [AUC(0-10)], 264.1 * 192.4 ng.h/mL, was nearly ten-fold higher than in the HeFH children (26.6 * 17.0 ng.h/mL). By 2, 4, 6, 12 and 24 months of treatment, the LDL cholesterol levels in the HeFH children had respectively decreased by 25%, 26%, 29%, 33%, and 32%. In the HeFH group, pravastatin treatment increased the markers of cholesterol absorption and decreased those of synthesis. High ratios of cholestanol to cholesterol were associated with the poor cholesterol-lowering efficacy of pravastatin. In cardiac transplant recipients, pravastatin 10 mg/day lowered the LDL cholesterol by approximately 19%. Compared with the patients without TxCAD, patients with TxCAD had significantly lower HDL cholesterol concentrations and higher apoB-100/apoA-I ratios at baseline (1.0 ± 0.3 mmol/L vs. 1.4 ± 0.3 mmol/L, P = 0.031; and 0.7 ± 0.2 vs. 0.5 ± 0.1, P = 0.034) and after one year of pravastatin use (1.0 ± 0.3 mmol/L vs. 1.4 ± 0.3 mmol/L, P = 0.013; and 0.6 ± 0.2 vs. 0.4 ± 0.1, P = 0.005). Compared with healthy controls, the transplant recipients exhibited elevated serum triglycerides at baseline (median 1.3 [range 0.6-3.2] mmol/L vs. 0.7 [0.3-2.4] mmol/L, P=0.0002), which negatively correlated with their HDL cholesterol concentration (r = -0.523, P = 0.022). Recipients also exhibited higher apoB-100/apoA1 ratios (0.6 ± 0.2 vs. 0.4 ± 0.1, P = 0.005). In addition, elevated triglyceride levels were still observed after one year of pravastatin use (1.3 [0.5-3.5] mmol/L vs. 0.7 [0.3-2.4] mmol/L, P = 0.0004). Clinically significant elevations in alanine aminotransferase, creatine kinase, or creatinine ocurred in neither group. Conclusions. Immunosuppressive medication considerably increased the plasma pravastatin concentrations. In both patient groups, pravastatin treatment was moderately effective, safe, and well tolerated. In the HeFH group, high baseline cholesterol absorption seemed to predispose patients to insufficient cholesterol-lowering efficacy of pravastatin. In the cardiac transplant recipients, low HDL cholesterol and a high apoB-100/apoA-I ratio were associated with development of TxCAD. Even though pravastatin in the transplant recipients effectively lowered serum total and LDL cholesterol concentrations, it failed to normalize their elevated triglyceride levels and, in some patients, to prevent the progression of TxCAD.
  • Karonen, Tiina (Helsingin yliopisto, 2012)
    Leukotriene receptor antagonists montelukast and zafirlukast are used for asthma and allergic rhinitis. Their marketing authorisations were granted over 15 years ago, before regulatory guidance for drug interaction studies existed. At that time the knowledge of their metabolic pathways was largely based on in vitro studies, in which the main enzymes catalysing the biotransformation of montelukast and zafirlukast were cytochrome P450 (CYP) 2C9 and CYP3A4. Since then the understanding of the importance of CYP enzymes in drug metabolism has increased markedly and also the role of CYP2C8 has been recognised. Montelukast and zafirlukast are both potent inhibitors of CYP2C8 in vitro, but neither of them has shown inhibitory effect on CYP2C8 in vivo. Montelukast has also been shown to fit well in the active site cavity of CYP2C8 in a crystallography study. These observations led to examine the role of CYP2C8 and CYP3A4 in the metabolism of montelukast in humans, as well as the role of CYP2C8, CYP2C9 and CYP3A4 in the metabolism of zafirlukast in humans. This work was carried out as four randomised, placebo-controlled cross-over drug interaction studies in healthy volunteers. The CYP2C8 inhibitor gemfibrozil resulted in over fourfold increase of the area under the plasma drug concentration-time curve (AUC) of montelukast, almost blocking the formation of its major metabolite M4. The CYP3A4 inhibitor itraconazole had no effect on the total elimination of montelukast, and decreased only the formation of a minor metabolite M5. The pharmacokinetics of zafirlukast was only affected by CYP2C9 inhibition by fluconazole, which resulted in a 1.6-fold increase of the AUC of zafirlukast. Inhibition of CYP2C8 and CYP3A4 had no effect on the pharmacokinetics of zafirlukast. The results of this work elucidated the biotransformation pathways of montelukast and zafirlukast in humans. CYP2C8 accounts for about 80% of the metabolism of montelukast, while CYP3A4 has no significant role in it. The main enzyme in the metabolism of zafirlukast in humans is CYP2C9. Concomitant use of CYP2C8 inhibitors with montelukast, or CYP2C9 inhibitors with zafirlukast may increase the risk of concentration dependent adverse drug reactions. With regard to drug interaction studies, a probe drug should be sensitive and relatively safe: according to the present findings montelukast is a promising candidate for a CYP2C8 probe substrate. These results also highlight the relevance of drug interaction studies and the regulatory guidelines related to them. Especially drugs that have been developed before the existence of these guidelines may be deficiently characterised with regard to their metabolism, leaving the possibility of unrecognized CYP-mediated interactions.
  • Arte, Sirpa (Helsingin yliopisto, 2001)
  • Tuominen, Esa (Helsingin yliopisto, 2011)
    The ability of the peripherally associated membrane protein cytochrome c (cyt c) to bind phospholipids in vitro was studied using fluorescence spectroscopy and large unilamellar liposomes. Previous work has shown that cyt c can bind phospholipids using two distinct mecha- nisms and sites, the A-site and the C-site. This binding is mediated by electrostatic or hydrophobic interactions, respectively. Here, we focus on the mechanism underlying these interactions. A chemically modified cyt c mutant Nle91 was used to study the ATP-binding site, which is located near the evolutionarily invariant Arg 91 on the protein surface. This site was also demonstrated to mediate phospholipid binding, possibly by functioning as a phospholipid binding site. Circular dichroism spectroscopy, time resolved fluorescence spectroscopy of zinc- porphyrin modified [Zn2+-heme] cyt c and liposome binding studies of the Nle91 mutant were used to demonstrate that ATP induces a conformational change in membrane- bound cyt c. The ATP-induced conformational changes were mediated by Arg 91 and were most pronounced in cyt c bound to phospholipids via the C-site. It has been previously reported that the hydrophobic interaction between phospho- lipids and cyt c (C-site) includes the binding of a phospholipid acyl chain inside the protein. In this mechanism, which is known as extended phospholipid anchorage, the sn-2 acyl chain of a membrane phospholipid protrudes out of the membrane surface and is able to bind in a hydrophobic cavity in cyt c. Direct evidence for this type of bind- ing mechanism was obtained by studying cyt c/lipid interaction using fluorescent [Zn2+- heme] cyt c and fluorescence quenching of brominated fatty acids and phospholipids. Under certain conditions, cyt c can form fibrillar protein-lipid aggregates with neg- atively charged phospholipids. These aggregates resemble amyloid fibrils, which are involved in the pathogenesis of many diseases. Congo red staining of these fibers con- firmed the presence of amyloid structures. A set of phospholipid-binding proteins was also found to form similar aggregates, suggesting that phospholipid-induced amyloid formation could be a general mechanism of amyloidogenesis.
  • Wadén, Johan (Helsingin yliopisto, 2010)
    Type 1 diabetes is associated with the risk for late diabetic complications which are divided into microvascular (retinopathy, nephropathy, and neuropathy) and macrovascular (cardiovascular disease, CVD) diseases. The risk for diabetic complication can be reduced by effective treatment, most importantly the glycaemic control. Glycaemia in type 1 diabetes is influenced by the interplay between insulin injections and lifestyle factors such as physical activity and diet. The effect of physical activity in patients with type 1 diabetes is not well known, however. The aim of this thesis was to investigate the physical activity and the physical fitness of patients with type 1 diabetes with special emphasis on glycaemic control and the diabetic complications. The patients included in the study were all part of the nationwide, multicenter Finnish Diabetic Nephropathy (FinnDiane) Study which aims to characterise genetic, clinical, and environmental factors that predispose to diabetic complications in patients with type 1 diabetes. In addition, subjects from the IDentification of EArly mechanisms in the pathogenesis of diabetic Late complications (IDEAL) Study were studied. Physical activity was assessed in the FinnDiane Study in 1945 patients by a validated questionnaire. Physical fitness was measured in the IDEAL Study by spiroergometry (cycle test with measurement of respiratory gases) in 86 young adults with type 1 diabetes and in 27 healthy controls. All patients underwent thorough clinical characterisation of their diabetic complication status. Four substudies were cross-sectional using baseline data and one study additionally used follow-up data. Physical activity, especially the intensity of activities, was reduced in patients affected by diabetic nephropathy, retinopathy, and CVD. Low physical activity was associated with poor glycaemic control, a finding most clear in women and evident also in patients with no signs of diabetic complications. Furthermore, low physical activity was associated with a higher HbA1c variability, which in turn was associated with the progression of renal disease and CVD during follow-up. A higher level of physical activity was also associated with better insulin sensitivity. The prevalence of the metabolic syndrome in type 1 diabetes was also lower the higher the physical activity. The aerobic physical fitness level of young adults with type 1 diabetes was reduced compared with healthy peers and in men an association between higher fitness level and lower HbA1c was observed. In patients with type 1 diabetes, a higher physical activity was associated with better glycaemic control and may thus be beneficial with respect to the prevention of diabetic complications.
  • Borodulin, Katja (Helsingin yliopisto, 2006)
    Physical inactivity, low cardiorespiratory fitness, and abdominal obesity are direct and mediating risk factors for cardiovascular disease (CVD). The results of recent studies suggest that individuals with higher levels of physical activity or cardiorespiratory fitness have lower CVD and all-cause mortality than those with lower activity or fitness levels regardless of their level of obesity. The interrelationships of physical activity, fitness, and abdominal obesity with cardiovascular risk factors have not been studied in detail. The aim of this study was to investigate the associations of different types of leisure time physical activity and aerobic fitness with cardiovascular risk factors in a large population of Finnish adults. In addition, a novel aerobic fitness test was implemented and the distribution of aerobic fitness was explored in men and women across age groups. The interrelationships of physical activity, aerobic fitness and abdominal obesity were examined in relation to cardiovascular risk factors. This study was part of the National FINRISK Study 2002, which monitors cardiovascular risk factors in a Finnish adult population. The sample comprised 13 437 men and women aged 25 to 74 years and was drawn from the Population Register as a stratified random sample according to 10-year age groups, gender and area. A separate physical activity study included 9179 subjects, of whom 5 980 participated (65%) in the study. At the study site, weight, height, waist and hip circumferences, and blood pressure were measured, a blood sample was drawn, and an aerobic fitness test was performed. The fitness test estimated maximal oxygen uptake (VO2max) and was based on a non-exercise method by using a heart rate monitor at rest. Waist-to-hip ratio (WHR) was calculated by dividing waist circumference with hip circumference and was used as a measure of abdominal obesity. Participants filled in a questionnaire on health behavior, a history of diseases, and current health status, and a detailed 12-month leisure time physical activity recall. Based on the recall data, relative energy expenditure was calculated using metabolic equivalents, and physical activity was divided into conditioning, non-conditioning, and commuting physical activity. Participants aged 45 to 74 years were later invited to take part in a 2-hour oral glucose tolerance test with fasting insulin and glucose measurements. Based on the oral glucose tolerance test, undiagnosed impaired glucose tolerance and type 2 diabetes were defined. The estimated aerobic fitness was lower among women and decreased with age. A higher estimated aerobic fitness and a lower WHR were independently associated with lower systolic and diastolic blood pressure, lower total cholesterol and triglyceride levels, and with higher high-density lipoprotein (HDL) cholesterol and HDL to total cholesterol ratio. The associations of the estimated aerobic fitness with diastolic blood pressure, triglycerides, and HDL to total cholesterol ratio were stronger in men with a higher WHR. High levels of conditioning and non-conditioning physical activity were associated with lower high-sensitivity C-reactive protein (CRP) levels. High levels of conditioning and overall physical activities were associated with lower insulin and glucose levels. The associations were stronger among women than men. A better self-rated physical fitness was associated with a higher estimated aerobic fitness, lower CRP levels, and lower insulin and glucose levels in men and women. In each WHR third, the risk of impaired glucose tolerance and type 2 diabetes was higher among physically inactive individuals who did not undertake at least 30 minutes of moderate-intensity physical activity on five days per week. These cross-sectional data show that higher levels of estimated aerobic fitness and regular leisure time physical activity are associated with a favorable cardiovascular risk factor profile and that these associations are present at all levels of abdominal obesity. Most of the associations followed a dose-response manner, suggesting that already low levels of physical activity or fitness are beneficial to health and that larger improvements in risk factor levels may be gained from higher activity and fitness levels. The present findings support the recommendation to engage regularly in leisure time physical activity, to pursue a high level of aerobic fitness, and to prevent abdominal obesity.
  • Hernelahti, Miika (Helsingin yliopisto, 2004)
  • Kaseva, Nina (Helsingin yliopisto, 2014)
    Advancements in neonatal care are resulting in increasing numbers of adult survivors after preterm birth at very low birth weight (VLBW, ≤ 1500 g). VLBW is associated with risk factors of non-communicable diseases, e.g. cardiovascular disease, osteoporosis and diabetes. We investigated mechanisms underlying the effects of preterm birth on later health in VLBW adults, with a focus on 1) physical activity, 2) nutrition and 3) stress response. The participants come from a follow-up cohort study, the Helsinki Study of Very Low Birth Weight Adults. Different subgroups from the original birth cohort (born in 1978-1985) have as young adults participated in the studies of this thesis. The controls, born at term, were group-matched for birth hospital, age and sex. We evaluated physical activity by self-report and objective measurement. The participants completed a physical activity questionnaire and underwent wrist-worn accelerometer measurement. To assess dietary intake, the participants completed a 3-day food record. For evaluation of stress response, the participants underwent the Trier Social Stress Test (TSST). In conjunction with TSST, we measured heart rate, salivary cortisol, plasma ACTH, cortisol, glucose, insulin, adrenalin and noradrenalin. 1) Based on self-report, healthy VLBW adults undertake approximately 50% less conditioning leisure-time physical activity, with lower yearly frequency, total time, total volume and energy expenditure than controls. We were unable to confirm our finding of lower physical activity with wrist-worn accelerometer measurement. 2) VLBW adults had lower consumption of vegetables, fruits, berries and milk products. This was combined with lower intake of calcium and vitamin D. 3) VLBW adults showed a lower hypothalamic-pituitary-adrenal axis (HPAA) response to stress than controls. This was accompanied by a lower insulin response. No evidence of a higher sympathetic-adrenal-medullary (SAM) system stress response was found. Furthermore, we observed a lower noradrenalin response to stress in VLBW women. This study showed that VLBW adults undertake less conditioning leisure-time physical activities and have unhealthier diets, both factors that negatively affect future health in this high-risk population. They may in part underlie the increased risk for chronic non-communicable diseases in VLBW individuals. Further, a lower HPAA response to stress was found in VLBW adults than in controls. For SAM stress response, the results were similar in VLBW and control groups, with lower noradrenalin response to stress in VLBW women only. These findings reinforce the supposition that stress response is programmed early in life. In sum, this study increased understanding of possible mechanisms linking preterm birth and adult risk of disease.
  • Kiilavuori, Kai (Helsingin yliopisto, 2000)
  • Lindholm, Harri (Helsingin yliopisto, 2013)
    Good recovery helps to restore functional reserves and facilitates the positive effects of stress at work. The risk of job stress is increased by excessive quantitative demands, low job control, poor recovery and disruption of biological rhythms. Stress causes changes in autonomic nervous system (ANS) function and in excretion of cortisol hormone regulated by hypothalamus-pituitary-adrenal cortex (HPA) axis. Melatonin and cortisol are diurnally oscillating hormones reflecting disruption of biological rhythms. Common symptoms of harmful stress are sleep problems, exhaustion and decrease in work efficiency. The risk of many common diseases increases. In this study perceived and physiologically measured stress and recovery were analysed in media work, which contains typical risk factors of job stress in 24/7 society. A standardized questionnaire was mailed to all employees of the Finnish Broadcasting Company with irregular shift work (ISW, n = 750) and to an equal number of randomly selected controls with regular 8-hour daytime work (RDW). The questionnaire included items of work characteristics, perceived stress, mental and physical health and lifestyle. The response rate was over 80 % in ISW group and about 35 % in RDW group. Seventy respondents from both groups were randomly selected for physiological measurements. Cortisol and melatonin hormones were analysed from five salivary samples taken while subjects were awake. Heart rate variability as indicator of ANS function was calculated from 24 hour ECG recordings. Body movements were monitored by actigraphy. Severe subjective stress, irregular shift work, and short actual sleep time were all significant explanatory variables of augmented morning cortisol response. The risk of daytime sleepiness was nearly twofold in the ISW group compared to the RDW group. The daytime sleepiness was associated with attenuated relaxation of ANS during sleep in ISW group. The changes in the afternoon and evening levels of cortisol and melatonin hormones might predispose to difficulties in initiating sleep. In all, about 40 % of workers reported high job control and 40% low job control. During the recovery period (from 18.00 to 06.00 hrs between working days), those who experienced high job control at work had significantly better recovery of ANS function than other workers. Depression, hypertension and poor general health were associated with many types of sleep disorders and they all increased the risk of nocturnal waking. Severe stress doubled the risk of difficulties initiating sleep and the risk of non-restorative sleep. Analysis of recovery should be included in the evaluation of workers health and well-being in 24/7 society. Simultaneously analysed autonomic nervous system function and neuroendocrine indicators provide additional information about stress and recovery. Ambulatory measurements in real life settings could offer new insights in occupational health studies, once their validation has been achieved.
  • Quintero, Ileana B. (Helsingin yliopisto, 2015)
    Prostatic acid phosphatase (PAP) has been linked to prostate cancer since the mid-1930s. The main research approach of PAP over that time has been based on its role in the human prostate. The regulatory mechanisms of expression of the PAP gene have also been studied, giving us information about the regulatory elements in the gene and the transcription factors that affect the gene expression in the prostatic tissue. However, little was known until recently about this protein s role and physiological function in other tissues. Our group generated and used a PAP-deficient mouse and was able to show that PAP is expressed in dorsal root ganglia (DRG) and spinal cord in mice. This is the same protein as thiamine monophosphatase (TMPase) whose enzymatic activity has been used for five decades to mark primary sensory neurons. In these tissues, PAP acts as an ecto-5'-nucleotidase and is able to dephosphorylate AMP to adenosine, and therefore produce an anti-nociceptive effect due to the binding of adenosine to the A1-adenosine receptor. We analyzed the ACPP gene, which enabled us to describe a new transmembrane isoform for PAP (TMPAP). This novel PAP isoform is produced by alternative splicing of the 11th exon of the ACPP gene. The alternative splicing is present in species such as the human, mouse and rat. The newly discovered isoform is widely expressed in human and mouse tissues and contains a tyrosine sequence (YxxΦ) in its carboxyl-terminus, which directs the protein to endocytosis. We have also corroborated its functionality by co-localization studies with different organelle markers in the endosomal/lysosomal pathway (I). The generation of a PAP-deficient mouse also enabled us to study the function/s of PAP in vivo. The lack of PAP in this mouse model led to the gradual changes in the mouse prostate that finally culminated with the development of prostate adenocarcinoma at the age of 12 months. Microarray analyses of different tissues that compared the PAP deficient mouse with the wild type (WT) mouse revealed changes in genes related to the vesicular trafficking, which support our previous results and led us to the conclusion that TMPAP could be involved in the regulation of the vesicular trafficking. We also detected the interaction between TMPAP and snapin, a SNARE (Soluble NSF Attachment Protein Receptor) associated protein, by yeast two-hybrid screening, co-localization and FRET (Förster resonance energy transfer) analysis. We concluded that, the disruption of this interaction in the PAP-deficient mouse leads to a disturbance in the vesicular transport of the cell and to the development of prostate adenocarcinoma in the PAP-deficient mouse prostate (II). Furthermore, we showed that the PAP-deficient mice present multiple behavioral and neurochemical alterations including increased size of brain lateral ventricles, hyperdopaminergic deregulation, and altered GABAergic transmission, symptoms that indicate that PAP also disturbes the normal function of the central nervous system (III). Snapin protein in the brain has been described as a protein important for the vesicular transport and for the fusion of vesicles with the plasma membrane, and we observed that the lack of PAP in GABAergic neurons leads to a change in the localization of snapin in the PAP-deficient mouse (III), which could indicate that as in the prostate a dysregulated vesicular trafficking could be the reason for the detected phenotype. The discovery of the new TMPAP and its localization in the endosomal/lysosomal pathway enabled an understanding of the phenotypic changes that occur in the PAP-deficient mouse. We hypothesized that TMPAP regulates vesicular trafficking by interacting with snapin, and its deficiency leads to a dysregulation of the endo-/exocytosis cycle, which produces the observed alterations in the mouse organs and tissues. The results obtained throughout this research project have opened up new lines of research related to PAP physiological function, and a deeper understanding of the expression, regulation and function of this protein could lead to new clinical applications.