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  • Kanerva, Mervi (Helsingin yliopisto, 2008)
    The objective of this study was to assess the utility of two subjective facial grading systems, to evaluate the etiologic role of human herpesviruses in peripheral facial palsy (FP), and to explore characteristics of Melkersson-Rosenthal syndrome (MRS). Intrarater repeatability and interrater agreement were assessed for Sunnybrook (SFGS) and House-Brackmann facial grading systems (H-B FGS). Eight video-recorded FP patients were graded in two sittings by 26 doctors. Repeatability for SFGS was from good to excellent and agreement between doctors from moderate to excellent by intraclass correlation coefficient and coefficient of repeatability. For H-B FGS, repeatability was from fair to good and agreement from poor to fair by agreement percentage and kappa coefficients. Because SFGS was at least as good in repeatability as H-B FGS and showed more reliable results in agreement between doctors, we encourage the use of SFGS over H-B FGS. Etiologic role of human herpesviruses in peripheral FP was studied by searching DNA of herpes simplex virus (HSV) -1 and -2, varicella-zoster virus (VZV), human herpesvirus (HHV) -6A, -6B, and -7, Epstein-Barr virus (EBV), and cytomegalovirus (CMV) by PCR/microarray methods in cerebrospinal fluid (CSF) of 33 peripheral FP patients and 36 controls. Three patients and five controls had HHV-6 or -7 DNA in CSF. No DNA of HSV-1 or -2, VZV, EBV, or CMV was found. Detecting HHV-7 and dual HHV-6A and -6B DNA in CSF of FP patients is intriguing, but does not allow etiologic conclusions as such. These DNA findings in association with FP and the other diseases that they accompanied require further exploration. MRS is classically defined as a triad of recurrent labial or oro-facial edema, recurrent peripheral FP, and plicated tongue. All three signs are present in the minority of patients. Edema-dominated forms are more common in the literature, while MRS with FP has received little attention. The etiology and true incidence of MRS are unknown. Characteristics of MRS were evaluated at the Departments of Otorhinolaryngology and Dermatology focusing on patients with FP. There were 35 MRS patients, 20 with FP and they were mailed a questionnaire (17 answered) and were clinically examined (14 patients). At the Department of Otorhinolaryngology, every MRS patient had FP and half had the triad form of MRS. Two patients, whose tissue biopsies were taken during an acute edema episode, revealed nonnecrotizing granulomatous findings typical for MRS, the other without persisting edema and with symptoms for less than a year. A peripheral blood DNA was searched for gene mutations leading to UNC-93B protein deficiency predisposing to HSV-1 infections; no gene mutations were found. Edema in most MRS FP patients did not dominate the clinical picture, and no progression of the disease was observed, contrary to existing knowledge. At the Department of Dermatology, two patients had triad MRS and 15 had monosymptomatic granulomatous cheilitis with frequent or persistent edema and typical MRS tissue histology. The clinical picture of MRS varied according to the department where the patient was treated. More studies from otorhinolaryngology departments and on patients with FP would clarify the actual incidence and clinical picture of the syndrome.
  • Laakkonen, Hanne (Helsingin yliopisto, 2011)
    Although improved outcomes for children on peritoneal dialysis (PD) have been seen in recent years, the youngest patients continue to demonstrate inferior growth, more frequent infections, more neurological sequelae, and higher mortality compared to older children. Also, maintain-ing normal intravascular volume status, especially in anuric patients, has proven difficult. This study was designed to treat and monitor these youngest PD patients, which are relatively many due to the high prevalence of congenital nephrotic syndrome of the Finnish type (CNF, NPHS1) in Finland, with a strict protocol, to evaluate the results and to improve metabolic balance, growth, and development. A retrospective analysis of 23 children under two years of age at onset of PD, treated between 1995 and 2000, was performed to obtain a control population for our prospective PD study. Respectively, 21 patients less than two years of age at the beginning of PD were enrolled in prospective studies between 2001 and 2005. Medication for uremia and nutrition were care-fully adjusted during PD. Laboratory parameters and intravascular volume status were regu-larly analyzed. Growth was analyzed and compared with midparental height. In a prospective neurological study, the risk factors for development and the neurological development was determined. Brain images were surveyed. Hearing was tested. In a retrospective neurological study, the data of six NPHS1 patients with a congruent neurological syndrome was analyzed. All these patients had a serious dyskinetic cerebral palsy-like syndrome with muscular dysto-nia and athetosis (MDA). They also had a hearing defect. Metabolic control was mainly good in both PD patient groups. Hospitalization time shortened clearly. The peritonitis rate diminished. Hypertension was a common problem. Left ventricular hypertrophy decreased during the prospective study period. None of the patients in either PD group had pulmonary edema or dialysis-related seizures. Growth was good and catch-up growth was documented in most patients in both patient groups during PD. Mortality was low (5% in prospective and 9% in retrospective PD patients). In the prospective PD patient group 11 patients (52%) had some risk factor for their neuro-development originating from the predialysis period. The neurological problems, detected be-fore PD, did not worsen during PD and none of the patients developed new neurological com-plications during PD. Brain infarcts were detected in four (19%) and other ischemic lesions in three patients (14%). At the end of this study, 29% of the prospectively followed patients had a major impairment of their neurodevelopment and 43% only minor impairment. In the NPHS1+MDA patients, no clear explanation for the neurological syndrome was found. The brain MRI showed increased signal intensity in the globus pallidus area. Kernic-terus was contemplated to be causative in the hypoproteinemic newborns but it could not be proven. Mortality was as high as 67%. Our results for young PD patients were promising. Metabolic control was acceptable and growth was good. However, the children were significantly smaller when compared to their midparental height. Although many patients were found to have neurological impairment at the end of our follow-up period, PD was a safe treatment whereby the neurodevelopment did not worsen during PD.
  • Wikstén, Eeva Johanna (Helsingin yliopisto, 2016)
    Peritonsillar abscess (PTA) is the most common otorhinolaryngological infection that requires special health care management. Its treatment varies greatly due to a lack of common clinical guidelines. Tonsillectomy (TE) is performed on a portion of PTA patients, yet it remains controversial as to which PTA patients would benefit from TE. Traditional bacterial culture is ineffective at defining the causative bacteria for PTA. Rapid microarray methods have been tested, for example on serum and joint fluid samples, but not yet on pus. Most of the bacteria found in PTA are susceptible to penicillin, but, to avoid complications, antibiotics, with unnecessary broad spectrum, are frequently used instead. The aim of the first study in this thesis was to explore the microbiology of adults with PTA using a modern identification method and to find cofactors among patients with different pathogens. Using a modern DNA-based microarray method, we examined the microbial findings in the pus aspirated from 180 PTA patients. Fusobacterium necrophorum proved to be the most prevalent bacteria, occurring more frequently in younger patients; group A Streptococcus was the second most common. The microarray method seemed to work well for identifying bacteria directly from pus. In the second study, the aim was to compare the treatment modalities for PTA in countries closely related to Finland. We sent an electronic questionnaire regarding PTA treatment to all central and university hospitals in Finland, Sweden, Norway and Denmark. The study revealed diversity among treatment modalities between the four countries. To identify factors predicting a doctor ́s decision for TE among PTA patients, in the third study, we retrieved data on 819 PTA patients from a national database, which included information on whether a TE was performed within five years after a PTA diagnosis and why. The study showed that young age and previous tonsillar infections increased the probability of having a TE performed. In the fourth study, the aim was to investigate whether combining metronidazole with penicillin enhances the recovery from PTA and whether metronidazole helps prevent PTA recurrences. A total of 200 prospectively collected patients were randomised to receive either penicillin and placebo or penicillin and metronidazole. The patients filled in an electronic diary daily for the first two weeks and then weekly for the following six weeks. Most patients (90 in each group) healed well without recurrence of PTA. Thus, metronidazole neither enhanced the recovery nor prevented recurrences; furthermore, it caused unwanted adverse effects (diarrhoea and nausea). These four explorations into PTA provided valuable insight. These results make a difference not just for one patient, but for the whole health care system; the treatment is evidence-based and can be offered to those whom it serves best.
  • Immonen, Sirpa (Helsingin yliopisto, 2012)
    Alcohol use plays a part in the life of older adults, and can be assumed to be increasingly prominent in the future as the baby boomers age. Understanding alcohol consumption patterns and factors associated with risky drinking in the general population of older adults aids in detecting older adults who may suffer from the hazardous use of alcohol or alcohol use disorders. The present study assessed some issues in alcohol consumption among older adults: the prevalence of consumption and associated factors, older adults own reasoning for their alcohol consumption, the drinking of alcohol for medicinal purposes, and potentially inappropriate drug alcohol interactions. The data were gathered using a postal questionnaire sent to a stratified random sample of older adults aged ≥65 years in the City of Espoo. The number of respondents was 1 395 (71.6%). The mean age was 78 years and 62.7% were women. The guidelines of the American Geriatrics Society were used to define the at-risk drinking limits. The prevalence of alcohol consumption was 71.5% and at-risk drinking was estimated to be 10.8%; 20.6% among older men and 4.2% among women. At-risk alcohol consumption was more common in the youngest age groups and among men; 25.4% of men and 7.7% of women aged 65 70 years exceeded the at-risk drinking limit. Although the frequency and quantity of alcohol use declined with age, 18.9% of males aged 71 80 years and 11.3% aged 81 90 years exceeded the at-risk drinking limit. The corresponding figures among females were 2.5% and 1.4%. The respondents most common reasons for drinking were for having fun, celebration (58.7%), and for social reasons (54.2%). Of the respondents, the younger age groups reported more often than the older age groups that they used alcohol for having fun, celebration, and for social reasons . The proportion reporting drinking alcohol for medicinal purposes increased with age. Alcohol was consumed with meals in all age groups, although this was more common in younger age groups. A larger proportion of the at-risk users than the moderate users indicated that they were using alcohol because of meaningless life, in relieving anxiety, relieving loneliness, and relieving depression , as a pastime and because everybody uses it . The medicinal consumption of alcohol was more common in the oldest age group. Both genders used this self-medication equally. The most common conditions for which alcohol was used as a medicine were cardiovascular diseases, sleep disturbances, a common cold and indigestion. The concomitant use of drugs that have potential interactions with alcohol was common. Of the drug users, 62.2% also used alcohol. Among the at-risk users and moderate users , 42.2% and 34.9% were on drugs potentially causing significant interactions with alcohol.
  • Hosia-Randell, Helka (Helsingin yliopisto, 2010)
    Prescribing for older patients is challenging. The prevalence of diseases increases with advancing age and causes extensive drug use. Impairments in cognitive, sensory, social and physical functioning, multimorbidity and comorbidities, as well as age-related changes in pharmacokinetics and pharmacodynamics all add to the complexity of prescribing. This study is a cross-sectional assessment of all long-term residents aged ≥ 65 years in all nursing homes in Helsinki, Finland. The residents’ health status was assessed and data on their demographic factors, health and medications were collected from their medical records in February 2003. This study assesses some essential issues in prescribing for older people: psychotropic drugs (Paper I), laxatives (Paper II), vitamin D and calcium supplements (Paper III), potentially inappropriate drugs for older adults (PIDs) and drug-drug interactions (DDIs)(Paper IV), as well as prescribing in public and private nursing homes. A resident was classified as a medication user if his or her medication record indicated a regular sequence for its dosage. Others were classified as non-users. Mini Nutritional Assessment (MNA) was used to assess residents’ nutritional status, Beers 2003 criteria to assess the use of PIDs, and the Swedish, Finnish, INteraction X-referencing database (SFINX) to evaluate their exposure to DDIs. Of all nursing home residents in Helsinki, 82% (n=1987) participated in studies I, II, and IV and 87% (n=2114) participated in the study III. The residents’ mean age was 84 years, 81% were female, and 70% were diagnosed with dementia. The mean number of drugs was 7.9 per resident; 40% of the residents used ≥ 9 drugs per day, and were thus exposed to polypharmacy. Eighty percent of the residents received psychotropics; 43% received antipsychotics, and 45% used antidepressants. Anxiolytics were prescribed to 26%, and hypnotics to 28% of the residents. Of those residents diagnosed with dementia, 11% received antidementia drugs. Fifty five percent of the residents used laxatives regularly. In multivariate analysis, those factors associated with regular laxative use were advanced age, immobility, poor nutritional status, chewing problems, Parkinson’s disease, and a high number of drugs. Eating snacks between meals was associated with lower risk for laxative use. Of all participants, 33% received vitamin D supplementation, 28% received calcium supplementation, and 20% received both vitamin D and calcium. The dosage of vitamin D was rather low: 21% received vitamin D 400 IU (10 µg) or more, and only 4% received 800 IU (20 µg) or more. In multivariate analysis, residents who received vitamin D supplementation enjoyed better nutritional status, ate snacks between meals, suffered no constipation, and received regular weight monitoring. Those residents receiving PIDs (34% of all residents) more often used psychotropic medication and were more often exposed to polypharmacy than residents receiving no PIDs. Residents receiving PIDs were less often diagnosed with dementia than were residents receiving no PIDs. The three most prevalent PIDs were short-acting benzodiazepine in greater dosages than recommended, hydroxyzine, and nitrofurantoin. These three drugs accounted for nearly 77% of all PID use. Of all residents, less than 5% were susceptible to a clinically significant DDI. The most common DDIs were related to the use of potassium-sparing diuretics, carbamazepine, and codeine. Residents exposed to potential DDIs were younger, had more often suffered a previous stroke, more often used psychotropics, and were more often exposed to PIDs and polypharmacy than were residents not exposed to DDIs. Residents in private nursing homes were less often exposed to polypharmacy than were residents in public nursing homes. Long-term residents in nursing homes in Helsinki use, on average, nearly eight drugs daily. The use of psychotropic drugs in our study was notably more common than in international studies. The prevalence of laxatives equaled other prior international studies. Regardless of the known benefit and recommendation of vitamin D supplementation for elderly residing mostly indoors, the proportion of nursing home residents receiving vitamin D and calcium was surprisingly low. The use of PIDs was common among nursing home residents. PIDs increased the likelihood of DDIs. However, DDIs did not seem a major concern among the nursing home population. Monitoring PIDs and potential drug interactions could improve the quality of prescribing.
  • Udd, Lina (Helsingin yliopisto, 2012)
    The Peutz-Jeghers Syndrome is a rare cancer predisposition condition, caused by mutations inactivating the LKB1 tumor suppressor kinase. This study aimed to further the understanding of this disease, provide potential means of treatment to Peutz-Jeghers patients, and to add to our understanding of cancer formation in general. These aims were pursued through exploring the molecular functions of the LKB1 kinase, studying the tumor formation upon loss of LKB1 function, and through intervening with this tumor formation process. The study was mainly performed in the Lkb1 knockout mouse or derived tissues and cells, but partly also with Peutz-Jeghers patients and patient materials. We found that the LKB1 kinase phosphorylates and thereby activates 13 kinases in the AMP-activated kinase family, any of which could putatively relay the tumor suppressor functions of LKB1. We also found that Cyclooxygenase-2 participates in tumorigenesis in Peutz-Jeghers syndrome by promoting the growth of gastric polyps, and that inhibitor treatment suppresses the polyp formation. We also observed that these Peutz-Jeghers polyps are less differentiated than previously thought, and that signs of poor differentiation can be seen in the gastric epithelium already prior to polyp formation. In addition, we found that the polyp formation process is likely to be enhanced by other genes in addition to LKB1 and Cycloxygenase-2, as alkylating mutagenesis increased polyp formation independently of the activity of the latter. Taken together, these results point to a wide array of molecules and processes interplaying in Peutz-Jeghers tumorigenesis beyond the LKB1 kinase. Both straight molecular targets of LKB1 activity, indirect mediators of LKB1-regulated tumorigenesis, and cooperating processes have been identified. One may expect that these findings will be of use for future studies both characterizing the Peutz-Jeghers syndrome and targeting treatments for this and related tumor diseases.
  • 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.