Many drugs exhibit major variability in both pharmacokinetic (PK) and pharmacodynamic (PD) parameters that prevents the use of the same dose for all patients. Variability can occur both between patients (IIV) as well as within patients over the course of time (IOV). In a drug with narrow therapeutic range and substantial IIV, dose selection may require individual adaptation. Adaptation can either be made before (a priori) or after (a posteriori) first drug administration. The former implies basing the dose on prior information known to be influential, such as kidney function indicators, weight or concomitant medication, whereas a posteriori dose adaptations are based on post-treatment observations. Often individualization cannot be based on the clinical outcome itself. In such cases, drug concentrations or biomarkers may be valuable for dose individualisation. In this thesis two therapeutic areas where dosing is critical have been investigated regarding the possibilities of a priori and a posteriori dose adaptation; anticancer treatment where myelosuppression is dose limiting, and tacrolimus used for immunosuppression in paediatric transplantation. In tacrolimus previously published models were found to be of little value for dose adaptation in the early critical days post-transplantation. New PK models were developed and used to suggest new dosing regimens tailored for the paediatric population, recognizing the changing pharmacokinetics in the early time post-transplantation. For several anticancer drugs covariates were identified that partly explained IIV in myelosuppression. IOV were found to be lower than IIV which implies that individual dose adaptations a posteriori can be valuable. Dose adaptation, using Bayesian principles in order to simultaneously minimise the risk of severe toxicity or subtherapeutic levels, was evaluated using simulations. Type and amount of data needed, as well as variability parameters influential on the outcome, were evaluated. Results show drug concentrations being of little value, if neutrophil counts are available. The models discussed in this thesis have been implemented in MS Excel macros for Bayesian forecasting, to allow widespread distribution to clinical settings without necessitating access to specific statistical software.

<p>Swedish Obesity Specialists examines how obesity is conceptualized as a medical condition by the staff working at an obesity clinic in Stockholm Sweden. Through eight weeks of participant observations and eight semi-structured interviews this thesis answers the question of how specialist working in the field of obesity construct obesity as a medical site. The thesis aims at understanding how obesity is becoming an issue for medicine, further how obesity’s entry into medicine creates new understandings of the body and medical treatments. Through the theoretical concepts of global assemblages and bio-power I argue that obesity as a disease is defined through seemingly objective criteria aimed at defining a population of sufferers, simultaneously for obesity to be viewed as disease scientifically valid treatments on an individual level must be put into place. By viewing obesity’s entry into medicine as a process of shared consensus, this thesis examines the relationship between global levels of knowledge production and their application and negotiation at one clinic treating obesity. Here expert knowledge and governance are integrated to create both treatment and an idea of what obesity as a medical condition is. In this thesis I argue that the application of expert knowledge and global criteria leads to unexpected views on what can be conceived as medical treatment. Further the thesis discusses how the body of the patient becomes reinterpreted once obesity becomes a medical condition.</p><p><strong> </strong></p><p><strong>Key words: </strong>Obesity, medical expertise, global assemblages, governance, lifestyle alteration</p>

<p>Cytochrome P450 (P450 or CYP) is an enzyme system involved in the oxygenation of a wide range of endogenous compounds as well as foreign chemicals and drugs. This thesis describes investigations of P450-catalyzed oxygenation of prostaglandins, linoleic and arachidoniacids.</p><p>The formation of bisallylic hydroxy metabolites of linoleic and arachidonic acids was studied with human recombinant P450s and with human liver microsomes. Several P450 enzymes catalyzed the formation of bisallylic hydroxy metabolites. Inhibition studies and stereochemic analysis of metabolites suggest that the enzyme CYP1A2 may contribute to the biosynthesis of bisallylic hydroxy fatty acid metabolites in adult human liver microsomes.</p><p>19<i>R</i>-Hydroxy-PGE and 20-hydroxy-PGE are major components of human and ovine semen, respectively. They are formed in the seminal vesicles, but the mechanism of their biosynthesis is unknown. Reverse transcription-polymerase chain reaction using degenerate primers for mammalian <i>CYP4</i> family genes, revealed expression of two nov P450 genes in human and ovine seminal vesicles. The full coding regions of the genes were cloned and the enzymes were expressed in a yeast system. The human enzyme was designated CYP4F8 and the ovine enzyme was designated CYP4F21. Comparison of their deduced protein sequenceshowed that they had 74 % amino acid identity. Recombinant CYP4F8 oxygenated two prostaglandin endoperoxides (PGH₁ and PGH₂) and three stable PGH₂ analogues int19-hydroxy metabolites. Oxygenation of these substrates was mirrored when incubated with microsomes isolated from human seminal vesicles. These results suggest that CYP4F8 is present in human seminal vesicles and that 19<i>R</i>-hydroxy-PGE is formed by CYP4F8-catalyze 19<i>R</i>-hydroxylation of PGH₁ and PGH₂, followed by PGE synthase-catalyzed isomerization. Studies of catalytic properties of recombinant CYP4F21 suggest that 20-hydroxy-PGE may be formed by similar mechanisms in ovine seminal vesicles. CYP4F8 is the first enzyme shown to hydroxylate prostaglandin endoperoxides.</p>

<p>Cholesterol is metabolized to a variety of important biological products in the body including bile acids and vitamin D. The present investigation is focused on enzymes that catalyze 7α-hydroxylation or 27-hydroxylation in the metabolism of cholesterol, oxysterols (side chain-hydroxylated derivatives of cholesterol) and vitamin D₃. The enzymes studied belong to the cytochrome P450 enzyme families CYP7 and CYP27.</p><p>The study describes purification of a cytochrome P450 enzyme fraction active in 7α-hydroxylation of 25-hydroxycholesterol, 27-hydroxycholesterol, dehydroepiandrosterone and pregnenolone from pig liver microsomes. Peptide sequence analysis indicated that this enzyme fraction contains an enzyme belonging to the CYP7B subfamily. The purified enzyme was not active towards cholesterol or testosterone. Purification and inhibition experiments suggested that hepatic microsomal 7α -hydroxylation of 27-hydroxycholesterol and dehydroepiandrosterone involves at least two enzymes, probably closely related.</p><p>The study shows that recombinantly expressed human and rat cholesterol 7α -hydroxylase (CYP7A) and partially purified pig liver cholesterol 7α -hydroxylase are active towards 20(<i>S</i>)-, 24-, 25- and 27-hydroxycholesterol. CYP7A was previously considered specific for cholesterol and cholestanol. The 7α -hydroxylation of 20(<i>S</i>)-, 25-, and 27-hydroxycholesterol in rat liver was significantly increased by treatment with cholestyramine, an inducer of CYP7A. Cytochrome P450 of renal origin showed 7α -hydroxylase activity towards 25- and 27-hydroxycholesterol, dehydroepiaundrosterone and pregnenolone but not towards 20(<i>S</i>)-, 24-hydroxycholesterol or cholesterol. The results indicate a physiological role for CYP7A as an oxysterol 7α -hydroxylase, in addition to the previously known human oxysterol 7α -hydroxylase CYP7B.</p><p>The role of renal sterol 27-hydroxylase (CYP27A) in the bioactivation of vitamin D₃ was studied with cytochrome P450 fractions purified from pig kidney mitochondria. Purification and inhibition experiments and experiments with a monoclonal antibody against CYP27A indicated that CYP27A plays a role in renal 25-hydroxyvitamin D₃ l α -hydroxylation.</p><p>The expression of CYP7A, CYP7B and CYP27A during development was studied. The levels of CYP27A in livers of newborn and six months old pigs were similar whereas the levels of CYP7A increased. The expression of CYP7B varied depending on the tissue. The expression of CYP7B increased with age in the liver whereas the CYP7B levels in kidney showed a marked age-dependent decrease.</p>

<p>Regularity is a key element of maintenance drug treatment; compliance is crucial for treatment success. Once- or twice-daily intake of a drug is always easier to comply with than regimens requiring more frequent dosing. Bronchodilating treatment was used as an example to illustrate how sustained duration of effect can be achieved by two different approaches: oral administration of the terbutaline prodrug bambuterol and inhalation of formoterol. Bioanalytical methods were employed to monitor the kinetic fate of bambuterol and formoterol in plasma, urine, or faeces. Generated terbutaline in plasma was used as a marker of effect for bambuterol. Established clinical laboratory tests were used to assess local and systemic effects of inhaled formoterol compared with salbutamol. </p><p>Recommended doses of bambuterol, 10-20 mg once daily in adults, normally produced plasma concentrations of the active moiety terbutaline within therapeutically relevant limits. Dose proportionality for terbutaline makes dosing with bambuterol predictable. Compared with adults, children should be given higher doses than indicated by their lower body weight. Pharmacokinetic analysis indicated that absorption of bambuterol was slow and multi-phasic and that slow biotransformation to terbutaline occurred both presystemically and systemically. </p><p>Systemically circulating formoterol was rapidly eliminated, the inactive (S;S)-formoterol more rapidly than the active (R;R)-formoterol. An inactive phenol glucuronide was the main metabolite, and a previously unknown sulphate metabolite was discovered. Duration of systemically mediated cardiovascular or metabolic side-effects of inhaled formoterol seemed not to differ from those of an inhaled systemically equieffective dose of salbutamol. There was a trend suggesting that the magnitude of systemic side-effects may be less pronounced after inhalation of formoterol compared with a locally equieffective dose of inhaled salbutamol. </p><p>Both approaches to sustaining stimulation of β₂-adrenoceptors have their pros and cons. Bambuterol can be dosed orally once daily, but full effect is reached slowly. The effect of formoterol is reached within a few minutes, but administration must occur via the lungs, often twice daily. Both treatments, however, give 24-h symptom relief during regular treatment.</p>

<p>The heptapeptide SP_1-7, a metabolite of the neuroactive peptide substance P (SP), is suggested to play a role in opioid addiction and memory function. These two dimensions are known to involve dopamine and glutamate transmissions mediated through dopamine receptors and N-methyl-D- aspartate (NNMA) receptors, respectively. Research on interactions between SP_1-7 and these two neurotransmitter systems may therefore be of importance to increase our understanding of the mechanisms behind opioid tolerance and dependence as well as memory processes. New knowledge in this area may lead to the discovery of new therapeutic routes for treatment of opioid addiction and other neuropsychiatric disorders, as well. </p><p>Studies described in this thesis include investigation of adaptive changes during morphine tolerance and withdrawal in brain levels of SP_1-7 and in the activity of substance P endopeptidase (SPE), an enzyme responsible for the generation of this fragment. In morphine tolerant and abstinent rats, the SP_1-7 level and SPE activity were significantly increased in discrete areas of the brain, which are crucial for the development of opioid tolerance and dependence. Furthermore, significant correlations between the SPE activity and some morphine withdrawal signs were observed. This finding was indicative of an endogenous modulatory mechanism involving both the enzyme and its active peptide product. </p><p>The effects of SP_1-7 on the expression of morphine withdrawal and its interaction with dopaminergic pathways were examined in this thesis by behavioural tests, microdialysis as well as Northern blot and autoradiography techniques. Pre-treatment of morphine dependent rats with SP_1-7 was found to stimulate dopamine release in nucleus accumbens and to inhibit the intensity of withdrawal behaviours. It was further shown to regulate both the dopamine D2 receptor gene transcript and the density of dopamine receptor proteins in mesolimbic dopamine pathways, confirming an interaction between SP_1-7 and the dopamine system. </p><p>The influence of SP_1-7 on glutamate transmission was investigated in morphine naive rats. The expression of the gene transcripts of the NMDA receptor subunits NR1, NR2A and NR2B was regulated in several brain regions involved in opioid withdrawal reactions and memory functions. The result is consistent with a possible decrease glutamate transmission in these areas. </p><p>It was concluded that SP_1-7 may function as an endogenous modulator of the expression of opioid withdrawal by influencing both dopaminergic and glutamatergic transmission. </p>

<p>A cytochrome P450 enzyme in pig kidney that catalyzes the hydroxylations of vitamin D₃ and C₂₇-sterols was cloned. DNA sequence analysis of the cDNA revealed that the enzyme belongs to the CYP27 family. The recombinant kidney CYP27A enzyme catalyzed the 25-hydroxylation of vitamin D₃ and the 27-oxygenation of C₂₇-sterols. It was shown that human embryonic kidney cells express CYP27A mRNA and are able to catalyze the same reactions. Microsomal vitamin D₃ 25-hydroxylase (CYP2D25), purified from pig liver, converted vitamin D₃ into 25- hydroxyvitamin D₃ in substrate concentrations which are within the physiological range. The enzyme also converted tolterodine, a substrate for CYP2D6, into its 5-hydroxymethyl metabolite. RT-PCR experiments revealed that CYP2D25 mRNA is expressed not only in liver and kidney but also in other organs. Experiments with human liver microsomes and recombinant human CYP2D6 indicate that the microsomal 25-hydroxylation of vitamin D₃ in human liver is catalyzed by an enzyme different from CYP2D6. Five residues in SRS-3 of CYP2D25 were simultaneously mutated to the equivalent residues in CYP2D6, an enzyme not active in 25-hydroxylation. Both wild-type and mutated CYP2D25 were expressed in the Saccharomyces cerevisiae W(R) strain. The 25-hydroxylase activity of recombinant mutant CYP2D25 was completely lost whereas the activity toward tolterodine remained unaffected. These results indicate that residues in SRS-3 of CYP2D25 are important determinants for its function in vitamin D₃ metabolism. A cDNA homologous with the hepatic CYP2D25 was cloned from pig kidney. The enzyme purified from pig kidney and the recombinant enzyme expressed in COS cells catalyzed 25-hydroxylation of vitamin D₃ and, in addition, lα-hydroxylation of 25-hydroxyvitamin D₃. Immunohistochemistry experiments indicate that CYP2D25 is expressed almost exclusively in the cells of cortical proximal tubules. The expression of CYP2D25 in kidney, but not in liver, was much higher in the adult pig than in the newborn. The results imply that CYP2D25 has a biological role in kidney. </p><p>Results from experiments with inhibitors in primary cultures of porcine hepatocytes suggest that both CYP2D25 and CYP27A1 contribute to the total 25-hydroxylation in hepatocytes and are equally important in the bioactivation of vitamin D₃. Phenobarbital treatment increased the CYP2D25 mRNA levels but did not affect the CYP27A1 mRNA levels. The rate of 25-hydroxylation by phenobarbital-treated hepatocytes was markedly reduced. These results show that primary cultures of porcine hepatocytes are suitable as a model to study the metabolism of vitamin D₃ and the regulation of the CYP enzymes involved in the 25-hydroxylation o vitamin D₃. </p>

<p>There is evidence of a link between peripheral signals of energy homeostasis and reward mechanisms. A hypothesis was formulated that the melanocortin (MC) system is part of this link. The hypothesis was tested by a series of receptor expression and neurochemical studies in rats.</p><p>A novel autoradiographical method was developed for quantification of MC₃ and MC₄ receptors in the rat brain. MC receptor levels were studied in three rat models combining underweight with an increased susceptibility for alcohol and drug consumption: alcohol preferring AA rats, nandrolone treated rats and food restricted rats. The results showed that MC receptors were differentially regulated in different brain regions in the three models. Interestingly, in all models the MC₃ receptor was down-regulated in the nucleus accumbens (ACB), a region involved in the reward system, thus possibly linking the MC₃ receptor to reward mechanisms.</p><p><i>In vivo</i> microdialysis indicated that the MC peptide α-MSH stimulates transmission of dopamine (DA), an important mediator of reward, in the ACB via an MC receptor mediated mechanism. Moreover, chronic treatment with an MC receptor agonist resulted in increased dopamine D₂ receptor levels in the VTA and decreased D₁ receptor levels in the ACB. The results show that melanocortins may have an important role for both acute and long term regulation of DA transmission.</p><p>These results support the hypothesis that the melanocortins may serve as an important link between reward and body weight homeostasis. The results add to the understanding of the frequent co-morbidity of eating disorders and substance abuse, and the similarities between eating disorders and addiction. The more detailed understanding of the relationship between metabolic status and reward may also generate novel possibilities to treat eating disorders as well as addictive conditions.</p>

<p>Misuse of anabolic-androgenic steroids (AAS), is attributed to elite athletes and body builders. The attentive involvement of AAS in acts of violence seen in society has raised interest to evaluate the importance of social, psychological and neurobiological mechanisms that underlie the psychiatric states associated with onset of controlled misuse, its maintenance, and via abuse its transition to addiction. The objective of this thesis is to examine whether misuse of AAS shares mechanisms with epidemiological and neurobiological models of psychotropic substances. </p><p>Epidemiological studies through a survey conducted in Uppsala, Sweden, suggest that misuse of doping agents, specifically AAS, has extended also to include adolescent males taking these agents in order to improve muscle mass, enhance sports performance, become intoxicated, braver, and because it is fun to try. Intake of AAS is in a subgroup highly connected to misuse of psychotropic substances. The adolescent AAS profile is highlighted in a multivariate model positing the factors high immigrant status, perceived average/bad school achievement, truancy, average/low self-esteem, strength training, heavy alcohol consumption and use of prescription tranquillisers to be independently associated with lifetime misuse. </p><p>Neurobiological studies indicate that chronic treatment with supra-therapeutic doses of the AAS nandrolone, significantly affects dopamine receptor density in the male rat brain and the corresponding gene transcripts in the mesocorticolimbic and nigrostriatal dopamine systems, in brain areas of importance for hedonia, reward-related learning, incentives and motoric behaviours. Identical treatment regimen affects the density of serotonin receptors in regions regulating anxiety, aggression, cognitive functions, impulsivity and its associated loss of inhibitory control. These alterations may reflect aversive conditions that could be linked to severe alleostatic states of addiction following chronic continuous "binge" intoxications of addictive drugs.</p><p>Thus, the AAS profile of misuse shares similarities with mechanisms of psychotropic substances regarding psychological and social models of onset and maintenance and with respect to AAS-induced neurobiological changes in the brain. This trend is alarming, strengthening the need of prevention and treatment programs targeting the specific subgroups of misusers. </p>

<p>Population pharmacokinetic (PK) – pharmacodynamic (PD) modelling, using nonlinear mixed effects models, is increasingly being applied to obtain PK-PD information in drug development. Covariate modelling, the establishment of relationships between model parameters and patient characteristics, is undertaken to explain PK-PD variability and facilitate dose adjustment decisions, and is consequently an important objective of population PK-PD.</p><p>The aims of this thesis were to increase the efficiency, predictability and robustness of covariate model building by examining in detail a number of aspects related to covariate modelling. The thesis demonstrates that the likelihood ratio (LR) test can be applied with confidence, in the assessment of statistical significance of parameter-covariate relationships (in NONMEM analyses), only if an estimation method appropriate for the data- and error-structure is utilised. Conversely, caution is needed in the interpretation of the LR test when variance or covariance parameters are modelled, since the type I error rate may be severely upward biased if the assumptions of normally distributed residuals and/or enough information in the data are violated. The two stepwise covariate model building procedures, using generalised additive models and NONMEM, were found to perform similarly in the examples examined. However, differences in performance may prevail in other situations, e.g. when sparse sampling precludes reliable individual parameter estimates. Stepwise selection was shown to result in over-estimated covariate effects (selection bias), but the imprecision in the estimates exceeded this bias. Important information about the PK-PD characteristics of a drug is obtainable on the application of covariate models for time-varying covariates that account for differences in variability between and within individuals, or estimate interindividual variability in the covariate effect. The knowledge gained in this thesis will contribute to the development of more predictable and robust covariate models, important both in individualisation of dosage and the development of new drugs.</p>