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Modeling is a valuable tool in drug development, to support decision making, improving study design, and aid in regulatory approval and labeling. This thesis describes the development of pharmacometric models for drugs used in anesthesia and analgesia. Models describing the effects on anesthetic depth, measured by the bispectral index (BIS), for a commonly used anesthetic, propofol, and for a novel anesthetic, AZD3043, were developed. The propofol model consisted of two effect-site compartments, and could describe the effects of propofol when the rate of infusion is changed during treatment. AZD3043 had a high clearance and a low volume of distribution, leading to a short half-life. The distribution to the effect site was fast, and together with the short plasma half-life leading to a fast onset and offset of effects. It was also shown that BIS after AZD3043 treatment is related to the probability of unconsciousness similar to propofol. In analgesia studies dropout due to lack of efficacy is common. This dropout is not at random and needs to be taken into consideration in order to avoid bias. A model was developed describing the PK, pain intensity and dropout hazard for placebo, naproxen and a novel analgesic compound, naproxcinod, after removal of a wisdom tooth. The model provides an opportunity to describe the effects of other doses or formulations. Visual predictive checks created by simultaneous simulations of PI and dropout provided a good way of assessing the goodness of fit when there is informative dropout. The performance of non-linear mixed effects models in the presence of informative dropout, with and without including models that describe such informative dropout was investigated by simulations and re-estimations. When a dropout model was not included there was in general more bias. The bias increased with decreasing number of observations per subject, increasing placebo effect and increasing dropout rate. Bias was relatively unaffected by the number o

Excessive and compulsive ethanol drinking is one of the most serious public health issues. Therefore, it is vital to increase the knowledge about risks and protection for alcohol use disorders (AUD) to optimize prevention and treatment strategies. Ethanol consumption commonly initiates during adolescence when extensive neuronal maturation and development also occurs. Early exposure to ethanol is a risk factor for AUD, but the effects of adolescent drinking and the basis for the individual susceptibility to AUD are not fully understood. The interactions between genotype and environmental factors determine the individual risk for AUD and this thesis aimed to examine the environmental impact. The specific aims were to investigate 1) how early-life conditions affect adolescent voluntary ethanol drinking, behavioural profiles, endogenous opioids and response to treatment with an opioid antagonist (naltrexone), and 2) whether alterations detected in the offspring may be mediated by variations in maternal behaviour. A rodent maternal separation (MS) model was used to mimic a protective and risk-inducing early-life environment, respectively, with the use of 15 min (MS15) or 360 min (MS360) of daily MS. The main findings were 1) the MS360, but not the MS15 rats, responded to naltrexone following adolescent ethanol drinking; all adolescent rats had a high voluntary ethanol intake independent of early environmental conditions whereas in the adult groups the MS360, but not the MS15 rats, increased their ethanol intake and preference over time; adolescent ethanol exposure resulted in higher dynorphin levels in hippocampus and higher Met-enkephalin-Arg6Phe7 in the amygdala, independently of rearing conditions, 2) behavioural profiling using the multivariate concentric square field™ test showed: the young MS360 rats had increased risk assessment and risk taking behaviour compared to the young MS15 rats; the young MS15 rats increased, whereas the young MS360 rats decreased, their r

Exposure to antibiotics is an important factor influencing the development of bacterial resistance. In an era where very few new antibiotics are being developed, a strategy for the development of optimal dosing regimen and combination treatment that reduces the rate of resistance development and overcome existing resistance is of utmost importance. In addition, the optimal dosing in subpopulations is often not fully elucidated. The aim of this thesis was to develop pharmacokinetic (PK) and pharmacokinetic-pharmacodynamic (PKPD) models that characterize the interaction of antibiotics with bacterial growth, killing and resistance over time, and can be applied to guide optimization of dosing regimens that enhance the efficacy of mono- and combination antibiotic therapy. A mechanism-based PKPD model that incorporates the growth, killing kinetics and adaptive resistance development in Escherichia coli against gentamicin was developed based on in vitro time-kill curve data. After some adaptations, the model was successfully applied for similar data on colistin and meropenem alone, and in combination, on one wild type and one meropenem-resistant strain of Pseudomonas aeruginosa. The developed population PK model for colistin and its prodrug colistin methanesulfonate (CMS) in combination with the PKPD model showed the benefits for applying a loading dose for this drug. Simulations predicted the variability in bacteria kill to be larger between dosing occasions than between patients. A flat-fixed loading dose followed by an 8 or 12 hourly maintenance dose with infusion duration of up to 2 hours was shown to result in satisfactory bacterial kill under these conditions. Pharmacometric models that characterize the time-course of drug concentrations, bacterial growth, antibacterial killing and resistance development were successfully developed. Predictions illustrated how PKPD models based on in vitro data can be utilized to guide development of antibiotic dosing, with example

Modeling is a valuable tool in drug development, to support decision making, improving study design, and aid in regulatory approval and labeling. This thesis describes the development of pharmacometric models for drugs used in anesthesia and analgesia. Models describing the effects on anesthetic depth, measured by the bispectral index (BIS), for a commonly used anesthetic, propofol, and for a novel anesthetic, AZD3043, were developed. The propofol model consisted of two effect-site compartments, and could describe the effects of propofol when the rate of infusion is changed during treatment. AZD3043 had a high clearance and a low volume of distribution, leading to a short half-life. The distribution to the effect site was fast, and together with the short plasma half-life leading to a fast onset and offset of effects. It was also shown that BIS after AZD3043 treatment is related to the probability of unconsciousness similar to propofol. In analgesia studies dropout due to lack of efficacy is common. This dropout is not at random and needs to be taken into consideration in order to avoid bias. A model was developed describing the PK, pain intensity and dropout hazard for placebo, naproxen and a novel analgesic compound, naproxcinod, after removal of a wisdom tooth. The model provides an opportunity to describe the effects of other doses or formulations. Visual predictive checks created by simultaneous simulations of PI and dropout provided a good way of assessing the goodness of fit when there is informative dropout. The performance of non-linear mixed effects models in the presence of informative dropout, with and without including models that describe such informative dropout was investigated by simulations and re-estimations. When a dropout model was not included there was in general more bias. The bias increased with decreasing number of observations per subject, increasing placebo effect and increasing dropout rate. Bias was relatively unaffected by the number o

Growth hormone (GH) stimulates growth and metabolism but also displays profound effects on the central nervous system (CNS). GH affects neurogenesis and neuroprotection, and has been shown to counteract drug-induced apoptosis in the brain. Anabolic androgenic steroids (AAS), mainly abused for their anabolic and performance-enhancing properties, can cause several adverse effects, such as cardiovascular complications, sterility, depression, and aggression. GH and AAS are both believed to interact with several signaling systems in the CNS. The aim of this thesis was to further investigate the impact of GH and AAS on neurochemistry and cognitive functions. Recombinant human GH (rhGH) and the steroid nandrolone decanoate (ND) were administered, separately and in combination with each other, to male rats. The results demonstrated that administration of GH improved spatial memory, assessed in a water maze test. Furthermore, GH induced alterations of the GABAB receptor mRNA expression, density, and functionality in the brain, for example in regions associated with cognition. GH also altered the mu opioid peptide (MOP) receptor, but not the delta opioid peptide (DOP) receptor functionality in the brain. Thus, some of the GH effects on cognition may involve effects on the GABAB receptors and MOP receptors. ND, on the contrary, seemed to induce impairments of memory and also altered the GABAB receptor mRNA expression in the brain. Furthermore, ND lowered the IGF-1 plasma concentrations and attenuated the IGF-1, IGF-2, and GHR mRNA expression in the pituitary. In addition, significant effects of GH and ND were found on plasma steroid concentrations, organ weight, as well as body weight. In conclusion, this thesis contributes with further knowledge on the cognitive and neurochemical consequences of GH and ND use. The findings regarding ND are worrying considering the common use of AAS among adolescents. GH improves memory functions and affects signaling systems in the brain asso

Exposure to antibiotics is an important factor influencing the development of bacterial resistance. In an era where very few new antibiotics are being developed, a strategy for the development of optimal dosing regimen and combination treatment that reduces the rate of resistance development and overcome existing resistance is of utmost importance. In addition, the optimal dosing in subpopulations is often not fully elucidated. The aim of this thesis was to develop pharmacokinetic (PK) and pharmacokinetic-pharmacodynamic (PKPD) models that characterize the interaction of antibiotics with bacterial growth, killing and resistance over time, and can be applied to guide optimization of dosing regimens that enhance the efficacy of mono- and combination antibiotic therapy. A mechanism-based PKPD model that incorporates the growth, killing kinetics and adaptive resistance development in Escherichia coli against gentamicin was developed based on in vitro time-kill curve data. After some adaptations, the model was successfully applied for similar data on colistin and meropenem alone, and in combination, on one wild type and one meropenem-resistant strain of Pseudomonas aeruginosa. The developed population PK model for colistin and its prodrug colistin methanesulfonate (CMS) in combination with the PKPD model showed the benefits for applying a loading dose for this drug. Simulations predicted the variability in bacteria kill to be larger between dosing occasions than between patients. A flat-fixed loading dose followed by an 8 or 12 hourly maintenance dose with infusion duration of up to 2 hours was shown to result in satisfactory bacterial kill under these conditions. Pharmacometric models that characterize the time-course of drug concentrations, bacterial growth, antibacterial killing and resistance development were successfully developed. Predictions illustrated how PKPD models based on in vitro data can be utilized to guide development of antibiotic dosing, with example

Growth hormone (GH) stimulates growth and metabolism but also displays profound effects on the central nervous system (CNS). GH affects neurogenesis and neuroprotection, and has been shown to counteract drug-induced apoptosis in the brain. Anabolic androgenic steroids (AAS), mainly abused for their anabolic and performance-enhancing properties, can cause several adverse effects, such as cardiovascular complications, sterility, depression, and aggression. GH and AAS are both believed to interact with several signaling systems in the CNS. The aim of this thesis was to further investigate the impact of GH and AAS on neurochemistry and cognitive functions. Recombinant human GH (rhGH) and the steroid nandrolone decanoate (ND) were administered, separately and in combination with each other, to male rats. The results demonstrated that administration of GH improved spatial memory, assessed in a water maze test. Furthermore, GH induced alterations of the GABAB receptor mRNA expression, density, and functionality in the brain, for example in regions associated with cognition. GH also altered the mu opioid peptide (MOP) receptor, but not the delta opioid peptide (DOP) receptor functionality in the brain. Thus, some of the GH effects on cognition may involve effects on the GABAB receptors and MOP receptors. ND, on the contrary, seemed to induce impairments of memory and also altered the GABAB receptor mRNA expression in the brain. Furthermore, ND lowered the IGF-1 plasma concentrations and attenuated the IGF-1, IGF-2, and GHR mRNA expression in the pituitary. In addition, significant effects of GH and ND were found on plasma steroid concentrations, organ weight, as well as body weight. In conclusion, this thesis contributes with further knowledge on the cognitive and neurochemical consequences of GH and ND use. The findings regarding ND are worrying considering the common use of AAS among adolescents. GH improves memory functions and affects signaling systems in the brain asso

Modeling is a valuable tool in drug development, to support decision making, improving study design, and aid in regulatory approval and labeling. This thesis describes the development of pharmacometric models for drugs used in anesthesia and analgesia. Models describing the effects on anesthetic depth, measured by the bispectral index (BIS), for a commonly used anesthetic, propofol, and for a novel anesthetic, AZD3043, were developed. The propofol model consisted of two effect-site compartments, and could describe the effects of propofol when the rate of infusion is changed during treatment. AZD3043 had a high clearance and a low volume of distribution, leading to a short half-life. The distribution to the effect site was fast, and together with the short plasma half-life leading to a fast onset and offset of effects. It was also shown that BIS after AZD3043 treatment is related to the probability of unconsciousness similar to propofol. In analgesia studies dropout due to lack of efficacy is common. This dropout is not at random and needs to be taken into consideration in order to avoid bias. A model was developed describing the PK, pain intensity and dropout hazard for placebo, naproxen and a novel analgesic compound, naproxcinod, after removal of a wisdom tooth. The model provides an opportunity to describe the effects of other doses or formulations. Visual predictive checks created by simultaneous simulations of PI and dropout provided a good way of assessing the goodness of fit when there is informative dropout. The performance of non-linear mixed effects models in the presence of informative dropout, with and without including models that describe such informative dropout was investigated by simulations and re-estimations. When a dropout model was not included there was in general more bias. The bias increased with decreasing number of observations per subject, increasing placebo effect and increasing dropout rate. Bias was relatively unaffected by the number o

Excessive and compulsive ethanol drinking is one of the most serious public health issues. Therefore, it is vital to increase the knowledge about risks and protection for alcohol use disorders (AUD) to optimize prevention and treatment strategies. Ethanol consumption commonly initiates during adolescence when extensive neuronal maturation and development also occurs. Early exposure to ethanol is a risk factor for AUD, but the effects of adolescent drinking and the basis for the individual susceptibility to AUD are not fully understood. The interactions between genotype and environmental factors determine the individual risk for AUD and this thesis aimed to examine the environmental impact. The specific aims were to investigate 1) how early-life conditions affect adolescent voluntary ethanol drinking, behavioural profiles, endogenous opioids and response to treatment with an opioid antagonist (naltrexone), and 2) whether alterations detected in the offspring may be mediated by variations in maternal behaviour. A rodent maternal separation (MS) model was used to mimic a protective and risk-inducing early-life environment, respectively, with the use of 15 min (MS15) or 360 min (MS360) of daily MS. The main findings were 1) the MS360, but not the MS15 rats, responded to naltrexone following adolescent ethanol drinking; all adolescent rats had a high voluntary ethanol intake independent of early environmental conditions whereas in the adult groups the MS360, but not the MS15 rats, increased their ethanol intake and preference over time; adolescent ethanol exposure resulted in higher dynorphin levels in hippocampus and higher Met-enkephalin-Arg6Phe7 in the amygdala, independently of rearing conditions, 2) behavioural profiling using the multivariate concentric square field™ test showed: the young MS360 rats had increased risk assessment and risk taking behaviour compared to the young MS15 rats; the young MS15 rats increased, whereas the young MS360 rats decreased, their r

Causal links between early-life stress, genes and later psychiatric diagnoses are not possible to fully address in human studies. Animal models therefore provide an important complement in which conditions can be well controlled and are here used to study and distinguish effects of early-life stress and alcohol exposure. The objective of this study was to investigate the impact of rearing conditions on behaviour in young rats and if these changes could be followed over time and to examine interaction effects between early-life environment and adolescent alcohol drinking on behaviour and immunoreactive levels of the opioid peptides dynorphin B, met-enkephalin-Arg(6)Phe(7) and beta-endorphin. We employed a rodent model, maternal separation, to study the impact of rearing conditions on behaviour, voluntary alcohol consumption and alcohol-induced effects. The consequences of short, 15 min (MS 15), and long, 360 min (MS 360), maternal separation in combination with adolescent voluntary alcohol consumption on behaviour and peptides were examined. A difference in the development of risk taking behaviour was found between the MS15 and MS360 while the development of general activity was found to differ between intake groups. Beta-endorphin levels in the pituitary and the periaqueductal gray area was found to be higher in the MS15 than the MS360. Adolescent drinking resulted in higher dynorphin B levels in the hippocampus and higher met-enkephalin-Arg(6)Phe(7) levels in the amygdala. Amygdala and hippocampus are involved in addiction processes and changes in these brain areas after adolescent alcohol drinking may have consequences for cognitive function and drug consumption behaviour in adulthood. The study shows that individual behavioural profiling over time in combination with neurobiological investigations provides means for studies of causality between early-life stress, behaviour and vulnerability to psychiatric disorders.

Our objectives were to document and compare plasma levels of Catecholamines, Cortisol,Glutathione and Malondialdehyde in camels after long term dehydration (20 days) in the presenceor absence of angiotensin II AT1 receptor blocker (Losartan) versus levels in non-dehydratedcamels; and to record the effects on glutathione and malondialdehyde activity in liver and kidneyhomogenate in the one-humped camel. Eighteen male camels were used in this study, sixcontrols, six dehydrated and treated with losartan (5mg/kg daily) and six were dehydrated withouttreatment. Our results revealed significant decrease (P<0.05) in plasma epinephrine level in bothtreated and dehydrated camels; while, Plasma norepinephrine showed significant increase in bothdehydrated groups (P< 0.01). Levels of plasma dopamine were also significantly increased (P<0.01) in both dehydrated groups compared to control camels.Plasma levels of cortisol increased significantly across dehydration with or without losartanadministration (P<0.01) compared with time-matched levels in control camels. Losartan had nosignificant modulating effect on the cortisol response to dehydration.Plasma, liver and kidney homogenates revealed significant increase (P<0.05) in glutathione levelsin both dehydrated groups compared to control.Plasma, liver and kidney homogenates for malondialdehyde levels in both treated and dehydratedcamels also showed significant increase (P<0.05 & P<0.01) compared to controls.In conclusion, our study demonstrates that the effect of dehydration with or without losartaninduced oxidative stress in these camels, leading to significant changes in plasma catecholaminesand cortisol levels, together with significant increments in glutathione and malondialdehydeactivities in plasma, liver and kidney homogenate to counter act the damaging effect of the freeradicals in the dehydrated camels.

The high-throughput brain slice method is a precise and robust technique for estimating the overall uptake of drugs into brain tissue through determination of the unbound volume of distribution in the brain (Vu,brain; ml·g brain-1). Vu,brain describes the relationship between the total drug concentration in the brain and the concentration of unbound drug in the brain interstitial fluid, regardless of blood–brain barrier function. The brain slice method is more physiologically based than the brain homogenate method with respect to the assessment of drug distribution in the brain because the cell-cell interactions, pH gradients and active transport systems are all conserved. The method provides information that is directly relevant to issues such as nonspecific binding to brain tissue, lysosomal trapping, and active uptake into the cells. For these reasons, the brain slice method is recommended for estimation of target-site pharmacokinetics in the early drug discovery process and fundamental pharmacological studies. This article provides a detailed protocol for the rat and mouse brain slice methods, with the aim of enabling simple, cost-effective profiling of compounds with diverse physicochemical properties. The procedure for assessing the viability of the brain slices after the 5 h incubation period is also described. The results are interpreted for a set of compounds covering a wide range of physicochemical properties and various pharmacological targets. Application of the method for evaluating the unbound intracellular-to-extracellular concentration ratio (Kp,uu,cell) and the unbound brain-to-plasma concentration ratio (Kp,uu,brain) is discussed.

The aim of this study was to characterize the blood–brain barrier (BBB) transport and pharmacokinetics–pharmacodynamics (PKPD) relationship of oxymorphone and to further elucidate its possible contribution to oxycodone analgesia. The BBB transport of oxymorphone was studied using microdialysis in male Sprague–Dawley rats. Samples from microdialysis blood and brain probes, brain tissue, and plasma were analyzed by liquid chromatography with tandem mass spectrometry. The effect was measured as tail-flick latency. The study consisted of a PKPD experiment with combined microdialysis and antinociceptive measurements (n = 8), and another antinociceptive effect experiment (n = 9) using a 10 times lower dose. The combined data were analyzed with an integrated PKPD model in nonlinear mixed effect modeling utilizing a specific method (M3) for handling missing PD observations. The concentration of unbound oxymorphone was higher in brain than in blood, with a ratio of 1.9 (RSE, 9.7%), indicating active uptake at the BBB. The integrated PKPD model described the oxymorphone BBB transport and PKPD relationship successfully, with an EC50 in the brain of 63 ng/mL, and the M3 method was able to address the issue of censored observations. Oxymorphone has active uptake transport at the BBB in rats, with moderate uptake clearance to the brain. Its contribution to analgesia after oxycodone administration is not significant.

Prolactin elevation is a side effect of all currently available D2 receptor antagonists used in the treatment of schizophrenia. Prolactin elevation is the result of a direct antagonistic D2 effect blocking the tonic inhibition of prolactin release by dopamine. The aims of this work were to assess the correlation between in vitro estimates of D2 receptor affinity and pharmacokinetic–pharmacodynamic model-based estimates obtained from analysis of clinical data using an agonist–antagonist interaction (AAI) model and to assess the value of such a correlation in early prediction of full prolactin time profiles. A population model describing longitudinal prolactin data was fitted to clinical data from 16 clinical phases 1 and 3 trials including five different compounds. Pharmacokinetic data were modeled for each compound and the prolactin model was both fitted in per-compound fits as well as simultaneously to all prolactin data. Estimates of prolactin elevating potency were compared to corresponding in vitro values and their predictability was evaluated through model-based simulations. The model successfully described the prolactin time course for all compounds. Estimates derived from experimental preclinical data and the model fit of the clinical data were strongly correlated (p < 0.001), and simulations adequately predicted the prolactin elevation in five out of six compounds. The AAI model has the potential to be used in drug development to predict prolactin response for a given exposure of D2 antagonists using routinely produced preclinical data.

Exposure to antibiotics is an important factor influencing the development of bacterial resistance. In an era where very few new antibiotics are being developed, a strategy for the development of optimal dosing regimen and combination treatment that reduces the rate of resistance development and overcome existing resistance is of utmost importance. In addition, the optimal dosing in subpopulations is often not fully elucidated. The aim of this thesis was to develop pharmacokinetic (PK) and pharmacokinetic-pharmacodynamic (PKPD) models that characterize the interaction of antibiotics with bacterial growth, killing and resistance over time, and can be applied to guide optimization of dosing regimens that enhance the efficacy of mono- and combination antibiotic therapy. A mechanism-based PKPD model that incorporates the growth, killing kinetics and adaptive resistance development in Escherichia coli against gentamicin was developed based on in vitro time-kill curve data. After some adaptations, the model was successfully applied for similar data on colistin and meropenem alone, and in combination, on one wild type and one meropenem-resistant strain of Pseudomonas aeruginosa. The developed population PK model for colistin and its prodrug colistin methanesulfonate (CMS) in combination with the PKPD model showed the benefits for applying a loading dose for this drug. Simulations predicted the variability in bacteria kill to be larger between dosing occasions than between patients. A flat-fixed loading dose followed by an 8 or 12 hourly maintenance dose with infusion duration of up to 2 hours was shown to result in satisfactory bacterial kill under these conditions. Pharmacometric models that characterize the time-course of drug concentrations, bacterial growth, antibacterial killing and resistance development were successfully developed. Predictions illustrated how PKPD models based on in vitro data can be utilized to guide development of antibiotic dosing, with example

1. We applied the regression offset approach to predict rat in vivo intrinsic clearance (CL(int)) for 54 new chemical acid entities with high plasma protein binding values and low renal clearance (CL). The prediction success was correlated to volume of distribution (V(d)), molecular weight (Mw) and CL. 2. A correlation between poor in vitro-in vivo extrapolation (IVIVE) and V(d) values distinct from the V(d) of albumin (0.1-0.2 L/kg) was revealed. For compounds with a V(d) value above 0.5 L/kg, 0% of the predictions of in vivo CL(int) was within twofold of the observed value, compared to 69% for compounds with a V(d) value below 0.5 L/kg. 3. Compounds with a Mw below 450 g/mol demonstrated more accurate in vivo CL(int) predictions than compounds with a Mw above 450 g/mol, i.e. 63% compared to 21% within twofold. For compounds with in vivo CL below 30% of the liver blood flow (LBF), 53% of the predictions was within twofold of the observed value, compared to 0% for compounds with CL above 30% of the LBF. 4. We show that accurate IVIVE for acidic compounds with high plasma protein binding and low renal CL can be associated with a low V(d) (i.e. around the V(d) of albumin) and with a low in vivo CL, and that Mw is an important optimization parameter for pharmacokinetic. This study also further demonstrates the advantages of the application of the regression method for identifying cases when metabolic CL is not the single major elimination pathway.

AZ'0908 is a novel microsomal prostaglandin E synthase-1 inhibitor intended for oral administration. Pharmacokinetic experiments in rats showed that bioavailability was much lower than anticipated and increased following pretreatment with the nonspecific cytochrome P450 (CYP) inhibitor 1-aminobenzotriazole, presumably by inhibition of intestinal metabolism. Stability experiments in rat liver and intestinal fractions revealed that the intrinsic clearance (Cl(int) ) was much higher in intestinal than in liver microsomes. Caco2 experiments showed that AZ'0908 was a substrate for breast cancer resistance protein. Permeability was generally high and the efflux component was saturable predicting good absorption. The Cl(int) values in human intestinal microsome and S9 fractions were low. A correlation occurred between in vitro intestinal metabolism and in vivo intestinal loss in rats and dogs. Enzyme identification experiments showed that human CYP2J2 was involved in the oxidation of AZ'0908. In rats, the major metabolic enzyme was not identified. However, rat CYP2J2 analogs were not investigated. Intestinal metabolism appeared to be a major occurrence, explaining intestinal loss of AZ'0908 in the rats. In view of good overall permeability, low in vitro intestinal turnover, and relative low intestinal abundance of CYP2J2, we predict that intestinal metabolism of AZ'0908 in human does not exert a major issue.

Emerging data indicate that growth hormone (GH) therapy could have a role in improving cognitive function. GH replacement therapy in experimental animals and human patients counteracts the dysfunction of many behaviours related to the central nervous system (CNS). Various behaviours, such as cognitive behaviours related to learning and memory, are known to be induced by GH; the hormone might interact with specific receptors located in areas of the CNS that are associated with the functional anatomy of these behaviours. GH is believed to affect excitatory circuits involved in synaptic plasticity, which alters cognitive capacity. GH also has a protective effect on the CNS, as indicated by its beneficial effects in patients with spinal cord injury. Data collected from animal models indicates that GH might also stimulate neurogenesis. This Review discusses the mechanisms underlying the interactions between GH and the CNS, and the data emerging from animal and human studies on the relationship between GH and cognitive function. In this article, particular emphasis is given to the role of GH as a treatment for patients with cognitive impairment resulting from deficiency of the hormone.

Excessive and compulsive ethanol drinking is one of the most serious public health issues. Therefore, it is vital to increase the knowledge about risks and protection for alcohol use disorders (AUD) to optimize prevention and treatment strategies. Ethanol consumption commonly initiates during adolescence when extensive neuronal maturation and development also occurs. Early exposure to ethanol is a risk factor for AUD, but the effects of adolescent drinking and the basis for the individual susceptibility to AUD are not fully understood. The interactions between genotype and environmental factors determine the individual risk for AUD and this thesis aimed to examine the environmental impact. The specific aims were to investigate 1) how early-life conditions affect adolescent voluntary ethanol drinking, behavioural profiles, endogenous opioids and response to treatment with an opioid antagonist (naltrexone), and 2) whether alterations detected in the offspring may be mediated by variations in maternal behaviour. A rodent maternal separation (MS) model was used to mimic a protective and risk-inducing early-life environment, respectively, with the use of 15 min (MS15) or 360 min (MS360) of daily MS. The main findings were 1) the MS360, but not the MS15 rats, responded to naltrexone following adolescent ethanol drinking; all adolescent rats had a high voluntary ethanol intake independent of early environmental conditions whereas in the adult groups the MS360, but not the MS15 rats, increased their ethanol intake and preference over time; adolescent ethanol exposure resulted in higher dynorphin levels in hippocampus and higher Met-enkephalin-Arg6Phe7 in the amygdala, independently of rearing conditions, 2) behavioural profiling using the multivariate concentric square field™ test showed: the young MS360 rats had increased risk assessment and risk taking behaviour compared to the young MS15 rats; the young MS15 rats increased, whereas the young MS360 rats decreased, their r

Causal links between early-life stress, genes and later psychiatric diagnoses are not possible to fully address in human studies. Animal models therefore provide an important complement in which conditions can be well controlled and are here used to study and distinguish effects of early-life stress and alcohol exposure. The objective of this study was to investigate the impact of rearing conditions on behaviour in young rats and if these changes could be followed over time and to examine interaction effects between early-life environment and adolescent alcohol drinking on behaviour and immunoreactive levels of the opioid peptides dynorphin B, met-enkephalin-Arg(6)Phe(7) and beta-endorphin. We employed a rodent model, maternal separation, to study the impact of rearing conditions on behaviour, voluntary alcohol consumption and alcohol-induced effects. The consequences of short, 15 min (MS 15), and long, 360 min (MS 360), maternal separation in combination with adolescent voluntary alcohol consumption on behaviour and peptides were examined. A difference in the development of risk taking behaviour was found between the MS15 and MS360 while the development of general activity was found to differ between intake groups. Beta-endorphin levels in the pituitary and the periaqueductal gray area was found to be higher in the MS15 than the MS360. Adolescent drinking resulted in higher dynorphin B levels in the hippocampus and higher met-enkephalin-Arg(6)Phe(7) levels in the amygdala. Amygdala and hippocampus are involved in addiction processes and changes in these brain areas after adolescent alcohol drinking may have consequences for cognitive function and drug consumption behaviour in adulthood. The study shows that individual behavioural profiling over time in combination with neurobiological investigations provides means for studies of causality between early-life stress, behaviour and vulnerability to psychiatric disorders.

Our objectives were to document and compare plasma levels of Catecholamines, Cortisol,Glutathione and Malondialdehyde in camels after long term dehydration (20 days) in the presenceor absence of angiotensin II AT1 receptor blocker (Losartan) versus levels in non-dehydratedcamels; and to record the effects on glutathione and malondialdehyde activity in liver and kidneyhomogenate in the one-humped camel. Eighteen male camels were used in this study, sixcontrols, six dehydrated and treated with losartan (5mg/kg daily) and six were dehydrated withouttreatment. Our results revealed significant decrease (P<0.05) in plasma epinephrine level in bothtreated and dehydrated camels; while, Plasma norepinephrine showed significant increase in bothdehydrated groups (P< 0.01). Levels of plasma dopamine were also significantly increased (P<0.01) in both dehydrated groups compared to control camels.Plasma levels of cortisol increased significantly across dehydration with or without losartanadministration (P<0.01) compared with time-matched levels in control camels. Losartan had nosignificant modulating effect on the cortisol response to dehydration.Plasma, liver and kidney homogenates revealed significant increase (P<0.05) in glutathione levelsin both dehydrated groups compared to control.Plasma, liver and kidney homogenates for malondialdehyde levels in both treated and dehydratedcamels also showed significant increase (P<0.05 & P<0.01) compared to controls.In conclusion, our study demonstrates that the effect of dehydration with or without losartaninduced oxidative stress in these camels, leading to significant changes in plasma catecholaminesand cortisol levels, together with significant increments in glutathione and malondialdehydeactivities in plasma, liver and kidney homogenate to counter act the damaging effect of the freeradicals in the dehydrated camels.

The high-throughput brain slice method is a precise and robust technique for estimating the overall uptake of drugs into brain tissue through determination of the unbound volume of distribution in the brain (Vu,brain; ml·g brain-1). Vu,brain describes the relationship between the total drug concentration in the brain and the concentration of unbound drug in the brain interstitial fluid, regardless of blood–brain barrier function. The brain slice method is more physiologically based than the brain homogenate method with respect to the assessment of drug distribution in the brain because the cell-cell interactions, pH gradients and active transport systems are all conserved. The method provides information that is directly relevant to issues such as nonspecific binding to brain tissue, lysosomal trapping, and active uptake into the cells. For these reasons, the brain slice method is recommended for estimation of target-site pharmacokinetics in the early drug discovery process and fundamental pharmacological studies. This article provides a detailed protocol for the rat and mouse brain slice methods, with the aim of enabling simple, cost-effective profiling of compounds with diverse physicochemical properties. The procedure for assessing the viability of the brain slices after the 5 h incubation period is also described. The results are interpreted for a set of compounds covering a wide range of physicochemical properties and various pharmacological targets. Application of the method for evaluating the unbound intracellular-to-extracellular concentration ratio (Kp,uu,cell) and the unbound brain-to-plasma concentration ratio (Kp,uu,brain) is discussed.

Modeling is a valuable tool in drug development, to support decision making, improving study design, and aid in regulatory approval and labeling. This thesis describes the development of pharmacometric models for drugs used in anesthesia and analgesia. Models describing the effects on anesthetic depth, measured by the bispectral index (BIS), for a commonly used anesthetic, propofol, and for a novel anesthetic, AZD3043, were developed. The propofol model consisted of two effect-site compartments, and could describe the effects of propofol when the rate of infusion is changed during treatment. AZD3043 had a high clearance and a low volume of distribution, leading to a short half-life. The distribution to the effect site was fast, and together with the short plasma half-life leading to a fast onset and offset of effects. It was also shown that BIS after AZD3043 treatment is related to the probability of unconsciousness similar to propofol. In analgesia studies dropout due to lack of efficacy is common. This dropout is not at random and needs to be taken into consideration in order to avoid bias. A model was developed describing the PK, pain intensity and dropout hazard for placebo, naproxen and a novel analgesic compound, naproxcinod, after removal of a wisdom tooth. The model provides an opportunity to describe the effects of other doses or formulations. Visual predictive checks created by simultaneous simulations of PI and dropout provided a good way of assessing the goodness of fit when there is informative dropout. The performance of non-linear mixed effects models in the presence of informative dropout, with and without including models that describe such informative dropout was investigated by simulations and re-estimations. When a dropout model was not included there was in general more bias. The bias increased with decreasing number of observations per subject, increasing placebo effect and increasing dropout rate. Bias was relatively unaffected by the number o

Growth hormone (GH) stimulates growth and metabolism but also displays profound effects on the central nervous system (CNS). GH affects neurogenesis and neuroprotection, and has been shown to counteract drug-induced apoptosis in the brain. Anabolic androgenic steroids (AAS), mainly abused for their anabolic and performance-enhancing properties, can cause several adverse effects, such as cardiovascular complications, sterility, depression, and aggression. GH and AAS are both believed to interact with several signaling systems in the CNS. The aim of this thesis was to further investigate the impact of GH and AAS on neurochemistry and cognitive functions. Recombinant human GH (rhGH) and the steroid nandrolone decanoate (ND) were administered, separately and in combination with each other, to male rats. The results demonstrated that administration of GH improved spatial memory, assessed in a water maze test. Furthermore, GH induced alterations of the GABAB receptor mRNA expression, density, and functionality in the brain, for example in regions associated with cognition. GH also altered the mu opioid peptide (MOP) receptor, but not the delta opioid peptide (DOP) receptor functionality in the brain. Thus, some of the GH effects on cognition may involve effects on the GABAB receptors and MOP receptors. ND, on the contrary, seemed to induce impairments of memory and also altered the GABAB receptor mRNA expression in the brain. Furthermore, ND lowered the IGF-1 plasma concentrations and attenuated the IGF-1, IGF-2, and GHR mRNA expression in the pituitary. In addition, significant effects of GH and ND were found on plasma steroid concentrations, organ weight, as well as body weight. In conclusion, this thesis contributes with further knowledge on the cognitive and neurochemical consequences of GH and ND use. The findings regarding ND are worrying considering the common use of AAS among adolescents. GH improves memory functions and affects signaling systems in the brain asso

Simultaneous optimization of in vitro ligand binding studies using an optimal design software package that can incorporate multiple design variables through non-linear mixed effect models and provide a general optimized design regardless of the binding site capacity and relative binding rates for a two binding system. Experimental design optimization was employed with D- and ED-optimality using PopED 2.8 including commonly encountered factors during experimentation (residual error, between experiment variability and non-specific binding) for in vitro ligand binding experiments: association, dissociation, equilibrium and non-specific binding experiments. Moreover, a method for optimizing several design parameters (ligand concentrations, measurement times and total number of samples) was examined. With changes in relative binding site density and relative binding rates, different measurement times and ligand concentrations were needed to provide precise estimation of binding parameters. However, using optimized design variables, significant reductions in number of samples provided as good or better precision of the parameter estimates compared to the original extensive sampling design. Employing ED-optimality led to a general experimental design regardless of the relative binding site density and relative binding rates. Precision of the parameter estimates were as good as the extensive sampling design for most parameters and better for the poorly estimated parameters. Optimized designs for in vitro ligand binding studies provided robust parameter estimation while allowing more efficient and cost effective experimentation by reducing the measurement times and separate ligand concentrations required and in some cases, the total number of samples.

Causal links between early-life stress, genes and later psychiatric diagnoses are not possible to fully address in human studies. Animal models therefore provide an important complement in which conditions can be well controlled and are here used to study and distinguish effects of early-life stress and alcohol exposure. The objective of this study was to investigate the impact of rearing conditions on behaviour in young rats and if these changes could be followed over time and to examine interaction effects between early-life environment and adolescent alcohol drinking on behaviour and immunoreactive levels of the opioid peptides dynorphin B, met-enkephalin-Arg(6)Phe(7) and beta-endorphin. We employed a rodent model, maternal separation, to study the impact of rearing conditions on behaviour, voluntary alcohol consumption and alcohol-induced effects. The consequences of short, 15 min (MS 15), and long, 360 min (MS 360), maternal separation in combination with adolescent voluntary alcohol consumption on behaviour and peptides were examined. A difference in the development of risk taking behaviour was found between the MS15 and MS360 while the development of general activity was found to differ between intake groups. Beta-endorphin levels in the pituitary and the periaqueductal gray area was found to be higher in the MS15 than the MS360. Adolescent drinking resulted in higher dynorphin B levels in the hippocampus and higher met-enkephalin-Arg(6)Phe(7) levels in the amygdala. Amygdala and hippocampus are involved in addiction processes and changes in these brain areas after adolescent alcohol drinking may have consequences for cognitive function and drug consumption behaviour in adulthood. The study shows that individual behavioural profiling over time in combination with neurobiological investigations provides means for studies of causality between early-life stress, behaviour and vulnerability to psychiatric disorders.

Our objectives were to document and compare plasma levels of Catecholamines, Cortisol,Glutathione and Malondialdehyde in camels after long term dehydration (20 days) in the presenceor absence of angiotensin II AT1 receptor blocker (Losartan) versus levels in non-dehydratedcamels; and to record the effects on glutathione and malondialdehyde activity in liver and kidneyhomogenate in the one-humped camel. Eighteen male camels were used in this study, sixcontrols, six dehydrated and treated with losartan (5mg/kg daily) and six were dehydrated withouttreatment. Our results revealed significant decrease (P<0.05) in plasma epinephrine level in bothtreated and dehydrated camels; while, Plasma norepinephrine showed significant increase in bothdehydrated groups (P< 0.01). Levels of plasma dopamine were also significantly increased (P<0.01) in both dehydrated groups compared to control camels.Plasma levels of cortisol increased significantly across dehydration with or without losartanadministration (P<0.01) compared with time-matched levels in control camels. Losartan had nosignificant modulating effect on the cortisol response to dehydration.Plasma, liver and kidney homogenates revealed significant increase (P<0.05) in glutathione levelsin both dehydrated groups compared to control.Plasma, liver and kidney homogenates for malondialdehyde levels in both treated and dehydratedcamels also showed significant increase (P<0.05 & P<0.01) compared to controls.In conclusion, our study demonstrates that the effect of dehydration with or without losartaninduced oxidative stress in these camels, leading to significant changes in plasma catecholaminesand cortisol levels, together with significant increments in glutathione and malondialdehydeactivities in plasma, liver and kidney homogenate to counter act the damaging effect of the freeradicals in the dehydrated camels.

The high-throughput brain slice method is a precise and robust technique for estimating the overall uptake of drugs into brain tissue through determination of the unbound volume of distribution in the brain (Vu,brain; ml·g brain-1). Vu,brain describes the relationship between the total drug concentration in the brain and the concentration of unbound drug in the brain interstitial fluid, regardless of blood–brain barrier function. The brain slice method is more physiologically based than the brain homogenate method with respect to the assessment of drug distribution in the brain because the cell-cell interactions, pH gradients and active transport systems are all conserved. The method provides information that is directly relevant to issues such as nonspecific binding to brain tissue, lysosomal trapping, and active uptake into the cells. For these reasons, the brain slice method is recommended for estimation of target-site pharmacokinetics in the early drug discovery process and fundamental pharmacological studies. This article provides a detailed protocol for the rat and mouse brain slice methods, with the aim of enabling simple, cost-effective profiling of compounds with diverse physicochemical properties. The procedure for assessing the viability of the brain slices after the 5 h incubation period is also described. The results are interpreted for a set of compounds covering a wide range of physicochemical properties and various pharmacological targets. Application of the method for evaluating the unbound intracellular-to-extracellular concentration ratio (Kp,uu,cell) and the unbound brain-to-plasma concentration ratio (Kp,uu,brain) is discussed.

Our objectives were to document and compare plasma levels of Catecholamines, Cortisol,Glutathione and Malondialdehyde in camels after long term dehydration (20 days) in the presenceor absence of angiotensin II AT1 receptor blocker (Losartan) versus levels in non-dehydratedcamels; and to record the effects on glutathione and malondialdehyde activity in liver and kidneyhomogenate in the one-humped camel. Eighteen male camels were used in this study, sixcontrols, six dehydrated and treated with losartan (5mg/kg daily) and six were dehydrated withouttreatment. Our results revealed significant decrease (P<0.05) in plasma epinephrine level in bothtreated and dehydrated camels; while, Plasma norepinephrine showed significant increase in bothdehydrated groups (P< 0.01). Levels of plasma dopamine were also significantly increased (P<0.01) in both dehydrated groups compared to control camels.Plasma levels of cortisol increased significantly across dehydration with or without losartanadministration (P<0.01) compared with time-matched levels in control camels. Losartan had nosignificant modulating effect on the cortisol response to dehydration.Plasma, liver and kidney homogenates revealed significant increase (P<0.05) in glutathione levelsin both dehydrated groups compared to control.Plasma, liver and kidney homogenates for malondialdehyde levels in both treated and dehydratedcamels also showed significant increase (P<0.05 & P<0.01) compared to controls.In conclusion, our study demonstrates that the effect of dehydration with or without losartaninduced oxidative stress in these camels, leading to significant changes in plasma catecholaminesand cortisol levels, together with significant increments in glutathione and malondialdehydeactivities in plasma, liver and kidney homogenate to counter act the damaging effect of the freeradicals in the dehydrated camels.

Causal links between early-life stress, genes and later psychiatric diagnoses are not possible to fully address in human studies. Animal models therefore provide an important complement in which conditions can be well controlled and are here used to study and distinguish effects of early-life stress and alcohol exposure. The objective of this study was to investigate the impact of rearing conditions on behaviour in young rats and if these changes could be followed over time and to examine interaction effects between early-life environment and adolescent alcohol drinking on behaviour and immunoreactive levels of the opioid peptides dynorphin B, met-enkephalin-Arg(6)Phe(7) and beta-endorphin. We employed a rodent model, maternal separation, to study the impact of rearing conditions on behaviour, voluntary alcohol consumption and alcohol-induced effects. The consequences of short, 15 min (MS 15), and long, 360 min (MS 360), maternal separation in combination with adolescent voluntary alcohol consumption on behaviour and peptides were examined. A difference in the development of risk taking behaviour was found between the MS15 and MS360 while the development of general activity was found to differ between intake groups. Beta-endorphin levels in the pituitary and the periaqueductal gray area was found to be higher in the MS15 than the MS360. Adolescent drinking resulted in higher dynorphin B levels in the hippocampus and higher met-enkephalin-Arg(6)Phe(7) levels in the amygdala. Amygdala and hippocampus are involved in addiction processes and changes in these brain areas after adolescent alcohol drinking may have consequences for cognitive function and drug consumption behaviour in adulthood. The study shows that individual behavioural profiling over time in combination with neurobiological investigations provides means for studies of causality between early-life stress, behaviour and vulnerability to psychiatric disorders.

The high-throughput brain slice method is a precise and robust technique for estimating the overall uptake of drugs into brain tissue through determination of the unbound volume of distribution in the brain (Vu,brain; ml·g brain-1). Vu,brain describes the relationship between the total drug concentration in the brain and the concentration of unbound drug in the brain interstitial fluid, regardless of blood–brain barrier function. The brain slice method is more physiologically based than the brain homogenate method with respect to the assessment of drug distribution in the brain because the cell-cell interactions, pH gradients and active transport systems are all conserved. The method provides information that is directly relevant to issues such as nonspecific binding to brain tissue, lysosomal trapping, and active uptake into the cells. For these reasons, the brain slice method is recommended for estimation of target-site pharmacokinetics in the early drug discovery process and fundamental pharmacological studies. This article provides a detailed protocol for the rat and mouse brain slice methods, with the aim of enabling simple, cost-effective profiling of compounds with diverse physicochemical properties. The procedure for assessing the viability of the brain slices after the 5 h incubation period is also described. The results are interpreted for a set of compounds covering a wide range of physicochemical properties and various pharmacological targets. Application of the method for evaluating the unbound intracellular-to-extracellular concentration ratio (Kp,uu,cell) and the unbound brain-to-plasma concentration ratio (Kp,uu,brain) is discussed.

Exposure to antibiotics is an important factor influencing the development of bacterial resistance. In an era where very few new antibiotics are being developed, a strategy for the development of optimal dosing regimen and combination treatment that reduces the rate of resistance development and overcome existing resistance is of utmost importance. In addition, the optimal dosing in subpopulations is often not fully elucidated. The aim of this thesis was to develop pharmacokinetic (PK) and pharmacokinetic-pharmacodynamic (PKPD) models that characterize the interaction of antibiotics with bacterial growth, killing and resistance over time, and can be applied to guide optimization of dosing regimens that enhance the efficacy of mono- and combination antibiotic therapy. A mechanism-based PKPD model that incorporates the growth, killing kinetics and adaptive resistance development in Escherichia coli against gentamicin was developed based on in vitro time-kill curve data. After some adaptations, the model was successfully applied for similar data on colistin and meropenem alone, and in combination, on one wild type and one meropenem-resistant strain of Pseudomonas aeruginosa. The developed population PK model for colistin and its prodrug colistin methanesulfonate (CMS) in combination with the PKPD model showed the benefits for applying a loading dose for this drug. Simulations predicted the variability in bacteria kill to be larger between dosing occasions than between patients. A flat-fixed loading dose followed by an 8 or 12 hourly maintenance dose with infusion duration of up to 2 hours was shown to result in satisfactory bacterial kill under these conditions. Pharmacometric models that characterize the time-course of drug concentrations, bacterial growth, antibacterial killing and resistance development were successfully developed. Predictions illustrated how PKPD models based on in vitro data can be utilized to guide development of antibiotic dosing, with example

Excessive and compulsive ethanol drinking is one of the most serious public health issues. Therefore, it is vital to increase the knowledge about risks and protection for alcohol use disorders (AUD) to optimize prevention and treatment strategies. Ethanol consumption commonly initiates during adolescence when extensive neuronal maturation and development also occurs. Early exposure to ethanol is a risk factor for AUD, but the effects of adolescent drinking and the basis for the individual susceptibility to AUD are not fully understood. The interactions between genotype and environmental factors determine the individual risk for AUD and this thesis aimed to examine the environmental impact. The specific aims were to investigate 1) how early-life conditions affect adolescent voluntary ethanol drinking, behavioural profiles, endogenous opioids and response to treatment with an opioid antagonist (naltrexone), and 2) whether alterations detected in the offspring may be mediated by variations in maternal behaviour. A rodent maternal separation (MS) model was used to mimic a protective and risk-inducing early-life environment, respectively, with the use of 15 min (MS15) or 360 min (MS360) of daily MS. The main findings were 1) the MS360, but not the MS15 rats, responded to naltrexone following adolescent ethanol drinking; all adolescent rats had a high voluntary ethanol intake independent of early environmental conditions whereas in the adult groups the MS360, but not the MS15 rats, increased their ethanol intake and preference over time; adolescent ethanol exposure resulted in higher dynorphin levels in hippocampus and higher Met-enkephalin-Arg6Phe7 in the amygdala, independently of rearing conditions, 2) behavioural profiling using the multivariate concentric square field™ test showed: the young MS360 rats had increased risk assessment and risk taking behaviour compared to the young MS15 rats; the young MS15 rats increased, whereas the young MS360 rats decreased, their r

Growth hormone (GH) stimulates growth and metabolism but also displays profound effects on the central nervous system (CNS). GH affects neurogenesis and neuroprotection, and has been shown to counteract drug-induced apoptosis in the brain. Anabolic androgenic steroids (AAS), mainly abused for their anabolic and performance-enhancing properties, can cause several adverse effects, such as cardiovascular complications, sterility, depression, and aggression. GH and AAS are both believed to interact with several signaling systems in the CNS. The aim of this thesis was to further investigate the impact of GH and AAS on neurochemistry and cognitive functions. Recombinant human GH (rhGH) and the steroid nandrolone decanoate (ND) were administered, separately and in combination with each other, to male rats. The results demonstrated that administration of GH improved spatial memory, assessed in a water maze test. Furthermore, GH induced alterations of the GABAB receptor mRNA expression, density, and functionality in the brain, for example in regions associated with cognition. GH also altered the mu opioid peptide (MOP) receptor, but not the delta opioid peptide (DOP) receptor functionality in the brain. Thus, some of the GH effects on cognition may involve effects on the GABAB receptors and MOP receptors. ND, on the contrary, seemed to induce impairments of memory and also altered the GABAB receptor mRNA expression in the brain. Furthermore, ND lowered the IGF-1 plasma concentrations and attenuated the IGF-1, IGF-2, and GHR mRNA expression in the pituitary. In addition, significant effects of GH and ND were found on plasma steroid concentrations, organ weight, as well as body weight. In conclusion, this thesis contributes with further knowledge on the cognitive and neurochemical consequences of GH and ND use. The findings regarding ND are worrying considering the common use of AAS among adolescents. GH improves memory functions and affects signaling systems in the brain asso

The high-throughput brain slice method is a precise and robust technique for estimating the overall uptake of drugs into brain tissue through determination of the unbound volume of distribution in the brain (Vu,brain; ml·g brain-1). Vu,brain describes the relationship between the total drug concentration in the brain and the concentration of unbound drug in the brain interstitial fluid, regardless of blood–brain barrier function. The brain slice method is more physiologically based than the brain homogenate method with respect to the assessment of drug distribution in the brain because the cell-cell interactions, pH gradients and active transport systems are all conserved. The method provides information that is directly relevant to issues such as nonspecific binding to brain tissue, lysosomal trapping, and active uptake into the cells. For these reasons, the brain slice method is recommended for estimation of target-site pharmacokinetics in the early drug discovery process and fundamental pharmacological studies. This article provides a detailed protocol for the rat and mouse brain slice methods, with the aim of enabling simple, cost-effective profiling of compounds with diverse physicochemical properties. The procedure for assessing the viability of the brain slices after the 5 h incubation period is also described. The results are interpreted for a set of compounds covering a wide range of physicochemical properties and various pharmacological targets. Application of the method for evaluating the unbound intracellular-to-extracellular concentration ratio (Kp,uu,cell) and the unbound brain-to-plasma concentration ratio (Kp,uu,brain) is discussed.

Our objectives were to document and compare plasma levels of Catecholamines, Cortisol,Glutathione and Malondialdehyde in camels after long term dehydration (20 days) in the presenceor absence of angiotensin II AT1 receptor blocker (Losartan) versus levels in non-dehydratedcamels; and to record the effects on glutathione and malondialdehyde activity in liver and kidneyhomogenate in the one-humped camel. Eighteen male camels were used in this study, sixcontrols, six dehydrated and treated with losartan (5mg/kg daily) and six were dehydrated withouttreatment. Our results revealed significant decrease (P<0.05) in plasma epinephrine level in bothtreated and dehydrated camels; while, Plasma norepinephrine showed significant increase in bothdehydrated groups (P< 0.01). Levels of plasma dopamine were also significantly increased (P<0.01) in both dehydrated groups compared to control camels.Plasma levels of cortisol increased significantly across dehydration with or without losartanadministration (P<0.01) compared with time-matched levels in control camels. Losartan had nosignificant modulating effect on the cortisol response to dehydration.Plasma, liver and kidney homogenates revealed significant increase (P<0.05) in glutathione levelsin both dehydrated groups compared to control.Plasma, liver and kidney homogenates for malondialdehyde levels in both treated and dehydratedcamels also showed significant increase (P<0.05 & P<0.01) compared to controls.In conclusion, our study demonstrates that the effect of dehydration with or without losartaninduced oxidative stress in these camels, leading to significant changes in plasma catecholaminesand cortisol levels, together with significant increments in glutathione and malondialdehydeactivities in plasma, liver and kidney homogenate to counter act the damaging effect of the freeradicals in the dehydrated camels.

Causal links between early-life stress, genes and later psychiatric diagnoses are not possible to fully address in human studies. Animal models therefore provide an important complement in which conditions can be well controlled and are here used to study and distinguish effects of early-life stress and alcohol exposure. The objective of this study was to investigate the impact of rearing conditions on behaviour in young rats and if these changes could be followed over time and to examine interaction effects between early-life environment and adolescent alcohol drinking on behaviour and immunoreactive levels of the opioid peptides dynorphin B, met-enkephalin-Arg(6)Phe(7) and beta-endorphin. We employed a rodent model, maternal separation, to study the impact of rearing conditions on behaviour, voluntary alcohol consumption and alcohol-induced effects. The consequences of short, 15 min (MS 15), and long, 360 min (MS 360), maternal separation in combination with adolescent voluntary alcohol consumption on behaviour and peptides were examined. A difference in the development of risk taking behaviour was found between the MS15 and MS360 while the development of general activity was found to differ between intake groups. Beta-endorphin levels in the pituitary and the periaqueductal gray area was found to be higher in the MS15 than the MS360. Adolescent drinking resulted in higher dynorphin B levels in the hippocampus and higher met-enkephalin-Arg(6)Phe(7) levels in the amygdala. Amygdala and hippocampus are involved in addiction processes and changes in these brain areas after adolescent alcohol drinking may have consequences for cognitive function and drug consumption behaviour in adulthood. The study shows that individual behavioural profiling over time in combination with neurobiological investigations provides means for studies of causality between early-life stress, behaviour and vulnerability to psychiatric disorders.

The use of nanoparticles, in many different fields, is rapidly increasing. What substances, and to what extent they exist in food and supplements, and the prevalence of such products on the market, is of interest to the Swedish National Food Agency. Answering those questions, as well as providing information regarding their potential toxicity, was the intent of this thesis. To understand the difficulties, and possibilities, in analyzing nanoparticles in food, a methods-section was included, covering the most common techniques, the theory behind them and when they may be used. It became abundantly clear that analyzing nanoparticles in food, and investigating their potential toxicity, is anything but simple, and requires a combination of many techniques. Quite surprisingly, it seems the occurrence of nanoparticles in food, as today, are not added on purpose, since not one nanosubstance to be used in food has been approved by EU. Rather, studies have revealed they originate from a nanosized fraction of food additives, such as TiO2 (E171), which has attracted particular attention in this report. This nanofraction may be as large as 40%, and the estimated total intake of TiO2 (as stated by EFSA) is 1.28 mg/kg/person, resulting in quite an amount of nano TiO2, provided the numbers are correct. The toxicity of silver has also been reviewed, since the colloidal form, which includes nanoparticles, is quite common as a health supplement. Toxicity studies are not unambiguous though, some indicate geno-and–cytotoxicity and others do not. Importantly, the toxicity studies on TiO2 have not been done on E171, but on many other forms of TiO2, whose relevance is difficult to predict. TiO2 is, however, classified as a possible carcinogen by IARC. Several groups have taken an interest in a potential relation between particle intake and various chronic inflammatory diseases, also discussed in this report. In conclusion, the size, modifications of E171 and its toxicity, requires more at

The use of nanoparticles, in many different fields, is rapidly increasing. What substances, and to what extent they exist in food and supplements, and the prevalence of such products on the market, is of interest to the Swedish National Food Agency. Answering those questions, as well as providing information regarding their potential toxicity, was the intent of this thesis. To understand the difficulties, and possibilities, in analyzing nanoparticles in food, a methods-section was included, covering the most common techniques, the theory behind them and when they may be used. It became abundantly clear that analyzing nanoparticles in food, and investigating their potential toxicity, is anything but simple, and requires a combination of many techniques. Quite surprisingly, it seems the occurrence of nanoparticles in food, as today, are not added on purpose, since not one nanosubstance to be used in food has been approved by EU. Rather, studies have revealed they originate from a nanosized fraction of food additives, such as TiO2 (E171), which has attracted particular attention in this report. This nanofraction may be as large as 40%, and the estimated total intake of TiO2 (as stated by EFSA) is 1.28 mg/kg/person, resulting in quite an amount of nano TiO2, provided the numbers are correct. The toxicity of silver has also been reviewed, since the colloidal form, which includes nanoparticles, is quite common as a health supplement. Toxicity studies are not unambiguous though, some indicate geno-and–cytotoxicity and others do not. Importantly, the toxicity studies on TiO2 have not been done on E171, but on many other forms of TiO2, whose relevance is difficult to predict. TiO2 is, however, classified as a possible carcinogen by IARC. Several groups have taken an interest in a potential relation between particle intake and various chronic inflammatory diseases, also discussed in this report. In conclusion, the size, modifications of E171 and its toxicity, requires more at

The blood-brain barrier (BBB) controls the movement of substances into and out of the brain. The tight junctions between endothelial cells and energy dependent transporters in the BBB influence rate and extent of drug distribution to the brain. The aim of this thesis was to study different methodological and pharmacokinetic aspects of drug transport at the BBB by characterizing possible active uptake and drug-drug interactions. Therefore, advanced tools for data acquisition and analysis were applied. The role of BBB transport in early drug development, with particular emphasis on in vitro-in vivo comparisons and species differences, was also investigated. Microdialysis in rats was used to study the BBB pharmacokinetics of oxymorphone, diphenhydramine (DPHM), oxycodone and morphine. Oxymorphone, DPHM and verapamil were all found to be actively taken up at the BBB, with brain to blood unbound drug ratios of 2, 5 and 2, respectively. The effect profile for oxycodone was successfully described using the modified M3 method for censored observations. In vitro experiments indicated a competitive interaction between DPHM and oxycodone on active uptake transport to the brain. No such interaction was observed in vivo due to much lower unbound concentrations achieved, compared with the in vitro Ki values. Active uptake of morphine at the BBB was not demonstrated even at very low concentrations as it was not possible to separate the active uptake transport process from active efflux by decreasing the morphine concentration. Mice carrying the human P-gp gene (hMDR1) were used to evaluate possible species differences in P-gp function. Differences were evident between the hMDR1 and normal mice in BBB penetration of various P-gp substrates and in the effect of blockers on P-gp function. Quantitative measurements of P-gp expression levels at the BBB and a comparison with human data are crucial for the future use of the hMDR1 model. In conclusion, this thesis reports active uptake of

New approaches quantifying the effect of treatment are needed in oncology to improve the drug development process and to enable treatment optimization for existing therapies. This thesis focuses on the development of pharmacometric models for biomarkers, side effects and efficacy in order to identify predictors of clinical response in anti-cancer drug therapy. The variability in myelosuppression was characterized in six different cytotoxic anticancer treatments to evaluate a model-based dose individualization approach utilizing neutrophil counts as a biomarker. The estimated impact of inter-occasion variability was relatively low in relation to the inter-individual variability, indicating that myelosuppression is predictable from one treatment course to another. The approach may thereby be useful for dose optimization within an individual. To further study and to identify predictors for the severe side effect febrile neutropenia (FN), the relationship between the shape of the myelosuppression time-course and the probability of FN was characterized. Patients with a rapid decline in neutrophil counts and high drug sensitivity were identified to have a higher probability of developing FN compared with other patients who experience grade 4 neutropenia. Predictors of clinical response in patients receiving sunitinib for the treatment of gastro-intestinal stromal tumor (GIST) were identified by the development of an integrated modeling framework. Drug exposure, biomarkers, tumor dynamics, side effects and overall survival (OS) were linked in a unified structure, and univariate and multivariate exposure variables were tested for their predictive capacities. The soluble biomarker, sVEGFR-3 and tumor size at start of treatment were found to be promising predictors of overall survival, with decreased sVEGFR-3 levels and smaller baseline tumor size being predictive of longer OS. Also hypertension and neutropenia was identified as predictors of OS. The developed modeling framew

Background Japanese encephalitis virus (JEV), a member of the Flaviviridae family, causes around 68,000 encephalitis cases annually, of which 20–30% are fatal, while 30–50% of the recovered cases develop severe neurological sequelae. Specific antivirals for JEV would be of great importance, particularly in those cases where the infection has become persistent. Being indispensable for flaviviral replication, the NS2B-NS3 protease is a promising target for design of anti-flaviviral inhibitors. Contrary to related flaviviral proteases, the JEV NS2B-NS3 protease is structurally and mechanistically much less characterized. Here we aimed at establishing a straightforward procedure for cloning, expression, purification and biochemical characterization of JEV NS2B(H)-NS3pro protease. Methodology/Principal Findings The full-length sequence of JEV NS2B-NS3 genotype III strain JaOArS 982 was obtained as a synthetic gene. The sequence of NS2B(H)-NS3pro was generated by splicing by overlap extension PCR (SOE-PCR) and cloned into the pTrcHisA vector. Hexahistidine-tagged NS2B(H)-NS3pro, expressed in E. coli as soluble protein, was purified to >95% purity by a single-step immobilized metal affinity chromatography. SDS-PAGE and immunoblotting of the purified enzyme demonstrated NS2B(H)-NS3pro precursor and its autocleavage products, NS3pro and NS2B(H), as 36, 21, and 10 kDa bands, respectively. Kinetic parameters, Km and kcat, for fluorogenic protease model substrates, Boc-GRR-amc, Boc-LRR-amc, Ac-nKRR-amc, Bz-nKRR-amc, Pyr-RTKR-amc and Abz-(R)4SAG-nY-amide, were obtained using inner filter effect correction. The highest catalytic efficiency kcat/Km was found for Pyr-RTKR-amc (kcat/Km: 1962.96±85.0 M−1 s−1) and the lowest for Boc-LRR-amc (kcat/Km: 3.74±0.3 M−1 s−1). JEV NS3pro is inhibited by aprotinin but to a lesser extent than DEN and WNV NS3pro. Conclusions/Significance A simplified procedure for the cloning, overexpression and purification of the NS2B(H)-NS3pro was establis

Viruses are a major threat to humans due to their unique adaptability, evolvability and capability to control their hosts as parasites and genetic elements. HIV/AIDS is the third largest cause of death by infectious diseases in the world, and drug resistance due to the viral mutations is still the leading cause of treatment failure. The flaviviruses, such as Dengue virus (DEN) and Japanese encephalitis virus (JEV), represent other major cause of morbidity and mortality, and the areas where these viruses are endemic are spreading rapidly. No curative therapy for any flavivirus could be made available as yet. The first part of this thesis focuses on the HIV-1 drug resistance caused by mutations in a major HIV drug target, the HIV-1 reverse transcriptase (RT) as a response to the largest class of clinically used anti-retrovirals, the NRTIs. A robust proteochemometric model was created to analyse the complex mutation patterns in RT drug resistance. The model identified more than ten frequently-occurring mutations, each conferring at least two-fold decrease in susceptibility for one or several NRTIs. Using our prediction server (hivdrc.org), the model can be applied to propose optimum combination therapy for patients harbouring mutated HIV variants. The second part of the thesis encompasses studies on a promising drug target, the NS2B(H)-NS3pro, in two flaviviruses, namely the dengue virus (DEN) and Japanese encephalitis virus (JEV). Functional determinants of DEN NS2B(H)-NS3pro were identified by site-directed mutagenesis. Further, peptide inhibitors were designed using proteochemometrics (PCM) and statistical molecular design (SMD), synthesized and assayed on DEN proteases, which resulted in some novel peptides with low micromolar or sub-micromolar inhibitor activity. The very poorly characterised JEV NS2B(H)-NS3pro was cloned, purified and the kinetic parameters of this attractive drug target were determined for a series of model substrates and inhibitor. The result

Early predictions of a potential drug candidate’s time-course of effect and side-effects, based on models describing drug concentrations, drug effects and disease progression, would be valuable to make drug development more efficient. Pharmacodynamic modeling can incorporate and propagate prior knowledge and be used for simulations of different scenarios. In this thesis three population pharmacodynamic models were developed to describe the antipsychotic effects and the side-effects prolactin elevation and Extra Pyramidal Symptoms (EPS) following administration of D2-receptor antagonists, commonly used in the treatment of schizophrenia. Model parameter estimates of prolactin elevating potencies of six compounds correlated with in vitro values of receptor affinities, and parameters related to diurnal prolactin variation and tolerance were similar for the different compounds. The developed prolactin model can thereby be used to predict the time-course of prolactin elevation in patients for a drug candidate using information on in vitro affinity to the D2-receptor. Furthermore, the clinical antipsychotic effect and the prolactin elevation was found to correlate on the individual level for the three antipsychotic compounds investigated and a quantitative relation between D2-receptor occupancy in the brain and prolactin elevation was established. These results support the use of prolactin concentrations as a biomarker in drug development or for individual dose adjustments in clinical care. The developed model for spontaneously reported EPS adverse events, following treatment with one of five antipsychotics drugs, characterized both the duration and severity of EPS. The model successfully described both the proportions and number of transitions between severity grades and was shown to adequately simulate longitudinal categorical EPS data. Complex pharmacodynamic models are often associated with long estimation times and non-normal distributions of individual parameters. A