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. 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. 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. 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.

The heptapeptide SP1-7, 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 SP1-7 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. Studies described in this thesis include investigation of adaptive changes during morphine tolerance and withdrawal in brain levels of SP1-7 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 SP1-7 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. The effects of SP1-7 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 SP1-7 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 SP1-7 and the dopamine system. The influence of SP1-7 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. It was concluded that SP1-7 may function as an endogenous modulator of the expression of opioid withdrawal by influencing both dopaminergic and glutamatergic transmission.

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. 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.

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. 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. In vivo 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. 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.

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. 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. 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. 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.

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. 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.

Diss. (sammanfattning) Linköping : Linköpings universitet, 2005. / Härtill tre uppsatser.

The main objective of the research described in this thesis has been the design and synthesis of inhibitors of the enzyme dihydrofolate reductase (DHFR) intended for local administration and devoid of systemic side-effects. The blocking of the enzymatic activity of DHFR is a key element in the treatment of many diseases, including cancer, bacterial and protozoal infections, and also opportunistic infections associated with AIDS (Pneumocystis carinii pneumonia, PCP). Recent research indicates that the enzyme also is involved in various autoimmune diseases, e.g., rheumatoid arthritis, inflammatory bowel diseases and psoriasis. Many useful antifolates have been developed to date although problems remain with toxicity and selectivity, e.g., the well-established, classical antifolate methotrexate exerts a high activity but also high toxicity. The new antifolates described herein were designed to retain the pharmacophore of methotrexate, but encompassing an ester group, so that they also would serve as substrates for the endogenous hydrolytic enzymes, e.g., esterases. Such antifolates would optimally comprise good examples of soft drugs because they in a controlled fashion would be rapidly and predictably metabolized to non-toxic metabolites after having exerted their biological effect at the site of administration. A preliminary screening of a large series of simpler aromatic esters as model compounds in a biological assay consisting of esterases from different sources was performed. The structural features of the least reactive ester were substituted for the methyleneamino bridge in methotrexate to produce analogues that were chemically stable but potential substrates for DHFR as well as for the esterases. The new inhibitor showed desirable activity towards rat liver DHFR, being only eight times less potent then methotrexate. Furthermore, the derived metabolites were found to be poor substrates for the same enzyme. The new compound showed good activity in a mice colitis model in vivo, but a pharmacokinetic study revealed that the half-life of the new compound was similar to methotrexate. A series of compounds characterized by a high lipophilicity and thus expected to provide better esterase substrates were designed and synthesized. One of these analogues in which three methoxy groups were substituted for the glutamic residue of methotrexate exhibited favorable pharmacokinetics. This compound is structurally similar to another potent DHFR inhibitor, trimetrexate, used in the therapy of PCP (vide supra). The new inhibitor that undergoes a fast metabolism in vivo is suitable as a model to further investigate the soft drug concept.

<p>Catalysis has a profound impact on all living species on the earth. Nature’s catalysts, the enzymes, have the ability to selectively promote a specific bio-chemical transformation, given the required substrate. As well as being highly selective, enzymes enhance the speed of these reactions, helping them to run at temperatures much lower than normally required, i.e. at body temperature. In comparison, reactions used in the production of new materials such as polymers, medicines, fragrances, petrochemicals, etc. are often catalyzed by transition metals. This thesis describes how the selectivity and activity of these catalysts can be influenced via two conceptually different methods: chelation control and microwave heating. The thesis primarily focuses on regio- and stereochemical aspects of the palladium-catalyzed arylation of olefins, i.e. the Heck reaction. Reaction rate enhancement of both palladium and enzyme (polymerase chain reaction [PCR]) catalysis by microwave heating is also discussed. </p><p>Novel chelation-controlled palladium-catalyzed multi- and asymmetric arylations of vinyl ethers were performed, resulting in tetra-substituted olefins as well as chiral quaternary carbon centers with excellent optical purity. In addition, a new synthetic route to diarylated ethanals, relying on a double chelation-controlled regioselective arylation followed by hydrolysis, has been discovered. High temperature conditions, using microwave heating, substantially reduce the reaction time for ligand-controlled asymmetric Heck arylations, while retaining levels of enantioselectivity in most cases. In addition, a potentially useful fast synthetic protocol for the employment of aryl boronic acids in oxidative Heck arylation was developed. Finally, microwave-assisted PCR was described for the first time; this method allows reductions in the run time of 50%.</p>

<p>Human Immunodeficiency Virus (HIV) is the causative agent of Acquired Immune Deficiency Syndrome (AIDS). The C₂-symmetric HIV-1 protease is one of the prime targets for chemotherapy in the treatment of the HIV infection. Inhibition of HIV-1 protease leads to immature and non-infectious viral particles. Design and synthesis of a number of C₂-symmetrical C-terminal duplicated HIV-1 protease inhibitors and subsequent biological evaluation is presented in this thesis.</p><p>A versatile three step synthetic route has been developed using a carbohydrate as an inexpensive chiral starting material thus allowing inhibitors with the desired stereochemistry to be obtained. By this efficient method a series of tailor-made P2/P2' modified inhibitors were synthesized, and these were evaluated on purified HIV-1 protease and in HIV-1 infected cell assays. Highly active HIV-1 protease inhibitors were identified among the tested compounds. Analyses of the X-ray crystal structures of two of the most active compounds, as complexes with the protease, guided the further design of P1/P1' elongated inhibitors. Substitutions in the para-position of the P1/P1' benzyl groups were promoted efficiently by microwave-irradiated of palladium-catalyzed reactions. Particular modifications in the P1/P1' region of the inhibitors resulted in a 40-fold increase of the anti-viral activity on HIV-1 infected cells. Furthermore, a fast, efficient, and general one-pot microwave enhanced synthesis protocol for transformations of organo-bromides to tetrazoles was developed and applied on the inhibitor scaffold. Attachment of linker molecules to the P1/P1' benzyl groups of one inhibitor was used to develop of sensitivity enhancer tools in surface plasmon resonance biosensor assays. These new assays enable the evaluation of low-molecular weight compounds as HIV-1 protease inhibitors.</p>