1.
Lindquist, Catarina
(författare)
Physiology and Pharmacology of GABAA receptors: The Brakes in the Brain
2005
Doktorsavhandling (övrigt vetenskapligt/konstnärligt) abstract
Inhibitory neurotransmission in the brain is mostly mediated by gamma-aminobutyric acid type A (GABAA) receptors. These receptors are involved in both phasic inhibition (point-to-point inhibition, synaptic transmission) and tonic inhibition (diffuse form of inhibition, brain homeostasis). In this thesis the functional and pharmacological properties of GABAA receptors expressed in brain slices or in Sf 9 cells were studied. GABAA receptors expressed extrasynaptically are believed to be involved in tonic inhibition. In hippocampal dentate gyrus granule cells we identified and characterized three types of extrasynaptic receptor types (GABARex) that varied in their affinity for GABA, maximal single-channel conductance and sensitivity to drugs. For the first time we showed how the GABA concentration determines the conductance of GABAA receptors in brain tissue. There is thus a direct link between the extracellular GABA concentration and the level of the tonic inhibition, providing dynamic control. It is only within the last ten years or so that the tonic inhibition was discovered and only recently has it gained widespread interest. One reason is that it has become quite clear that the first site of action and probably often the most important site of action of drugs are the extrasynaptic receptors. We found that a drug now in clinical trials (THIP) at the clinically relevant concentration activates these extrasynaptic receptors. It has been assumed that spontaneous openings of the receptors are only functionally significant in receptor complexes containing the epsilon-subunit or mutations. We show that alpha/beta and alpha/beta/gamma?receptors can open spontaneously and be modified by drugs. The capacity to open spontaneously may be vital for fast responses such as at synapses. This suggests that the functional properties of receptors located at synapses and outside of synapses (extrasynaptic receptors: GABARex) differ. Those at synapses open rapidly (ms) whereas those at extrasynaptic sites open after a delay of ten to hundreds of seconds. This functional difference is very important in terms of brain function as it ensures fast flow of information (synaptic transmission) but in a controlled way that is set by the gain and the time window for synaptic transmission integration via the tonic inhibition. By using the compound SR95531, we constructed a model that accounts for activation and inhibition of both phasic- and tonic-like currents in an expression system. This model can be used to calculate what concentrations of the inhibitor to use to specifically block certain GABARex receptors in brain tissue to study how a particular population of GABARex contributes to the tonic inhibition and how it affects both excitatory and inhibitory synaptic transmission.
2.
Ahlin, Gustav, 1977-
(författare)
In vitro and in silico prediction of drug-drug interactions with transport proteins
2009
Doktorsavhandling (övrigt vetenskapligt/konstnärligt) abstract
Drug transport across cells and cell membranes in the human body is crucial for the pharmacological effect of drugs. Active transport governed by transport proteins plays an important role in this process. A vast number of transport proteins with a wide tissue distribution have been identified during the last 15 years. Several important examples of their role in drug disposition and drug-drug interactions have been described to date. Investigation of drug-drug interactions at the transport protein level are therefore of increasing interest to the academic, industrial and regulatory research communities. The gene expression of transport proteins involved in drug transport was investigated in the jejunum, liver, kidney and colon to better understand their influence on the ADMET properties of drugs. In addition, the gene and protein expression of transport proteins in cell lines, widely used for predictions of drug transport and metabolism, was examined. The substrate and inhibitor heterogeneity of many transport proteins makes it difficult to foresee whether the transport proteins will cause drug-drug interactions. Therefore, in vitro assays for OCT1 and OATP1B1, among the highest expressed transport proteins in human liver, were developed to allow investigation of the inhibitory patterns of these proteins. These assays were used to investigate two data sets, consisting of 191 and 135 registered drugs and drug-like molecules for the inhibition of OCT1 and OATP1B1, respectively. Numerous new inhibitors of the transport proteins were identified in the data sets and the properties governing inhibition were determined. Further, antidepressant drugs and statins displayed strong inhibition of OCT1 and OATP1B1, respectively. The inhibition data was used to develop predictive in silico models for each of the two transport proteins. The highly polymorphic nature of some transport proteins has been shown to affect drug response and may lead to an increased risk of drug-drug interactions, and therefore, the OCT1 in vitro assay was used to study the effect of common genetic variants of OCT1 on drug inhibition and drug-drug interactions. The results indicated that OCT1 variants with reduced function were more susceptible to inhibition. Further, a drug-drug interaction of potential clinical significance in the genetic OCT1 variant M420del was proposed. In summary, gene expression of transport proteins was investigated in human tissues and cell lines. In vitro assays for two of the highest expressed liver transport proteins were used to identify previously unknown SLC transport protein inhibitors and to develop predictive in silico models, which may detect previously known drug-drug interactions and enable new ones to be identified at the transport protein level. In addition, the effect of genetic variation on inhibition of the OCT1 was investigated.
3.
Alenius, Malin, 1974-
(författare)
Treatment Response in Psychotic Patients in a Naturalistic Setting : Classification, Genes, Drugs, Insight and Social Networks
2009
Doktorsavhandling (övrigt vetenskapligt/konstnärligt) abstract
Many patients with psychotic symptoms respond poorly to treatment. Various approaches have been made to classify these patients according to treatment response. However, existing classifications have been criticized for various reasons and a new classification system is needed. Further, no satisfactory explanation of the poor treatment response has been apparent. The general aim of this thesis was therefore to develop and validate a new classification method of functional remission in a naturalistic population of patients with psychosis and to utilize this classification to investigate the population from genetic, drug treatment, insight and social network points of view. Data for this cross-sectional study of patients (n=123) attending the Psychosis Outpatient Care clinic in the county of Jönköping, Sweden, were obtained from patient interviews, blood samples and information from patient files. The new classification method CANSEPT, which combines the CAN rating scale (CAN), the UKU side effect rating scale (SE) and the patient’s previous treatment history (PT), showed validity in discriminating the patients and was accepted well by the patients. CANSEPT was used to group the patients in the other studies in this thesis. The results indicated that the gene polymorphism ABCB1 3435T, was related to worse significant social and clinical needs for patients on olanzapine, while the polymorphism DRD2 Taq1 A1 was related to a greater risk of significant side effects; especially if male, or taking strong dopamine D2-receptor antagonistic drugs. Drug treatment factors were also related to treatment response; longer duration of untreated prodromal and early psychosis was seen for patients with current significant social and clinical needs and non-adherence to treatment was associated with worse significant side effects. Worse treatment outcomes also appeared to be associated with smaller social network groups, worse insight into illness, poorer knowledge of warning signs and worse coping strategies. In summary, CANSEPT was shown to be a useful valid, multidimensional tool for classification of treatment response. Gene polymorphisms, duration of untreated illness, non-adherence to treatment, social networks and knowledge should be taken into consideration when investigating inadequate treatment response.
4.
Annas, Anita
(författare)
Metabolism-dependent activation of food and environmental mutagens in endothelial cells
2000
Doktorsavhandling (övrigt vetenskapligt/konstnärligt) abstract
The endothelial cells of blood vessels have been proposed as a target for toxic effects ofxenobiotics in the cardiovascular system. In the present studies, induction of cytochromeP450 1A (CYP1A) enzymes and metabolic activation of food and environmentalmutagens were examined in endothelial cells of rodents, birds, and humans. Theheterocyclic amine 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) and thepolycyclic aromatic hydrocarbons 7,12-dimethylbenz[a]anthracene (DMBA) andbenzo[a]pyrene were used as models for food and environmental mutagens.The results showed that Trp-P-1 was activated into tissue-binding metabolites in endothelial cells, preferentially of capillaries and veins, in rodents pretreated with the aryl hydrocarbon (Ah) receptor agonist β-naphthoflavone (BNF), whereas similar activationdid not occur in vehicle-treated animals. Similarly, exposure to BNF increased the tissue-binding of Trp-P-1 and DMBA in cultured human umbilical vein endothelial cells (HUVEC), as compared with vehicle-treated cells. In contrast, exposure to BNF did not increase the binding of the mutagens in human umbilical artery endothelial cells (HUAEC). The formation of reactive metabolites of Trp-P-1 and DMBA correlated with induction of CYP1A1 protein and/or CYP1A-dependent catalytic activities, such as 7-ethoxyresorufin O-deethylase (EROD) and DMBA hydroxylase, in endothelial cells of rodents and cultured HUVEC. Moreover, exposure to BNF increased the activation ofbenzo[a]pyrene into genotoxic metabolites in HUVEC as compared with vehicle-treated cells.In chicken and eider duck embryos, BNF induced EROD and activation of Trp-P-1 to tissue binding metabolites in the endothelial linings, preferentially of capillaries and veins, in heart and chorioallantoic membrane (CAM). In vehicle-treated embryos, these activities were low.Overall, the present studies show that CYP1A-dependent activation of xenobiotics into tissue binding or genotoxic metabolites can be induced in blood vessel endothelia in various species following exposure to Ah receptor agonists. The results also show that there is a differential response to Ah receptor agonists within the vascular tree; CYP1A and enzymatic activities are preferentially induced in endothelial cells of veins and capillaries, and to lesser extent in arteries. The results suggest that certain endothelial cellsmay be targets for CYP1A-dependent activation of xenobiotics in individuals exposed to Ah receptor agonists.
5.
Azarbayjani, Faranak
(författare)
Common mechanism for teratogenicity of antiepileptic drugs : Drug-induced embryonic arrhythmia and hypoxia-reoxygenation damage
2001
Doktorsavhandling (övrigt vetenskapligt/konstnärligt) abstract
The Antiepilptic drugs (AEDs) phenytoin (PHT), carbamazepine (CBZ), phenobarbital (PB), tri- and dimethadione (TMD and DMD) are known teratogens having a common malformation pattern in human and animal studies. This thesis was designed chiefly to test a hypothesis correlating the teratogenicity of these AEDs to episodes of pharmacologically induced embryonic arrhythmia and hypoxia-reoxygenation damage.Effects on the embryonic heart were studied both after maternal administration in mice and inmouse embryos cultured in vitro. Only AEDs, correlated with the same type of malformation as could be induced by episodes of interrupted oxygen supply to the embryo (e.g. cleft palate) caused concentration dependent bradycardia and arrhythmia. PHT and DMD had the highest potential and affected embryonic heart at clinically relevant concentration, followed by CBZ, TMD and PB. Valproate and vigabatrin not associated with hypoxia-related malformations caused neither arrhythmia nor severe bradycardia.The results showed that the embryonic heart is extremely susceptible to PHT and DMD onlyduring a restricted period of development, between gestational days 9-13 (weeks 5-9 of human pregnancy).An observed genetic susceptibility to react with arrhythmia at low concentrations when exposed to PHT or to external stress, could explain why A/J strain of mice is more susceptible to develop cleft palate compared to other strains. High activities of reactive oxygen species (ROS) capturing antioxidant enzymes observed in untreated A/J embryos supported this assumption. The potential to cause embryonic arrythmia by an AED was related to the potential to inhibit the rapid component of the delayed rectifier potassium channel (I kr ).A marked I kr blocking activity (70%)of DMD in voltage clamping studies was observed. The I kr inhibition occurred at similar concentrations, which causes severe arrhythmia.The idea of a relation between teratogenicity and arrhythmia, resulting in ischemia followed by reperfusion and generation of ROS was supported by mechanistic studies. Pre-treatment with the spin-trapping agent PBN, which has the capacity to capture ROS, markedly reduced the incidence of PHT and DMD-induced cleft palate. In utero exposure to teratogenic doses of DMD and PHT resulted in hemorrhages in the embryonic palatal region. The same type of haemorrhage in the palatal region precedes orofacial clefts induced by episodic hypoxia.
6.
Bardage, Carola
(författare)
Cardiovascular disease and hypertension : Population-based studies on self-rated health and health-related quality of life in Sweden
2000
Doktorsavhandling (övrigt vetenskapligt/konstnärligt) abstract
The aim with this thesis was to study cardiovascular disease and hypertension, use of drugs and health from an epidemiological perspective. Various methods - self-rated health (SRH), health related quality of life (HRQL) - the 36-item short form questionnaire (SF-36) - and health utility measurements - the rating scale (RS) and the time-trade off (TTO) methods - were employed.Data from the Swedish Adoption/Twin Study of Aging (SATSA) in 1984, 1987, 1990, and 1993 as well as a general population survey conducted in Uppsala County in 1995 were used.Persons who have cardiovascular disease, both with and without drug treatment, were found to have a lower SRH as compared to others in the population. Longitudinal analyses showed that SRH was relatively stable over time among persons with cardiovascular disease. Both having a low SRH and having cardiovascular disease were associated with a higher mortality rate.Hypertensives were found to have a lower HRQL than do others in the general population as measured by the SF-36. The lowest scoring was found in the general health perception scale (GH), whereas role emotional (RE) and mental health (MH) were the scales least affected by hypertension.Nearly 20 percent of the antihypertensive drug users reported side effects.The pattern of side effects was similar to that reported in clinical trials. Both hypertension itself and the drug treatment were found to have an impact on the patient's health-state utility as measured by the RS. Comparative analyses showed that health utilities and psychometric quality-of-life instruments were only moderately correlated among hypertensives. The results also showed that inequalities in HRQL were present with respect to several sociodemographic factors. In summary, this thesis revealed that persons with cardiovascular disease and/or with hypertension experience poorer health than others in the population. The poor health may be caused both by the disease and/or the drug treatment. The results in this thesis also suggested that special attention and care should be directed to persons with cardiovascular disease and/or hypertension reporting ill health. This especially is important given that low HRQL can be a riskfactor for subsequent cardiovascular events or complications which in turn might result in higher mortality rate.
7.
Bengtsson, Jörgen, 1976-
(författare)
Developmental Aspects of Drug Transport Across the Blood-Brain Barrier
2009
Doktorsavhandling (övrigt vetenskapligt/konstnärligt) abstract
The developmental aspect of drug transport across the blood-brain barrier (BBB) was investigated. Microdialysis was used to study unbound morphine BBB transport at different ages in sheep. An in vitro study was performed to find differentially expressed genes in brain capillary-rich fractions of the brain in rats of different ages. Microdialysis and brain-to-plasma ratios were used to study the contribution of breast cancer resistance protein (Bcrp) to the transport of nitrofurantoin (NTF) across the BBB of rats during development as well as in adult rats and mice. A method of analysing morphine and its metabolites in plasma and microdialysis samples was developed and validated. The in vivo recovery of deuterated morphine, used as a calibrator in microdialysis experiments, was not affected by the presence of morphine in the tissue. A net influx of morphine was observed in premature lambs and adult sheep, in contrast to the efflux seen in other species. This influx decreased with age, indicating that the morphine transport across the BBB changes with age. In contrast, the transport of the morphine metabolite morphine-3-glucuronide (M3G) did not change with age. Microarray data indicated that several active transporters are differentially expressed with age. Moreover, the mRNA expression levels of Abcg2 (Bcrp) and Slc22a8 (organic anion transporter 3) changed with age when quantified using real-time polymerase chain reaction. In contrast, the expression of Abcb1 (P-glycoprotein) and occludin (a tight junction protein) did not change with age. In rats, the brain distribution of NTF decreased with age due to increased protein binding in plasma. The concentration ratio of unbound NTF across the BBB was low in the adult rat, due to intra-brain metabolism and/or efflux by other transporters. Bcrp did not appear to have a significant contribution in the developing rat or in knock-out mice compared to wild-type controls with regard to NTF BBB transport. In conclusion, in vitro studies showed that the expression levels of some genes changed with age, presumably affecting subsequent drug distribution to the brain. Further, in vivo studies showed that distribution across the BBB changed with age for morphine but not for M3G or NTF.
8.
Berggren, Jonas
(författare)
Engineering of Pharmaceutical Particles : Modulation of Particle Structural Properties, Solid-State Stability and Tabletting Behaviour by the Drying Process
2003
Doktorsavhandling (övrigt vetenskapligt/konstnärligt) abstract
Relationships between stresses during the drying process, particle structural and functional properties, and particle engineering by the drying process were addressed in this thesis. In the first part, the importance of the drying phase and the effect of the drying rate on the intragranular porosity of microcrystalline cellulose pellets were investigated. Differences in porosities of dried pellets could be explained by liquid-related differences in densification during convective drying rather than by differences in densification during wet agglomeration. An increased drying rate gave more porous pellets with a lower compression shear strength, and thereby stronger tablets. The next part dealt with modulation of solid-state stability and tabletting behaviour of amorphous lactose by incorporation of different polymers by spray drying. Increased content and molecular weight of poly(vinylpyrrolidone) (PVP) resulted in an increased resistance to crystallisation provoked by heat and moisture. The stabilising effect was even more evident after long-term storage. However, the glass transition temperature was almost unaffected and may, therefore, be questioned as a stability indicator for these types of materials. The presence of the polymers resulted in somewhat less deformable particles. Incorporation of PVP increased the compactability, whilst a surfactant decreased it, which could be shown to be related to differences in particle-particle adhesivity between the different particles. This thesis contributes to increased mechanistic understanding in the area of particle engineering that may lead to better prediction and optimisation of the functionality of pharmaceutical particles, which is of the utmost importance in the development and production of solid dosage forms.
9.
Bergman, Ebba, 1977-
(författare)
The Hepatobiliary Transport of Rosuvastatin In Vivo
2009
Doktorsavhandling (övrigt vetenskapligt/konstnärligt) abstract
In vivo studies of hepatobiliary disposition are challenging. The hepatobiliary system is complex, as its physiological localization, complex cellular structure with numerous transporters and enzymes, and the interindividual variability in protein expression and biliary flow will all affect the in vivo disposition of a drug under investigation. The research included in this thesis has focused on the involvement of hepatic transport proteins in the hepatobiliary disposition of rosuvastatin. The impact that several transport inhibitors had on the pharmacokinetics of rosuvastatin was investigated in healthy volunteers and in pigs. The effects were considerable, following inhibition of sinusoidal transport proteins by cyclosporine and rifampicin. These inhibitors significantly reduced the hepatic extraction of rosuvastatin by 50 and 35%, respectively, and the plasma exposure increased by factors of 9.1 and 6.3, respectively. Drug-drug interactions (DDI) resulting in markedly higher plasma exposures are important from a drug safety perspective as increased extrahepatic exposure of statins is associated with an increased risk of severe side-effects, such as myopathy which in rare cases could develop into rhabdomyolysis. The DDI caused by cyclosporine and rifampicin can probably be attributed to inhibition of hepatic uptake transporters. In contrast, inhibition of canalicular transporters by imatinib did not significantly affect the pharmacokinetics of rosuvastatin, which suggests that the intracellular concentration of the inhibitor in the hepatocyte was insufficient to affect the transport of rosuvastatin, or that imatinib is not a sufficiently potent inhibitor in vivo. Furthermore, gemfibrozil administered as a single dose into the jejunum in healthy volunteers and pigs did not affect the plasma or biliary pharmacokinetics of rosuvastatin. The previously reported DDI in humans upon repeated dosing with gemfibrozil might be explained by the accumulation of metabolites able to affect the disposition of rosuvastatin. The investigations presented in this thesis conclude that transport proteins are of considerable importance for the hepatobiliary disposition of rosuvastatin in vivo. The Loc-I-Gut catheter can be applied for the investigation of biliary accumulation and to determine bile specific metabolites, however it has limitations when conducting quantitative measurements. In the porcine model, hepatic bile can be collected for up to six hours and enables the determination of the hepatic extraction in vivo.
10.
Bergström, Christel A. S., 1973-
(författare)
Computational and Experimental Models for the Prediction of Intestinal Drug Solubility and Absorption
2003
Doktorsavhandling (övrigt vetenskapligt/konstnärligt) abstract
New effective experimental techniques in medicinal chemistry and pharmacology have resulted in a vast increase in the number of pharmacologically interesting compounds. However, the number of new drugs undergoing clinical trial has not augmented at the same pace, which in part has been attributed to poor absorption of the compounds.The main objective of this thesis was to investigate whether computer-based models devised from calculated molecular descriptors can be used to predict aqueous drug solubility, an important property influencing the absorption process. For this purpose, both experimental and computational studies were performed. A new small-scale shake flask method for experimental solubility determination of crystalline compounds was devised. This method was used to experimentally determine solubility values used for the computational model development and to investigate the pH-dependent solubility of drugs. In the computer-based studies, rapidly calculated molecular descriptors were used to predict aqueous solubility and the melting point, a solid state characteristic of importance for the solubility. To predict the absorption process, drug permeability across the intestinal epithelium was also modeled.The results show that high quality solubility data of crystalline compounds can be obtained by the small-scale shake flask method in a microtiter plate format. The experimentally determined pH-dependent solubility profiles deviated largely from the profiles predicted by a traditionally used relationship, highlighting the risk of data extrapolation. The in silico solubility models identified the non-polar surface area and partitioned total surface areas as potential new molecular descriptors for solubility. General solubility models of high accuracy were obtained when combining the surface area descriptors with descriptors for electron distribution, connectivity, flexibility and polarity. The used descriptors proved to be related to the solvation of the molecule rather than to solid state properties. The surface area descriptors were also valid for permeability predictions, and the use of the solubility and permeability models in concert resulted in an excellent theoretical absorption classification. To summarize, the experimental and computational models devised in this thesis are improved absorption screening tools applicable to the lead optimization in the drug discovery process.
