Thesis (doctoral)--Lund University, 1994.

Thesis (doctoral)--Lund University, 1994.

The adenosine system has become a promising target for the treatment of schizophrenia due to its unique relationship with dopamine D2 receptors. Dopamine D2 receptors display heightened sensitivity in schizophrenia, and inhibition of these receptors has been shown to alleviate some of the psychotic symptoms of the disorder. Inhibition of adenosine A(2A) receptors has been shown to decrease dopamine D2 receptor sensitivity, making this receptor a potential target for treatment of the disorder. This effect occurs because adenosine A(2A) receptors form a mutually inhibitory heterodimeric complex with dopamine D2 receptors. The present study looked at the effects of an adenosine agonist on prepulse inhibition (PPI) and cyclic-AMP response binding element protein (CREB) concentrations in the nucleus accumbens (NAc) using a rodent model of schizophrenia (NQ model) that presents with increased D2 receptor sensitivity. Results showed that the A(2A) agonist was effective in improving PPI in NQ-treated animals. The agonist was also effective in reducing increased CREB concentrations in the NAc of NQ-treated animals to control levels. The effectiveness of the agonist suggests that the adenosine system may be a viable target for the treatment of some of the psychotic symptoms associated with schizophrenia.

Pyrrolobenzodiazepine (PBD) derivatives interact with the minor-groove of DNA to form mono-adducts (monomers) or cross-links (dimers). They show remarkable activity in vitro and in vivo in a wide range of tumour types and one dimer, SJG-136 is currently in clinical development. Preclinical studies have shown that SJG-136 is a P-gp substrate limiting its anti-tumour activity. The work presented in this thesis identifies key physicochemical properties influencing both the interaction of PBDs with ABC transporters P-gp, MRP1 and BCRP and their growth inhibitory potency. A testable hypothesis for further optimisation of PBDs is proposed. The biological activity of 4 dimers and 12 monomers was assessed using several in vitro models presenting differential expression of ABC transporters. Biological endpoints were the growth inhibitory effect determined using a sulforhodamine B assay and γ-H2AX foci formation. In addition PBD transport was evaluated using a Caco-2 transwell assay. P-gp substrate specificity was restricted to dimers. The MW, the number of (N+O) atoms (>8), a polar surface area (>75 Ǻ2) and hydrogen bonding energy (>10) could discriminate substrates among the PBDs. P-gp polymorphism was also evaluated. The mutation in position 2677 (G/T) was associated with reduced sensitivity to the PBDs. When combined mutations in position 3435/2677 were linked, the transporter abrogated this apparent gain of function. The impact of MRP1 was identified for all dimers and 1/12 monomers. In addition, the cooperative role of glutathione in the resistance mediated by MRP1 to the PBDs was revealed. The presence of a carbonyl moiety at the extremity was shown to discriminate the 7 substrate for MRP1 among the monomers. A structure-activity-relationship study showed that negatively charged (N+O) atoms and a greater number of aromatic rings confer greater dependency to BCRP. BCRP polymorphism was also evaluated. The T482 mutant was associated with an increase in drug transport. The cytotoxicity of the PBDs correlated to the interaction of the DNA as measured by ΔTm. Compounds, being non surface active, with a greater polar surface area and number of aromatic rings and a lower solvent accessible surface area were associated with a greater cytotoxicity. Van-der-waals energy and the electrostatic forces were identified in silico as predictable features involved in the DNA binding. New PBDs were designed and were predicted to be associated with a greater affinity for DNA and with minimal interaction with ABC transporters.

No description available.

No description available.

No description available.

No description available.

No description available.

Pulmonary fibrosis (PF) is a serious lung disease, as its life expectancy is only 3-5 years upon occurrence and more than 50 % of the cases are idiopathic, i.e., unknown cause. Two drugs, pirfenidone (PIR) and nintedanib, have recently been approved; however, their efficacies are moderate without evidence of prolonged survival. While this is primarily due to our insufficient knowledge about key PF pathogenesis, inductions of oxidative stress and transforming growth factor-b1 (TGF-b1) have been suggested in PF lungs. Hence, anti-oxidative melatonin (MEL) and curcumin (CUR) have been studied yet their efficacies remain moderate without clear understanding about the mechanisms of action. Accordingly, this project hypothesized that a novel hybrid molecule of MEL and CUR, AM24, was a more potent inhibitor against oxidative stress and TGF-b1 induced PF pathobiologic events than MEL or CUR, so that its pulmonary delivery enabled therapeutic intervention in an animal model of PF. Free radical scavenging activity and various in vitro lung cell-based anti-fibrotic activities of AM24 were determined and compared with those of MEL and CUR as well as their admixture (MEL+CUR) and PIR. Pulmonary administration of AM24 was then examined for therapeutic intervention in a rat model of bleomycin (BLM)-induced experimental PF. AM24 was equipotent to MEL, but less potent than CUR in the hydrogen peroxide-induced free radical (ABTS) scavenging assay, ranked with the half-maximal inhibitory concentration (IC50) of 25.7, 32.0 and 11.4 uM, respectively. However, in the in vitro human lung fibroblast systems, AM24 was shown to be more potent than MEL or CUR and notably than MEL+CUR or PIR in the TGF-b1 induced 1) collagen synthesis by the picrosirius red assay, 2) proliferation by the MTT assay; and 3) differentiation to myofibroblast by western blot analysis of a myofibroblast marker, a-smooth muscle actin (a-SMA). In detail, at 10 uM, AM24 inhibited TGF-b1 induced 1) collagen synthesis by 90 %; 2) proliferation by ~72 %; and 3) differentiation to myofibroblast completely, while MEL, CUR, MEL+CUR and PIR resulted in 30-55 % or insignificant inhibition. In addition, in the in vitro human lung alveolar epithelial cell system, AM24 at 10 uM almost completely inhibited TGF-b1 induced epithelial-mesenchymal transition (EMT), as measured with western blot expressions of an epithelial marker, E-cadherin, and a mesenchymal marker, vimentin. Again, MEL, CUR, MEL+CUR and PIR exerted much less inhibitory activities. Hence, all these results consistently suggested that AM24 was a unique hybrid molecule of MEL and CUR and possessed highly potent anti-fibrotic activities in addition to the free radical scavenging activity. AM24 was then examined for therapeutic intervention in an in vivo rat model of BLM-induced PF. BLM was orotracheally spray-dosed to the lungs at 0.6 mg/kg on day 1 to develop experimental PF in 14 days. Lung administrations of AM24 at 0.1 mg/kg commenced at 6 hours of BLM induction on day 1 and continued thrice weekly over two weeks. Functional treadmill exercise endurance was measured on day 12 and 15; and lungs were harvested upon sacrifice on day 16. Overall, AM24 showed significant intervention activities as follows: 1) exercise endurance was reduced only ~20%, much lower than 78% of the untreated PF rats; 2) reduced fibrotic tissue area and alveolar structural destruction were seen by histological examinations; and 3) lung’s induced collagen deposition was inhibited by ~78 %. However, unlike the literature, the lung’s TGF-b1, PCNA (a cell proliferation marker), and a-SMA (a differentiation marker), were not largely induced in the BLM-induced PF model, so that the intervention activities of AM24 to these markers were not clearly shown. In contrast, induced EMT was seen in the BLM-induced model, represented by increased mesenchymal marker, vimentin, and by decreased epithelial marker, E-cadherin; and AM24 appeared to counter this induced EMT. Accordingly, while the BLM-induced PF model may need further optimizations for clearer pathogenic changes, AM24 exerted certain degree of in vivo efficacies with a lung dose of 0.1 mg/kg, which was much lower than the effective doses of MEL, CUR, PIR and nintedanib seen in the literature with BLM induced PF model. In conclusion, this thesis study has provided an early proof-of-concept for AM24, a novel MEL-CUR hybrid molecule, being potently anti-oxidative and anti-fibrotic in the in vitro lung cell-based assessments. As a result, AM24 enabled therapeutic intervention just with a lung dose of 0.1 mg/kg in the BLM-induced rat model of experimental PF.