Characterization of inhibition of platelet function by paracetamol and its interaction with diclofenac and parecoxib

Aim: To characterize the inhibition of platelet function by paracetamol in vivo and in vitro, and to evaluate the possible interaction of paracetamol and diclofenac or valdecoxib in vivo. To assess the analgesic effect of the drugs in an experimental pain model. Methods: Healthy volunteers received increasing doses of intravenous paracetamol (15, 22.5 and 30 mg/kg), or the combination of paracetamol 1 g and diclofenac 1.1 mg/kg or valdecoxib 40 mg (as the pro-drug parecoxib). Inhibition of platelet function was assessed with photometric aggregometry, the platelet function analyzer (PFA-100), and release of thromboxane B2. Analgesia was assessed with the cold pressor test. The inhibition coefficient of platelet aggregation by paracetamol was determined as well as the nature of interaction between paracetamol and diclofenac by an isobolographic analysis in vitro. Results: Paracetamol inhibited platelet aggregation and TxB2-release dose-dependently in volunteers and concentration-dependently in vitro. The inhibition coefficient was 15.2 mg/L (95% CI 11.8 - 18.6). Paracetamol augmented the platelet inhibition by diclofenac in vivo, and the isobole showed that this interaction is synergistic. Paracetamol showed no interaction with valdecoxib. PFA-100 appeared insensitive in detecting platelet dysfunction by paracetamol, and the cold-pressor test showed no analgesia. Conclusions: Paracetamol inhibits platelet function in vivo and shows synergism when combined with diclofenac. This effect may increase the risk of bleeding in surgical patients with an impaired haemostatic system. The combination of paracetamol and valdecoxib may be useful in patients with low risk for thromboembolism. The PFA-100 seems unsuitable for detection of platelet dysfunction and the cold-pressor test seems unsuitable for detection of analgesia by paracetamol.

Ischemic Stroke in Young Adults

Stroke is the second leading cause of death and the leading cause of disability worldwide. Of all strokes, up to 80% to 85% are ischemic, and of these, less than 10% occur in young individuals. Stroke in young adults—most often defined as stroke occurring under the age of 45 or 50—can be particularly devastating due to long expected life-span ahead and marked socio-economic consequences. Current basic knowledge on ischemic stroke in this age group originates mostly from rather small and imprecise patient series. Regarding emergency treatment, systematic data on use of intravenous thrombolysis are absent. For this Thesis project, we collected detailed clinical and radiological data on all consecutive patients aged 15 to 49 with first-ever ischemic stroke between 1994 and 2007 treated at the Helsinki University Central Hospital. The aims of the study were to define demographic characteristics, risk factors, imaging features, etiology, and long-term mortality and its predictors in this patient population. We additionally sought to investigate, whether intravenous thrombolysis is safe and beneficial for the treatment of acute ischemic stroke in the young. Of our 1008 patients, most were males (ratio 1.7:1), who clearly outnumbered females after the age of 44, but females were preponderant among those aged <30. Occurrence increased exponentially. The most frequent risk factors were dyslipidemia (60%), smoking (44%), and hypertension (39%). Risk factors accumulated in males and along aging. Cardioembolism (20%) and cervicocerebral artery dissection (15%) were the most frequent etiologic subgroups, followed by small-vessel disease (14%), and large-artery atherosclerosis (8%). A total of 33% had undetermined etiology. Left hemisphere strokes were more common in general. Posterior circulation infarcts were more common among those aged <45. Multiple brain infarcts were present in 23% of our patients, 13% had silent infarcts, and 5% had leukoaraiosis. Of those with silent brain infarcts, majority (54%) had only a single lesion, and most of the silent strokes were located in basal ganglia (39%) and subcortical regions (21%). In a logistic regression analysis, type 1 diabetes mellitus in particular predicted the presence of both silent brain infarcts (odds ratio 5.78, 95% confidence interval 2.37-14.10) and leukoaraiosis (9.75; 3.39-28.04). We identified 48 young patients with hemispheric ischemic stroke treated with intravenous tissue plasminogen activator, alteplase. For comparisons, we searched 96 untreated control patients matched by age, gender, and admission stroke severity, as well as 96 alteplase-treated older controls aged 50 to 79 matched by gender and stroke severity. Alteplase-treated young patients recovered more often completely (27% versus 10%, P=0.010) or had only mild residual symptoms (40% versus 22%, P=0.025) compared to age-matched controls. None of the alteplase-treated young patients had symptomatic intracerebral hemorrhage or died within 3-month follow-up. Overall long-term mortality was low in our patient population. Cumulative mortality risks were 2.7% (95% confidence interval 1.5-3.9%) at 1 month, 4.7% (3.1-6.3%) at 1 year, and 10.7% (9.9-11.5%) at 5 years. Among the 30-day survivors who died during the 5-year follow-up, more than half died due to vascular causes. Malignancy, heart failure, heavy drinking, preceding infection, type 1 diabetes, increasing age, and large-artery atherosclerosis causing the index stroke independently predicted 5-year mortality when adjusted for age, gender, relevant risk factors, stroke severity, and etiologic subtype. In sum, young adults with ischemic stroke have distinct demographic patterns and they frequently harbor traditional vascular risk factors. Etiology in the young is extremely diverse, but in as many as one-third the exact cause remains unknown. Silent brain infarcts and leukoaraiosis are not uncommon brain imaging findings in these patients and should not be overlooked due to their potential prognostic relevance. Outcomes in young adults with hemispheric ischemic stroke can safely be improved with intravenous thrombolysis. Furthermore, despite their overall low risk of death after ischemic stroke, several easily recognizable factors—of which most are modifiable—predict higher mortality in the long term in young adults.

Modern computed tomography techniques in stroke management

Technological development of fast multi-sectional, helical computed tomography (CT) scanners has allowed computed tomography perfusion (CTp) and angiography (CTA) in evaluating acute ischemic stroke. This study focuses on new multidetector computed tomography techniques, namely whole-brain and first-pass CT perfusion plus CTA of carotid arteries. Whole-brain CTp data is acquired during slow infusion of contrast material to achieve constant contrast concentration in the cerebral vasculature. From these data quantitative maps are constructed of perfused cerebral blood volume (pCBV). The probability curve of cerebral infarction as a function of normalized pCBV was determined in patients with acute ischemic stroke. Normalized pCBV, expressed as a percentage of contralateral normal brain pCBV, was determined in the infarction core and in regions just inside and outside the boundary between infarcted and noninfarcted brain. Corresponding probabilities of infarction were 0.99, 0.96, and 0.11, R² was 0.73, and differences in perfusion between core and inner and outer bands were highly significant. Thus a probability of infarction curve can help predict the likelihood of infarction as a function of percentage normalized pCBV. First-pass CT perfusion is based on continuous cine imaging over a selected brain area during a bolus injection of contrast. During its first passage, contrast material compartmentalizes in the intravascular space, resulting in transient tissue enhancement. Functional maps such as cerebral blood flow (CBF), and volume (CBV), and mean transit time (MTT) are then constructed. We compared the effects of three different iodine concentrations (300, 350, or 400 mg/mL) on peak enhancement of normal brain tissue and artery and vein, stratified by region-of-interest (ROI) location, in 102 patients within 3 hours of stroke onset. A monotonic increasing peak opacification was evident at all ROI locations, suggesting that CTp evaluation of patients with acute stroke is best performed with the highest available concentration of contrast agent. In another study we investigated whether lesion volumes on CBV, CBF, and MTT maps within 3 hours of stroke onset predict final infarct volume, and whether all these parameters are needed for triage to intravenous recombinant tissue plasminogen activator (IV-rtPA). The effect of IV-rtPA on the affected brain by measuring salvaged tissue volume in patients receiving IV-rtPA and in controls was investigated also. CBV lesion volume did not necessarily represent dead tissue. MTT lesion volume alone can serve to identify the upper size limit of the abnormally perfused brain, and those with IV-rtPA salvaged more brain than did controls. Carotid CTA was compared with carotid DSA in grading of stenosis in patients with stroke symptoms. In CTA, the grade of stenosis was determined by means of axial source and maximum intensity projection (MIP) images as well as a semiautomatic vessel analysis. CTA provides an adequate, less invasive alternative to conventional DSA, although tending to underestimate clinically relevant grades of stenosis.

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.

Direct bone marrow HSC transplantation enhances local engraftment at the expense of systemic engraftment in NSG mice

Direct bone marrow (BM) injection has been proposed as a strategy to bypass homing inefficiencies associated with intravenous (IV) hematopoietic stem cell (HSC) transplantation. Despite physical delivery into the BM cavity, many donor cells are rapidly redistributed by vascular perfusion, perhaps compromising efficacy. Anchoring donor cells to 3-dimensional (3D) multicellular spheroids, formed from mesenchymal stem/stromal cells (MSC) might improve direct BM transplantation. To test this hypothesis, relevant combinations of human umbilical cord blood-derived CD34(+) cells and BM-derived MSC were transplanted into NOD/SCID gamma (NSG) mice using either IV or intrafemoral (IF) routes. IF transplantation resulted in higher human CD45(+) and CD34(+) cell engraftment within injected femurs relative to distal femurs regardless of cell combination, but did not improve overall CD45(+) engraftment at 8 weeks. Analysis within individual mice revealed that despite engraftment reaching near saturation within the injected femur, engraftment at distal hematopoietic sites including peripheral blood, spleen and non-injected femur, could be poor. Our data suggest that the retention of human HSC within the BM following direct BM injection enhances local chimerism at the expense of systemic chimerism in this xenogeneic model.

Proton magnetic resonance spectroscopy of a boron neutron capture therapy 10B-carrier, L-p-boronophenylalanine-fructose complex

Boron neutron capture therapy (BNCT) is a radiotherapy that has mainly been used to treat malignant brain tumours, melanomas, and head and neck cancer. In BNCT, the patient receives an intravenous infusion of a 10B-carrier, which accumulates in the tumour area. The tumour is irradiated with epithermal or thermal neutrons, which result in a boron neutron capture reaction that generates heavy particles to damage tumour cells. In Finland, boronophenylalanine fructose (BPA-F) is used as the 10B-carrier. Currently, the drifting of boron from blood to tumour as well as the spatial and temporal accumulation of boron in the brain, are not precisely known. Proton magnetic resonance spectroscopy (1H MRS) could be used for selective BPA-F detection and quantification as aromatic protons of BPA resonate in the spectrum region, which is clear of brain metabolite signals. This study, which included both phantom and in vivo studies, examined the validity of 1H MRS as a tool for BPA detection. In the phantom study, BPA quantification was studied at 1.5 and 3.0 T with single voxel 1H MRS, and at 1.5 T with magnetic resonance imaging (MRSI). The detection limit of BPA was determined in phantom conditions at 1.5 T and 3.0 T using single voxel 1H MRS, and at 1.5 T using MRSI. In phantom conditions, BPA quantification accuracy of ± 5% and ± 15% were achieved with single voxel MRS using external or internal (internal water signal) concentration references, respectively. For MRSI, a quantification accuracy of <5% was obtained using an internal concentration reference (creatine). The detection limits of BPA in phantom conditions for the PRESS sequence were 0.7 (3.0 T) and 1.4 mM (1.5 T) mM with 20 × 20 × 20 mm3 single voxel MRS, and 1.0 mM with acquisition-weighted MRSI (nominal voxel volume 10(RL) × 10(AP) × 7.5(SI) mm3), respectively. In the in vivo study, an MRSI or single voxel MRS or both was performed for ten patients (patients 1-10) on the day of BNCT. Three patients had glioblastoma multiforme (GBM), and five patients had a recurrent or progressing GBM or anaplastic astrocytoma gradus III, and two patients had head and neck cancer. For nine patients (patients 1-9), MRS/MRSI was performed 70-140 min after the second irradiation field, and for one patient (patient 10), the MRSI study began 11 min before the end of the BPA-F infusion and ended 6 min after the end of the infusion. In comparison, single voxel MRS was performed before BNCT, for two patients (patients 3 and 9), and for one patient (patient 9), MRSI was performed one month after treatment. For one patient (patient 10), MRSI was performed four days before infusion. Signals from the tumour spectrum aromatic region were detected on the day of BNCT in three patients, indicating that in favourable cases, it is possible to detect BPA in vivo in the patient’s brain after BNCT treatment or at the end of BPA-F infusion. However, because the shape and position of the detected signals did not exactly match the BPA spectrum detected in the in vitro conditions, assignment of BPA is difficult. The opportunity to perform MRS immediately after the end of BPA-F infusion for more patients is necessary to evaluate the suitability of 1H MRS for BPA detection or quantification for treatment planning purposes. However, it could be possible to use MRSI as criteria in selecting patients for BNCT.

Medicalising Prohibition : Harm reduction in Finnish and International Drug Policy

The increase in drug use and related harms in the late 1990s in Finland has come to be referred to as the second drug wave. In addition to using criminal justice as a basis of drug policy, new kinds of drug regulation were introduced. Some of the new regulation strategies were referred to as "harm reduction". The most widely known practices of harm reduction include needle and syringe exchange programmes for intravenous drug users and medicinal substitution and maintenance treatment programmes for opiate users. The purpose of the study is to examine the change of drug policy in Finland and particularly the political struggle surrounding harm reduction in the context of this change. The aim is, first, to analyse the content of harm reduction policy and the dynamics of its emergence and, second, to assess to what extent harm reduction undermines or threatens traditional drug policy. The concept of harm reduction is typically associated with a drug policy strategy that employs the public health approach and where the principal focus of regulation is on drug-related health harms and risks. On the other hand, harm reduction policy has also been given other interpretations, relating, in particular, to human rights and social equality. In Finland, harm reduction can also be seen to have its roots in criminal policy. The general conclusion of the study is that rather than posing a threat to a prohibitionist drug policy, harm reduction has come to form part of it. The implementation of harm reduction by setting up health counselling centres for drug users with the main focus on needle exchange and by extending substitution treatment has implied the creation of specialised services based on medical expertise and an increasing involvement of the medical profession in addressing drug problems. At the same time the criminal justice control of drug use has been intensified. Accordingly, harm reduction has not entailed a shift to a more liberal drug policy nor has it undermined the traditional policy with its emphasis on total drug prohibition. Instead, harm reduction in combination with a prohibitionist penal policy constitutes a new dual-track drug policy paradigm. The study draws on the constructionist tradition of research on social problems and movements, where the analysis centres on claims made about social problems, claim-makers, ways of making claims and related social mobilisation. The research material mainly consists of administrative documents and interviews with key stakeholders. The doctoral study consists of five original articles and a summary article. The first article gives an overview of the strained process of change of drug policy and policy trends around the turn of the millennium. The second article focuses on the concept of harm reduction and the international organisations and groupings involved in defining it. The third article describes the process that in 1996 97 led to the creation of the first Finnish national drug policy strategy by reconciling mutually contradictory views of addressing the drug problem, at the same as the way was paved for harm reduction measures. The fourth article seeks to explain the relatively rapid diffusion of needle exchange programmes after 1996. The fifth article assesses substitution treatment as a harm reduction measure from the viewpoint of the associations of opioid users and their family members.

Oncolytic Adenoviruses with Radiation Therapy for Treatment of Prostate Cancer

Prostate cancer is the most common cancer in males. Although many patients with localized disease can be cured with surgery and radiotherapy, advanced disease and especially castration resistant metastatic disease remains incurable, with a median life expectancy of less than 18 months. Oncolytic adenoviruses (Ads) are a new promising treatment against cancer due to their innate capacity to kill cancer cells. Viral replication in tumor cells leads to oncolysis and production of a multiplicity of new virions that are capable of further destroying cancerous tissue. Oncolytic Ads can be modified for tumor targeted infection and replication and be armed with therapeutic transgenes to maximize the oncolytic effect. Worldwide, clinical trials with oncolytic Ads have demonstrated good safety while the antitumor efficacy remains to be improved. Importantly, the best responses have been reported when oncolytic adenoviruses have been combined with standard cancer treatments, such as chemotherapy and radiation. Further, a challenge in many virotherapy approaches has been the monitoring of virus replication in vivo. Reporter genes have been extensively used as transgenes to evaluate the biodistribution of the virus and activity of specific promoters. However, these techniques are often limited to preclinical evaluation and not amenable to human use. The aim of the thesis was to find and develop new oncolytic Ads with maximum efficacy against metastatic, castration resistant prostate cancer and study them in vitro and in vivo combined to different forms of radiation therapy. Using combination therapy, we were aiming for better antitumor efficacy with reduced side effects. Capsid modified Ads for enhanced transduction were studied. Serotype 3 targeted chimera, Ad5/3, was found to have enhanced infectivity for prostate cancer and was used for developing new viruses for the study. Correlation between Ad-encoded marker peptide secretion and simultaneous viral replication was evaluated and the effects of radiotherapy on viral replication were studied in detail. We found that the repair of double strand breaks caused by ionizing radiation was inhibited by adenoviral proteins and led to autophagic cell death. Both subcutaneous models and intrapulmonary tumor models mimicking metastatic, aggressive disease were used in vivo. Virus efficacy was evaluated by intratumoral injections. Also, intravenous administration was evaluated to study the effectiveness in metastatic disease. Oncolytic adenovirus treatment led to significant tumor growth control and increased the survival rate of the mice. These results were further improved when oncolytic Ads were combined with radiation therapy. Oncolytic Ads expressing human sodium/iodide transporter (hNIS) as a transgene were evaluated for their oncolytic potency and for the functionality of hNIS in vitro and in vivo. Monitoring of viral replication was also assessed using different imaging modalities relative to clinical use. SPECT imaging of tumor-bearing mice was evaluated and combined with simultaneous CT-scanning to obtain important anatomical information on biodistribution, also in a three-dimensional form. It was shown that hNIS-expressing adenoviruses could harbour a bi-functional transgene allowing for localization and imaging of viral replication. Targeted radiotherapy was applied by systemic radioiodide administration and resulted in iodide accumulation into Ad-infected tumor. The combination treatment showed significantly enhanced antitumor efficacy in mice bearing prostate cancer tumors. In summary, the results presented above aim to provide new treatment modalities for castration resistant prostate cancer. Molecular insights were provided for better understanding of the benefits of combined radiation therapy and oncolytic adenoviruses, which will hopefully facilitate the translation of the approach into clinical use for humans.

Simplified Treatment of Childhood Acute Bone and Joint Infections

Acute childhood osteomyelitis (OM), septic arthritis (SA), and their combination osteomyelitis with adjacent septic arthritis (OM+SA), are treated with long courses of antimicrobials and immediate surgery. We conducted a prospective multi-center randomized trial among Finnish children at age 3 months to 15 years in 1983-2005. According to the two-by-two factorial study design, children with OM or OM+SA received 20 or 30 days of antimicrobials, whereas those with SA were treated for 10 or 30 days. In addition, the whole series was randomized to be treated with clindamycin or a first-generation cephalosporin. Cases were included only if the causative agent was isolated. The treatment was instituted intravenously, but only for the first 2-4 days. Percutaneous aspiration was done to obtain a representative sample for bacteriology, but all other surgical intervention was kept at a minimum. A total of 265 patients fulfilled our strict inclusion criteria and were analyzed; 106 children had OM, 134 SA, and 25 OM+SA. In the OM group, one child in the long and one child in the short-term treatment group developed sequelae. One child with SA twice developed a late re-infection of the same joint, but the causative agents differed. Regarding surgery, diagnostic arthrocentesis or corticotomy was the only surgical procedure performed in most cases. Routine arthrotomy was not required even in hip arthritis. Serum C-reactive protein (CRP) proved to be a reliable laboratory index in the diagnosis and monitoring of osteoarticular infections. The recovery rate was similar regardless of whether clindamycin or a first-generation cephalosporin was used. We conclude that a course of 20 days of these well-absorbing antimicrobials is sufficient for OM or OM+SA, and 10 days for SA in most cases beyond the neonatal age. A short intravenous phase of only 2-5 days often suffices. CRP gives valuable information in monitoring the course of illness. Besides diagnostic aspiration, surgery should be reserved for selected cases.

Ketoprofeenin biologinen käytettävyys ja farmakokinetiikka sioilla eri antotavoilla ja ketoprofeeniyliannoksen farmakokinetiikka ja vaikutus munuaisiin lampailla

Ketoprofeeni on yleisesti käytetty ei-steroidinen tulehduskipulääke (NSAID) lampaiden ja sikojen kivunlievityksessä. Tietoa ketoprofeenin oikeista annosmääristä eri eläinlajeilla on saatavilla rajallisesti. Oikeaa lääkeainemäärää ei voida luotettavasti ekstrapoloida toisten eläinlajien tai ihmisten perusteella. Epäillyissä tulehduskipulääkemyrkytyksissä ongelmana on tietää, oliko eläimen saama lääkeannos toksinen. Lampailla tehdyn tutkimuksen tavoitteena oli selvittää, muuttuuko ketoprofeenin kinetiikka kymmenkertaisella yliannoksella, tutkia yliannoksen vaikutusta munuaisiin ja löytää yksinkertainen tapa diagnosoida yliannos virtsasta. Sioilla tehdyn tutkimuksen tavoitteena oli selvittää ketoprofeenin biologista käytettävyyttä ja ketoprofeenin farmakokinetiikkaa sioilla intravaskulaarisella, intramuskulaarisella ja peroraalisella annolla. Keskeiset tutkimuksessa määritettävät muuttujat olivat AUC0-_, Cmax ja tmax. Hyötyosuus laskettiin i.v. -annon perusteella. Kuudelle lampaalle annettiin 30 mg/kg i.v. -ketoprofeenia. Ketoprofeenin pitoisuuksia seurattiin 24 tunnin ajan plasmanäytteillä, joiden perusteella määritettiin farmakokineettiset parametrit. Veri- ja virtsanäytteistä tutkittiin muun muassa mahdollisesta munuaisvauriosta kertovia entsyymejä. 24 tunnin kuluttua lääkkeenannosta lampaat lopetettiin ja munuaiset tutkittiin histologisesti. Tutkittaville kahdeksalle sialle annosteltiin 3 mg/kg intravaskulaarista, intramuskulaarista ja oraalista ketoprofeenia sekä 6 mg/kg oraalista ketoprofeenia. Tutkimus suoritettiin satunnaistettuna vaihtovuorotutkimuksena. Ketoprofeenin pitoisuuksia seurattiin plasmanäytteillä 48 tunnin ajan lääkkeenannosta ja kaikille antotavoille laskettiin farmakokineettiset parametrit. Lisäksi tutkittiin valmisteiden biologinen samanarvoisuus. Molempien tutkimusten in vivo -kokeet suoritettiin Eläinlääketieteellisessä tiedekunnassa. Samoin munuaisten histologinen tutkimus ja virtsasta ja verestä tehdyt määritykset, lukuun ottamatta ketoprofeeninpitoisuuden analysointia. Plasman ketoprofeenipitoisuus analysoitiin korkean erotuskyvyn nestekromatografialla (HPLC). Ketoprofeenimääritykset ja farmakokineettinen analyysi suoritettiin Farmasian tiedekunnassa. Lampaiden kymmenkertainen ketoprofeeniyliannos oli toksinen. Seerumin urea- ja kreatiniinipitoisuus nousivat ja histologisissa näytteissä näkyi akuutti munuaistiehyen vaurio. Useiden entsyymien pitoisuus nousi virtsassa. Selvimmin ja nopeimmin nousi virtsan laktaattidehydrogenaasipitoisuus, jonka määrittäminen vaikuttaa potentiaaliselta tavalta diagnosoida ketoprofeenin toksinen annos. Ketoprofeenin eliminaation puoliintumisaika toksisella annoksella oli samaa suuruusluokkaa kuin aiemmissa tutkimuksissa terapeuttisella annoksella, joten yliannos ei muuttanut ketoprofeenin kinetiikkaa. AUC- ja Cmax -arvot olivat suhteessa suurempia kuin terapeuttisella annoksella, joten tutkimuksen perusteella kyseiset arvot eivät nousseet lineaarisesti annoksen noustessa toksiseksi. Sioille annetut ketoprofeenivalmisteet eivät olleet biologisesti samanarvoisia keskenään. Hyötyosuus oli erittäin hyvä kaikilla antotavoilla. tmax oli kaikilla antotavoilla hieman yli tunnin kuluttua lääkkeenannosta. Oraalisen 3 mg/kg -annoksen Cmax oli 5,1 mg/l ja AUC 32 mg l-1 h ja intramuskulaarisen vastaavat arvot olivat 7,6 mg/l ja 37 mg l-1 h. Oraalisen ketoprofeenin annostasojen AUC- ja Cmax -arvot korreloivat keskenään, joten ketoprofeenin kinetiikka oli lineaarista. Intravaskulaarisen ja oraalisen annon puoliintumisajoissa oli tilastollisesti merkitsevä ero. Ketoprofeenin jakautumistilavuudessa ja puhdistumassa ei ollut tilastollisesti merkitsevää eroa eri antotapojen välillä.

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.

Protection of adult but not newborn mice against lethal intracerebral challenge with Japanese encephalitis virus by adoptively transferred virus-specific cytotoxic T lymphocytes: Requirement for L3T4(+) T cells

The protective ability of cytotoxic T cells (CTL) raised in vitro against Japanese encephalitis virus (JEV) was examined by adoptive transfer experiments. Adoptive transfer of anti-JEV effecters by intracerebral (i.c.) but not by intraperitoneal (i.p.) or intravenous (i.v.) routes protected adult BALB/c mice against lethal i.c. JEV challenge. In contrast to adult mice, adoptive transfer of anti-JEV effecters into newborn (4-day-old) and suckling (8-14-day-old) mice did not confer protection. However, virus-induced death was delayed in suckling mice compared to newborn mice upon adoptive transfer. The specific reasons for lack of protection in newborn mice are not clear but virus load was found to be higher in newborn mice brains compared to those of adults and virus clearance was observed only in adult mice brains but not in newborn mice brains upon adoptive transfer. Specific depletion of Lyt 2.2(+), L3T4(+) or Thy-1(+) T cell populations before adoptive transfer abrogated the protective ability of transferred effecters. However, when Lyt 2.2(+) cell-depleted and L3T4(+) cell-depleted effecters were mixed and transferred into adult mice the protective activity was retained, demonstrating that both Lyt 2.2(+) and L3T4(+) T cells are necessary to confer protection. Although the presence of L3T4(+) T cells in adoptively transferred effector populations enhanced virus-specific serum neutralizing antibodies, the presence of neutralizing antibodies alone without Lyt 2.2(+) cells was not sufficient to confer protection.

Benzothiophene Carboxamide Derivatives as Novel Antimalarials

Bromo-benzothiophene carboxamide derivatives have been shown in the preceding article to inhibit Plasmodium falciparum Enoyl-ACP reductase. Here, we report bromo-benzothiophene carboxamide derivatives as potent inhibitors of Plasmodium asexual blood-stages in vitro as well as in vivo in the mouse model. These compounds specifically impair the development of metabolically active trophozoite stage of intraerythrocytic cycle and the intravenous administration of 3-bromo-N-(4-fluorobenzyl)-benzo[b]thiophene-2-carboxamide (compound 6) enhances the longevity of P. berghei infected mice by 2 weeks compared to disease control animals thereby preventing the onset of ataxia and convulsions in treated mice. These compounds thus hold promise for the development of potent antimalarials.(C) 2011 IUBMB IUBMB Life, 63(12): 1111-1115, 2011

Inhibition of the Interaction Between NS3 Protease and HCV IRES With a Small Peptide: A Novel Therapeutic Strategy

Recently, we have demonstrated that the protease domain of NS3 alone can bind specifically to hepatitis C virus (HCV) internal ribosome entry site (IRES) near the initiator AUG, dislodges human La protein and inhibits translation in favor of viral RNA replication. Here, by using a computational approach, the contact points of the protease on the HCV IRES were putatively mapped. A 30-mer NS3 peptide was designed from the predicted RNA-binding region that retained RNA-binding ability and also inhibited IRES-mediated translation. This peptide was truncated to 15 mer and this also demonstrated ability to inhibit HCV RNA-directed translation as well as replication. More importantly, its activity was tested in an in vivo mouse model by encapsulating the peptide in Sendai virus virosomes followed by intravenous delivery. The study demonstrates for the first time that the HCV NS3-IRES RNA interaction can be selectively inhibited using a small peptide and reports a strategy to deliver the peptide into the liver.

Improvement in Bone Properties by Using Risedronate Adsorbed Hydroxyapatite Novel Nanoparticle Based Formulation in a Rat Model of Osteoporosis

A superior drug formulation capable of achieving efficient osteogenesis is in imperative demand for the treatment of osteoporosis. In the present study we investigated the potential of using novel risedronate-hydroxyapatite (HA) nanoparticle based formulation in an animal model of established osteoporosis. Nanoparticles of HA loaded with risedronate (NHLR) of various sizes (80-130 nm) were generated for bone targeted drug delivery. Three months after ovariectomy, 36 ovariectomized (OVX) rats were divided into 6 equal groups and treated with various doses of NHLR (500,350 and 250 mu g/kg intravenous single dose) and sodium risedronate (500 mu g/kg, intravenous single dose). Untreated OVX and sham OVX served as controls. One month after drug administration, the left tibia and femur were tested for bone mechanical properties and histology, respectively. In the right femur, bone density was measured by method based on Archimedes principle and bone porosity analyses were performed using X-ray imaging. NHLR (250 mu g/kg) showed a significant increase in bone density and reduced bone porosity when compared with OVX control. Moreover, NHLR (250 mu g/kg) significantly increased bone mechanical properties and bone quality when compared with OVX control. The results strongly suggest that the NHLR, which is a novel nanoparticle based formulation, has a therapeutic advantage over risedronate sodium monotherapy for the treatment of osteoporosis in a rat model of postmenopausal osteoporosis.