Liquid Chromatography-Mass Spectrometry in Studies of Drug Metabolism and Permeability

Poor pharmacokinetics is one of the reasons for the withdrawal of drug candidates from clinical trials. There is an urgent need for investigating in vitro ADME (absorption, distribution, metabolism and excretion) properties and recognising unsuitable drug candidates as early as possible in the drug development process. Current throughput of in vitro ADME profiling is insufficient because effective new synthesis techniques, such as drug design in silico and combinatorial synthesis, have vastly increased the number of drug candidates. Assay technologies for larger sets of compounds than are currently feasible are critically needed. The first part of this work focused on the evaluation of cocktail strategy in studies of drug permeability and metabolic stability. N-in-one liquid chromatography-tandem mass spectrometry (LC/MS/MS) methods were developed and validated for the multiple component analysis of samples in cocktail experiments. Together, cocktail dosing and LC/MS/MS were found to form an effective tool for increasing throughput. First, cocktail dosing, i.e. the use of a mixture of many test compounds, was applied in permeability experiments with Caco-2 cell culture, which is a widely used in vitro model for small intestinal absorption. A cocktail of 7-10 reference compounds was successfully evaluated for standardization and routine testing of the performance of Caco-2 cell cultures. Secondly, cocktail strategy was used in metabolic stability studies of drugs with UGT isoenzymes, which are one of the most important phase II drug metabolizing enzymes. The study confirmed that the determination of intrinsic clearance (Clint) as a cocktail of seven substrates is possible. The LC/MS/MS methods that were developed were fast and reliable for the quantitative analysis of a heterogenous set of drugs from Caco-2 permeability experiments and the set of glucuronides from in vitro stability experiments. The performance of a new ionization technique, atmospheric pressure photoionization (APPI), was evaluated through comparison with electrospray ionization (ESI), where both techniques were used for the analysis of Caco-2 samples. Like ESI, also APPI proved to be a reliable technique for the analysis of Caco-2 samples and even more flexible than ESI because of the wider dynamic linear range. The second part of the experimental study focused on metabolite profiling. Different mass spectrometric instruments and commercially available software tools were investigated for profiling metabolites in urine and hepatocyte samples. All the instruments tested (triple quadrupole, quadrupole time-of-flight, ion trap) exhibited some good and some bad features in searching for and identifying of expected and non-expected metabolites. Although, current profiling software is helpful, it is still insufficient. Thus a time-consuming largely manual approach is still required for metabolite profiling from complex biological matrices.

Cytochrome P450-mediated drug interactions affecting lidocaine

Lidocaine is a widely used local anaesthetic agent that also has anti-arrhythmic effects. It is classified as a type Ib anti-arrhythmic agent and is used to treat ventricular tachycardia or ventricular fibrillation. Lidocaine is eliminated mainly by metabolism, and less than 5% is excreted unchanged in urine. Lidocaine is a drug with a medium to high extraction ratio, and its bioavailability is about 30%. Based on in vitro studies, the earlier understanding was that CYP3A4 is the major cytochrome P450 (CYP) enzyme involved in the metabolism of lidocaine. When this work was initiated, there was little human data on the effect of inhibitors of CYP enzymes on the pharmacokinetics of lidocaine. Because lidocaine has a low therapeutic index, medications that significantly inhibit lidocaine clearance (CL) could increase the risk of toxicity. These studies investigated the effects of some clinically important CYP1A2 and CYP3A4 inhibitors on the pharmacokinetics of lidocaine administered by different routes. All of the studies were randomized, double-blind, placebo-controlled cross-over studies in two or three phases in healthy volunteers. Pretreatment with clinically relevant doses of CYP3A4 inhibitors erythromycin and itraconazole or CYP1A2 inhibitors fluvoxamine and ciprofloxacin was followed by a single dose of lidocaine. Blood samples were collected to determine the pharmacokinetic parameters of lidocaine and its main metabolites monoethylglycinexylidide (MEGX) and 3-hydroxylidocaine (3-OH-lidocaine). Itraconazole and erythromycin had virtually no effect on the pharmacokinetics of intravenous lidocaine, but erythromycin slightly prolonged the elimination half-life (t½) of lidocaine (Study I). When lidocaine was taken orally, both erythromycin and itraconazole increased the peak concentration (Cmax) and the area under the concentration-time curve (AUC) of lidocaine by 40-70% (Study II). Compared with placebo and itraconazole, erythromycin increased the Cmax and the AUC of MEGX by 40-70% when lidocaine was given intravenously or orally (Studies I and II). The pharmacokinetics of inhaled lidocaine was unaffected by concomitant administration of itraconazole (Study III). Fluvoxamine reduced the CL of intravenous lidocaine by 41% and prolonged the t½ of lidocaine by 35%. The mean AUC of lidocaine increased 1.7-fold (Study IV). After oral administration of lidocaine, the mean AUC of lidocaine in-creased 3-fold and the Cmax 2.2-fold by fluvoxamine (Study V). During the pretreatment with fluvoxamine combined with erythromycin, the CL of intravenous lidocaine was 53% smaller than during placebo and 21% smaller than during fluvoxamine alone. The t½ of lidocaine was significantly longer during the combination phase than during the placebo or fluvoxamine phase. The mean AUC of intravenous lidocaine increased 2.3-fold and the Cmax 1.4-fold (Study IV). After oral administration of lidocaine, the mean AUC of lidocaine increased 3.6-fold and the Cmax 2.5-fold by concomitant fluvoxamine and erythromycin. The t½ of oral lidocaine was significantly longer during the combination phase than during the placebo (Study V). When lidocaine was given intravenously, the combination of fluvoxamine and erythromycin prolonged the t½ of MEGX by 59% (Study IV). Compared with placebo, ciprofloxacin increased the mean Cmax and AUC of intravenous lidocaine by 12% and 26%, respectively. The mean plasma CL of lidocaine was reduced by 22% and its t½ prolonged by 7% (Study VI). These studies clarify the principal role of CYP1A2 and suggest only a modest role of CYP3A4 in the elimination of lidocaine in vivo. The inhibition of CYP1A2 by fluvoxamine considerably reduces the elimination of lidocaine. Concomitant use of fluvoxamine and the CYP3A4 inhibitor erythromycin further increases lidocaine concentrations. The clinical implication of this work is that clinicians should be aware of the potentially increased toxicity of lidocaine when used together with inhibitors of CYP1A2 and particularly with the combination of drugs inhibiting both CYP1A2 and CYP3A4 enzymes.

Studies on CYP2C8-mediated drug interactions

Useiden lääkkeiden yhtäaikainen käyttö on nykyään hyvin yleistä, mikä lisää lääkeaineiden haitallisten yhteisvaikutusten riskiä. Lääkeaineiden poistumisessa elimistöstä ovat tärkeässä osassa niitä hajottavat (metaboloivat) maksan sytokromi P450 (CYP) entsyymit. Vasta aivan viime vuosina on havaittu, että CYP2C8-entsyymillä voi olla tärkeä merkitys mm. lääkeaineyhteisvaikutuksissa. Eräät lääkeaineet voivat estää (inhiboida) CYP2C8-entsyymin kautta tapahtuvaa metaboliaa. Tässä työssä selvitettiin CYP2C8-entsyymiä estävien lääkkeiden vaikutusta sellaisten lääkeaineiden pitoisuuksiin, joiden aikaisemman tiedon perusteella arveltiin metaboloituvan CYP2C8-välitteisesti. Näiden lääkeaineiden metaboliaa tutkittiin myös koeputkiolosuhteissa (in vitro -menetelmillä). Lisäksi CYP2C8-entsyymiä estävän lipidilääke gemfibrotsiilin yhteisvaikutusmekanismia tutkittiin selvittämällä interaktion säilymistä koehenkilöillä gemfibrotsiilin annostelun lopettamisen jälkeen. Yhteisvaikutuksia tutkittiin terveillä vapaaehtoisilla koehenkilöillä käyttäen vaihtovuoroista koeasetelmaa. Koehenkilöille annettiin CYP2C8-entsyymiä estävää lääkitystä muutaman päivän ajan ja tämän jälkeen kerta-annos tutkimuslääkettä. Koehenkilöiltä otettiin useita verinäytteitä, joista määritettiin lääkepitoisuudet nestekromatografisilla tai massaspektrometrisillä menetelmillä. Gemfibrotsiili nosti ripulilääke loperamidin pitoisuudet keskimäärin kaksinkertaiseksi. Gemfibrotsiili lisäsi, mutta vain hieman, kipulääke ibuprofeenin pitoisuuksia, eikä sillä ollut mitään vaikutusta unilääke tsopiklonin pitoisuuksiin toisin kuin aiemman kirjallisuuden perusteella oli odotettavissa. Toinen CYP2C8-estäjä, mikrobilääke trimetopriimi, nosti diabeteslääke pioglitatsonin pitoisuuksia keskimäärin noin 40 %. Gemfibrotsiili nosti diabeteslääke repaglinidin pitoisuudet 7-kertaiseksi ja tämä yhteisvaikutus säilyi lähes yhtä voimakkaana vielä 12 tunnin päähän viimeisestä gemfibrotsiiliannoksesta. Tehdyt havainnot ovat käytännön lääkehoidon kannalta merkittäviä ja ne selvittävät CYP2C8-entsyymin merkitystä useiden lääkkeiden metaboliassa. Gemfibrotsiilin tai muiden CYP2C8-entsyymiä estävien lääkkeiden yhteiskäyttö loperamidin kanssa voi lisätä loperamidin tehoa tai haittavaikutuksia. Toisaalta CYP2C8-entsyymin osuus tsopiklonin ja ibuprofeenin metaboliassa näyttää olevan pieni. Trimetopriimi nosti kohtalaisesti pioglitatsonin pitoisuuksia, ja kyseisten lääkkeiden yhteiskäyttö voi lisätä pioglitatsonin annosriippuvaisia haittavaikutuksia. Gemfibrotsiili-repaglinidi-yhteisvaikutuksen päämekanismi in vivo näyttää olevan CYP2C8-entsyymin palautumaton esto. Tämän vuoksi gemfibrotsiilin estovaikutus ja yhteisvaikutusriski säilyvät pitkään gemfibrotsiilin annostelun lopettamisen jälkeen, mikä tulee ottaa huomioon käytettäessä sitä CYP2C8-välitteisesti metaboloituvien lääkkeiden kanssa.

Levosimendan in patients with ischaemic heart disease

Levosimendan is a drug developed for the treatment of heart failure. Its mechanism of action includes calcium sensitization of contractile proteins and the opening of ATP-sensitive potassium channels. The combination of positive inotropy with possible anti-ischaemic effects via potassium channel opening may offer benefits in comparison with currently available intravenous inotropes, which are contraindicated in patients with ongoing myocardial ischaemia. The active levosimendan metabolite OR-1896 significantly prolongs the duration of the haemodynamic effects of levosimendan. The aims of the present study were to investigate: 1) the clinical effects and safety of intravenous and oral levosimendan and 2) the pharmacodynamics and pharmacokinetics of intravenous and oral levosimendan and its metabolites in patients with ischaemic heart disease. Levosimendan was administered intravenously or orally in four studies to 557 patients with ischaemic heart disease with or without concomitant heart failure. One study included patients with acute myocardial infarction, while the other three studies included stable ischaemic patients. Non-invasive haemodynamic measurements were used in all studies, and blood samples for pharmacokinetics were drawn in three studies. Safety was followed by ECG recordings, adverse event inquiries and laboratory assessments. Intravenous levosimendan, administered as a 6-hour infusion did not cause clinically significant hypotension or ischaemia in comparison with placebo and reduced worsening heart failure and short- and long-term mortality. Increase in incidence of hypotension and ischaemia was seen only with the highest dose (0.4 µg/kg/min). Both intravenous and oral levosimendan possessed a moderate positive inotropic effect. Vasodilatory effect was more pronounced with intravenous levosimendan. A chronotropic effect was seen in all studies; however, it was not accompanied by any increase in arrhythmic events. The formation of levosimendan metabolites after oral dosing increased linearly with the daily dose of the parent drug, leading to increased inotropic and chronotropic response. Levosimendan was well tolerated in all studies. In conclusion, levosimendan was safe and effective in the treatment of patients with acute or chronic ischaemia. The risk-benefit ratio of intravenous levosimendan is favourable up to the dose of 0.2 µg/kg/min. The daily dose of oral levosimendan in patients with ischaemic heart failure should not exceed 4 mg due to an increase in chronotropic response.

Individualized immunosuppression after renal transplantation in childhood

Pediatric renal transplantation (TX) has evolved greatly during the past few decades, and today TX is considered the standard care for children with end-stage renal disease. In Finland, 191 children had received renal transplants by October 2007, and 42% of them have already reached adulthood. Improvements in treatment of end-stage renal disease, surgical techniques, intensive care medicine, and in immunosuppressive therapy have paved the way to the current highly successful outcomes of pediatric transplantation. In children, the transplanted graft should last for decades, and normal growth and development should be guaranteed. These objectives set considerable requirements in optimizing and fine-tuning the post-operative therapy. Careful optimization of immunosuppressive therapy is crucial in protecting the graft against rejection, but also in protecting the patient against adverse effects of the medication. In the present study, the results of a retrospective investigation into individualized dosing of immunosuppresive medication, based on pharmacokinetic profiles, therapeutic drug monitoring, graft function and histology studies, and glucocorticoid biological activity determinations, are reported. Subgroups of a total of 178 patients, who received renal transplants in 1988 2006 were included in the study. The mean age at TX was 6.5 years, and approximately 26% of the patients were <2 years of age. The most common diagnosis leading to renal TX was congenital nephrosis of the Finnish type (NPHS1). Pediatric patients in Finland receive standard triple immunosuppression consisting of cyclosporine A (CsA), methylprednisolone (MP) and azathioprine (AZA) after renal TX. Optimal dosing of these agents is important to prevent rejections and preserve graft function in one hand, and to avoid the potentially serious adverse effects on the other hand. CsA has a narrow therapeutic window and individually variable pharmacokinetics. Therapeutic monitoring of CsA is, therefore, mandatory. Traditionally, CsA monitoring has been based on pre-dose trough levels (C0), but recent pharmacokinetic and clinical studies have revealed that the immunosuppressive effect may be related to diurnal CsA exposure and blood CsA concentration 0-4 hours after dosing. The two-hour post-dose concentration (C2) has proved a reliable surrogate marker of CsA exposure. Individual starting doses of CsA were analyzed in 65 patients. A recommended dose based on a pre-TX pharmacokinetic study was calculated for each patient by the pre-TX protocol. The predicted dose was clearly higher in the youngest children than in the older ones (22.9±10.4 and 10.5±5.1 mg/kg/d in patients <2 and >8 years of age, respectively). The actually administered oral doses of CsA were collected for three weeks after TX and compared to the pharmacokinetically predicted dose. After the TX, dosing of CsA was adjusted according to clinical parameters and blood CsA trough concentration. The pharmacokinetically predicted dose and patient age were the two significant parameters explaining post-TX doses of CsA. Accordingly, young children received significantly higher oral doses of CsA than the older ones. The correlation to the actually administered doses after TX was best in those patients, who had a predicted dose clearly higher or lower (> ±25%) than the average in their age-group. Due to the great individual variation in pharmacokinetics standardized dosing of CsA (based on body mass or surface area) may not be adequate. Pre-Tx profiles are helpful in determining suitable initial CsA doses. CsA monitoring based on trough and C2 concentrations was analyzed in 47 patients, who received renal transplants in 2001 2006. C0, C2 and experienced acute rejections were collected during the post-TX hospitalization, and also three months after TX when the first protocol core biopsy was obtained. The patients who remained rejection free had slightly higher C2 concentrations, especially very early after TX. However, after the first two weeks also the trough level was higher in the rejection-free patients than in those with acute rejections. Three months after TX the trough level was higher in patients with normal histology than in those with rejection changes in the routine biopsy. Monitoring of both the trough level and C2 may thus be warranted to guarantee sufficient peak concentration and baseline immunosuppression on one hand and to avoid over-exposure on the other hand. Controlling of rejection in the early months after transplantation is crucial as it may contribute to the development of long-term allograft nephropathy. Recently, it has become evident that immunoactivation fulfilling the histological criteria of acute rejection is possible in a well functioning graft with no clinical sings or laboratory perturbations. The influence of treatment of subclinical rejection, diagnosed in 3-month protocol biopsy, to graft function and histology 18 months after TX was analyzed in 22 patients and compared to 35 historical control patients. The incidence of subclinical rejection at three months was 43%, and the patients received a standard rejection treatment (a course of increased MP) and/or increased baseline immunosuppression, depending on the severity of rejection and graft function. Glomerular filtration rate (GFR) at 18 months was significantly better in the patients who were screened and treated for subclinical rejection in comparison to the historical patients (86.7±22.5 vs. 67.9±31.9 ml/min/1.73m2, respectively). The improvement was most remarkable in the youngest (<2 years) age group (94.1±11.0 vs. 67.9±26.8 ml/min/1.73m2). Histological findings of chronic allograft nephropathy were also more common in the historical patients in the 18-month protocol biopsy. All pediatric renal TX patients receive MP as a part of the baseline immunosuppression. Although the maintenance dose of MP is very low in the majority of the patients, the well-known steroid-related adverse affects are not uncommon. It has been shown in a previous study in Finnish pediatric TX patients that steroid exposure, measured as area under concentration-time curve (AUC), rather than the dose correlates with the adverse effects. In the present study, MP AUC was measured in sixteen stable maintenance patients, and a correlation with excess weight gain during 12 months after TX as well as with height deficit was found. A novel bioassay measuring the activation of glucocorticoid receptor dependent transcription cascade was also employed to assess the biological effect of MP. Glucocorticoid bioactivity was found to be related to the adverse effects, although the relationship was not as apparent as that with serum MP concentration. The findings in this study support individualized monitoring and adjustment of immunosuppression based on pharmacokinetics, graft function and histology. Pharmacokinetic profiles are helpful in estimating drug exposure and thus identifying the patients who might be at risk for excessive or insufficient immunosuppression. Individualized doses and monitoring of blood concentrations should definitely be employed with CsA, but possibly also with steroids. As an alternative to complete steroid withdrawal, individualized dosing based on drug exposure monitoring might help in avoiding the adverse effects. Early screening and treatment of subclinical immunoactivation is beneficial as it improves the prospects of good long-term graft function.

Inhibition of CYP1A2-mediated drug metabolism in vitro and in humans : With special emphasis on rofecoxib and other NSAIDs

The cytochrome P450 1A2 (CYP1A2) is one of the major metabolizing enzymes. The muscle relaxant tizanidine is a selective substrate of CYP1A2, and the non-steroidal anti-inflammatory drug (NSAID) rofecoxib was thought to modestly in-hibit it. Cases suggesting an interaction between tizanidine and rofecoxib had been reported, but the mechanism was unknown. Also other NSAIDs are often used in combination with muscle relaxants. The aims of this study were to investigate the effect of rofecoxib, several other NSAIDs and female sex steroids on CYP1A2 ac-tivity in vitro and in vivo, and to evaluate the predictability of in vivo inhibition based on in vitro data. In vitro, the effect of several NSAIDs, female sex steroids and model inhibitors on CYP1A2 activity was studied in human liver microsomes, without and with preincubation. In placebo controlled, cross-over studies healthy volunteers ingested a single dose of tizanidine after a pretreament with the inhibitor (rofecoxib, tolfenamic acid or celecoxib) or placebo. Plasma (and urine) concentrations of tizanidine and its metabolites were measured, and the pharmacodynamic effects were recorded. A caffeine test was also performed. In vitro, fluvoxamine, tolfenamic acid, mefenamic acid and rofecoxib potently in-hibited CYP1A2. Ethinylestradiol, celecoxib, desogestrel and zolmitriptan were moderate, and etodolac, ciprofloxacin, etoricoxib and gestodene were weak inhibi-tors of CYP1A2. At 100 µM, other tested NSAIDs and steroids inhibited CYP1A2 less than 35%. Rofecoxib was found to be a mechanism-based inhibitor of CYP1A2. In vivo, rofecoxib greatly increased the plasma concentrations (over ten-fold) and the pharmacodynamic effects of tizanidine. Also the metabolism of caf-feine was impaired by rofecoxib. Despite the relatively strong in vitro CYP1A2 inhibitory effects, tolfenamic acid and celecoxib did not have a significant effect on tizanidine and caffeine concentrations in humans. Competitive inhibition model and the free plasma concentration of the inhibitor predicted well the effect of fluvoxam-ine and the lack of effect of tolfenamic acid and celecoxib on tizanidine concentra-tions in humans, and mechanism-based inhibition model explained the effects of rofecoxib. However, the effects of ciprofloxacin and oral contraceptives were un-derestimated from the in vitro data. Rofecoxib is a potent mechanism-based inhibitor of CYP1A2 in vitro and in vivo. This mechanism may be involved in the adverse cardiovascular effects of rofecoxib. Tolfenamic acid and celecoxib seem to be safe in combination with tizanidine, but mefenamic acid might have some effect on tizanidine concentrations in vivo. Con-sidering the mechanism of inhibition, and using the free plasma concentration of the inhibitor, many but not all CYP1A2 interactions can be predicted from in vitro data.

Just say no? The influence of personal goals, beliefs, psychosocial adjustment, and parental relationships in drug use by adolescents

The study in its entirety focused on factors related to adolescents decisions concerning drug use. The term drug use is taken here to include the use of tobacco products, alcohol, narcotics, and other addictive substances. First, the reasons given for drug use (attributions) were investigated. Secondly, the influence of personal goals, the beliefs involved in decision making, psychosocial adjustment including body image and involvement with peers, and parental relationships on drug use were studied. Two cohorts participated in the study. In 1984, a questionnaire on reasons for drug use was administered to a sample of adolescents aged 14-16 (N=396). A further questionnaire was administered to another sample of adolescents aged 14-16 (N=488) in 1999. The results for both cohorts were analyzed in Articles I and II. In Articles III and IV further analysis was carried out on the second cohort (N=488). The research report presented here provides a synthesis of all four articles, together with material from a further analysis. In a comparison of the two cohorts it was found that the attributions for drug use had changed considerably over the intervening fifteen-year period. In relation to alcohol and narcotics use an increase was found in reasons involving inner subjective experiences, with mention of the good feeling and fun resulting from alcohol and narcotics use. In addition, the goals of alcohol consumption were increasingly perceived as drinking to get drunk, and for its own sake. The attributions for the adolescents own smoking behavior were quite different from the attributions for smoking by others. The attributions were only weakly influenced by the participants gender or by their smoking habits, either in 1984 or 1999. In relation to participants own smoking, the later questionnaire elicited more mention of inner subjective experiences involving "good feeling. In relation to the perceived reasons for other people s smoking, it elicited more responses connected with the notion of "belonging. In the second sample, the results indicated that the levels of body satisfaction among adolescent girls are lower than those among adolescent boys. Overall, dissatisfaction with one's physical appearance seemed to relate to drug use. Girls were also found to engage in more discussions than boys; this applied to (i) discussion with peers (concerning both intimate and general matters), and (ii) discussion with parents (concerning general matters). However, more than a quarter of the boys (out of the entire population) reported only low intimacy with both parents and peers. If both drinking and smoking were considered, it seemed that girls in particular who reported drinking and smoking also reported high intimacy with parents and peers. Boys who reported drinking and smoking reported only medium intimacy with parents and peers. In addition, having an intimate relationship with one's peers was associated with a greater tendency to drink purely in order to get drunk. Overall, the results seemed to suggest that drug use is connected with a close relationship with peers and (surprisingly) with a close relationship with parents. Nevertheless, there were also indications that to some extent peer relationships can also protect adolescents from smoking and alcohol use. The results, which underline the complexity of adolescent drug use, are taken up in the Discussion section. It may be that body image and/or other identity factors play a more prominent role in all drug use than has previously been acknowledged. It does appear that in the course of planning support campaigns for adolescents at risk of drug use, we should focus more closely on individuals and their inner world. More research on this field is clearly needed, and therefore some ideas for future research are also presented.

Cyclin dependent protein kinases as drug targets – potential in cardiovascular diseases

Complications of atherosclerosis such as myocardial infarction and stroke are the primary cause of death in Western societies. The development of atherosclerotic lesions is a complex process, including endothelial cell dysfunction, inflammation, extracellular matrix alteration and vascular smooth muscle cell (VSMC) proliferation and migration. Various cell cycle regulatory proteins control VSMC proliferation. Protein kinases called cyclin dependent kinases (CDKs) play a major role in regulation of cell cycle progression. At specific phases of the cell cycle, CDKs pair with cyclins to become catalytically active and phosphorylate numerous substrates contributing to cell cycle progression. CDKs are also regulated by cyclin dependent kinase inhibitors, activating and inhibitory phosphorylation, proteolysis and transcription factors. This tight regulation of cell cycle is essential; thus its deregulation is connected to the development of cancer and other proliferative disorders such as atherosclerosis and restenosis as well as neurodegenerative diseases. Proteins of the cell cycle provide potential and attractive targets for drug development. Consequently, various low molecular weight CDK inhibitors have been identified and are in clinical development. Tylophorine is a phenanthroindolizidine alkaloid, which has been shown to inhibit the growth of several human cancer cell lines. It was used in Ayurvedic medicine to treat inflammatory disorders. The aim of this study was to investigate the effect of tylophorine on human umbilical vein smooth muscle cell (HUVSMC) proliferation, cell cycle progression and the expression of various cell cycle regulatory proteins in order to confirm the findings made with tylophorine in rat cells. We used several methods to determine our hypothesis, including cell proliferation assay, western blot and flow cytometric cell cycle distribution analysis. We demonstrated by cell proliferation assay that tylophorine inhibits HUVSMC proliferation dose-dependently with an IC50 value of 164 nM ± 50. Western blot analysis was used to determine the effect of tylophorine on expression of cell cycle regulatory proteins. Tylophorine downregulates cyclin D1 and p21 expression levels. The results of tylophorine’s effect on phosphorylation sites of p53 were not consistent. More sensitive methods are required in order to completely determine this effect. We used flow cytometric cell cycle analysis to investigate whether tylophorine interferes with cell cycle progression and arrests cells in a specific cell cycle phase. Tylophorine was shown to induce the accumulation of asynchronized HUVSMCs in S phase. Tylophorine has a significant effect on cell cycle, but its role as cell cycle regulator in treatment of vascular proliferative diseases and cancer requires more experiments in vitro and in vivo.

Treatment of illicit opioid and γ-hydroxybutyrate overdose by Helsinki emergency medical services

Aims: To examine the characteristics, incidence, treatment and outcome of presumed opioid, γ-hydroxybutyrate (GHB) and γ-butyrolactone (GBL) overdoses involving users of illicit drugs in Helsinki. GHB/GBL were included in this study, despite not being opioids, due to the relative ease with which they can cause potentially fatal respiratory depression. The incidence and time interval of recurrent opioid toxicity after prehospital administration of naloxone, an opioid antagonist, was studied in presumed heroin overdose patients. Naloxone has been reported to have many adverse effects and the effects of naloxone administered during an opioid overdose on the cardiovascular system and catecholamine levels in piglets were studied. Materials and methods: Patients included in these published retrospective studies were from the following time periods: Study I: 1995-2002, II: 1997-2000, III: 1995-2000, V: 2006-2007. Presumed opioid overdose patients were examined in studies I, II and III. GHB/GBL overdoses among injecting drug users was examined in study V. Recurrent opioid toxicity after prehospital naloxone administration in heroin overdose patients was examined in study III. The effects of naloxone (80 μg/kg i.v.) on the cardiovascular system and catecholamine levels administered during morphine overdose (8mg/kg i.v.) and under propofol anesthesia with spontaneous breathing were studied in eight piglets (IV). In this thesis, previously unpublished data on the incidence of opioid overdose between 2001-2007 and comparison of the characteristics of buprenorphine and heroin overdose patients encountered in 1995-2005 are also included. Results: Helsinki Emergency Medical Service (EMS) ambulances were dispatched annually to 34,153- 45,118 calls from 1995 to 2007. Of them, 7-8% were coded as intoxications or overdoses. During this time, 436 patients were treated by the EMS for presumed opioid overdose. The peak incidence of opioid overdoses was in the year 2000 (113 cases), after which they declined to 6-26 cases annually. The annual incidence of buprenorphine related overdoses increased from 4 (4% of opioid overdoses) in the year 2000 to 8 (30% of opioid overdoses) in 2007. The annual number of GHB related overdose patients treated by Helsinki EMS increased from 21 to 73 between 2004-2007. There appeared to be a peak in the incidence of both GHB/GBL and opioid related overdoses on Saturdays. Characteristics of opioid overdose patients The median age of opioid overdose patients was 28 years (22;33, 25- and 75-percentiles), and 84% were male. Buprenorphine overdose patients had more polydrug, such as alcohol and/or benzodiazepines, use in comparison with heroin overdose patients, 70% versus 33%, respectively. Severe respiratory depression was reported less often with buprenorphine overdoses compared to heroin overdoses, in 67.0% versus 85.4%, respectively. Outcome of heroin overdose patients with cardiac arrest Ninety four patients suffered cardiac arrest due to acute drug poisoning/overdose and were thus considered for resuscitation. Resuscitation was attempted in 72 cases. Cardiac arrest was caused by heroin overdose for 19 patients of which three (16%) were discharged alive. Other agents also induced cardiac arrest in 53 patients, of which six (11%) were discharged alive. The arrest was either EMS witnessed or occurring after the emergency call for all survivors of heroin induced cardiac arrest. Characteristics of GHB/GBL overdose patients The records of 100 GHB/GBL related overdose patients from 2006-2007 were retrieved. The median age of GHB/GBL overdose patients encountered on weekend nights was 24 years (22;27, 25- and 75-percentiles) and 49% were male. Polydrug use was reported in 62-80% of the cases. Thirty nine patients were encountered on Friday-Saturday or Saturday-Sunday night between 11 pm-6 am. The remaining sixty one patients were outside this time frame. There was a statistically significant difference between these two groups in history of chronic injecting drug use (33% vs. 59%, respectively, p=0.012). Recurrent heroin toxicity after prehospital naloxone administration Study III included 145 presumed heroin overdose patients. After prehospital care, 84 patients refused further care and were not transported to an Emergency Department (ED). Seventy one (85%) of them were administered naloxone by the EMS. During a 12-h follow up period, none of these patients developed severe recurrent opioid toxicity. The remaining 61 patients were transported to an ED. Prior to transportation, 52 (85%) patients were administered naloxone by the EMS. Fifteen of them were administered naloxone also in the ED and recurrent opioid toxicity was evident either on arrival at the ED or shortly thereafter. Prehospital naloxone was administered either intravenously, intramuscularly (i.m.) or subcutaneously (s.c.). There was a tendency for more frequent recurrent heroin toxicity among the patients with only intravenous administration of prehospital naloxone (13/36) compared with the patients with intramuscular or subcutaneous prehospital naloxone (2/16), p=0.106. The effects of naloxone on the cardiovascular system and catecholamine levels in piglets The administration of morphine to piglets resulted in an obvious respiratory depression, which was reversed by naloxone. Two severely hypoxemic piglets developed cardiac arrest after naloxone administration. In the other six animals, the administration of naloxone did not provoke arrhythmias, cardiac ischemia or visible evidence of pulmonary edema. There was a statistically significant (p=0.012) increase in norepinephrine levels after morphine administration and before naloxone administration: from 1.9 (1.3-2.3) ng/ml at baseline, to 31.7 (8.3-83.0) ng/ml (median, 25 and 75 percentiles parentheses) after morphine administration. After the administration of naloxone, the catecholamine levels continued to increase in only one of the animals. Conclusions: The incidence of buprenorphine related overdoses increased during the study period, but was still lower in comparison to those involving heroin. Injecting drug users have also started to use GHB/GBL. While recreational drug users use GHB/GBL during weekend nights, a GHB/GBL overdose patient encounter during weekdays has a more probable history of injecting drug use. Patients with cardiac arrest after heroin overdose have a poor prognosis. It appears to be safe to leave heroin overdose patients on scene after prehospital treatment with naloxone. Although no statistically significant difference was observed, it seems prudent to administer part of the total naloxone dose s.c. or i.m. to reduce the risk of recurrent respiratory depression. If transported to an ED, an observation period of one to two hours after the last naloxone dose seems adequate. The treating physician must be vigilant, however, due to the high prevalence of polydrug use and high morbidity after non fatal heroin overdose. Furthermore, care should be taken regarding possible chronic disorders and drug rehabilitation should be addressed. In the experimental animal study, two animals developed cardiac arrest after receiving naloxone while in hypoxemia and bradycardia. Further studies are required to assess the effect of naloxone during opioid-induced hypercapnia and hypoxemia in animals addicted to opioids.

Mechanism-based inhibition of CYP2C8 by gemfibrozil in humans : characterisation of time and dose relationships

Drug-drug interactions may cause serious, even fatal clinical consequences. Therefore, it is important to examine the interaction potential of new chemical entities early in drug development. Mechanism-based inhibition is a pharmacokinetic interaction type, which causes irreversible loss of enzyme activity and can therefore lead to unusually profound and long-lasting consequences. The in vitro in vivo extrapolation (IVIVE) of drug-drug interactions caused by mechanism-based inhibition is challenging. Consequently, many of these interactions have remained unrecognised for many years. The concomitant use of the fibrate-class lipid-lowering agent gemfibrozil increases the concentrations of some drugs and their effects markedly. Even fatal cases of rhabdomyolysis occurred in patients administering gemfibrozil and cerivastatin concomitantly. One of the main mechanisms behind this effect is the mechanism-based inhibition of the cytochrome P450 (CYP) 2C8 enzyme by a glucuronide metabolite of gemfibrozil leading to increased cerivastatin concentrations. Although the clinical use of gemfibrozil has clearly decreased during recent years, gemfibrozil is still needed in some special cases. To enable safe use of gemfibrozil concomitantly with other drugs, information concerning the time and dose relationships of CYP2C8 inhibition by gemfibrozil should be known. This work was carried out as four in vivo clinical drug-drug interaction studies to examine the time and dose relationships of the mechanism-based inhibitory effect of gemfibrozil on CYP2C8. The oral antidiabetic drug repaglinide was used as a probe drug for measuring CYP2C8 activity in healthy volunteers. In this work, mechanism-based inhibition of the CYP2C8 enzyme by gemfibrozil was found to occur rapidly in humans. The inhibitory effect developed to its maximum already when repaglinide was given 1-3 h after gemfibrozil intake. In addition, the inhibition was shown to abate slowly. A full recovery of CYP2C8 activity, as measured by repaglinide metabolism, was achieved 96 h after cessation of gemfibrozil treatment. The dose-dependency of the mechanism-based inhibition of CYP2C8 by gemfibrozil was shown for the first time in this work. CYP2C8 activity was halved by a single 30 mg dose of gemfibrozil or by twice daily administration of less than 30 mg of gemfibrozil. Furthermore, CYP2C8 activity was decreased over 90% by a single dose of 900 mg gemfibrozil or twice daily dosing of approximately 100 mg gemfibrozil. In addition, with the application of physiological models to the data obtained in the dose-dependency studies, the major role of mechanism-based inhibition of CYP2C8 in the interaction between gemfibrozil and repaglinide was confirmed. The results of this work enhance the proper use of gemfibrozil and the safety of patients. The information related to time-dependency of CYP2C8 inhibition by gemfibrozil may also give new insights in order to improve the IVIVE of the drug-drug interactions of new chemical entities. The information obtained by this work may be utilised also in the design of clinical drug-drug interaction studies in the future.