Effects of some 5-hydroxytryptamine and related ligands in anxiety models

Drugs acting at 5-HT receptors were evaluated on three animal models of anxiety. On the elevated X-maze test the majority of 5-HT1 agonists were found to be anxiogenic. However, ipsapirone was anxiolytic and buspirone and gepirone were inactive. The 5-HT2 agonist DOI and the 5-HT2 antagonist ritanserin were anxiolytic while ICI 169,369, a 5-HT2 antagonist was inactive. All 5-HT3 antagonists tested were inactive in this test, while the indirect serotomimetics zimeldine and fenfluramine were anxiogenic. Neither beta-adrenoceptor agonists nor antagonists had reproducible effects on anxiety in this model. Combined beta-1/beta-2 adrenoceptor antagonists reversed the anxiogenic effects of 8-OH-DPAT while selective beta-1 or beta-2 antagonists did not. On the social interaction model the 5-HT1 agonists 8-OH-DPAT, RU 24969 and 5-MeODMT were anxiogenic and ipsapirone was anxiolytic. The 5-HT2 agonist DOI and the beta-adrenoceptor- and 5-HT- antagonist pindolol were anxiolytic, while the 5-HT2 and 5-HT3 antagonists were inactive. In the marble burying test, the 5-HT upake inhibitors zimeldine, fluvoxamine, indalpine and citalopram, the 5-HT1B/5-HT1C agonists mCPP and TFMPP and the 5-HT2/5-HT1C agonist DOI reduced marble burying without affecting locomotor activity. 5-HT1A agonists and the 5-HT2 and 5-HT3 antagonists were without effect. Lesions of the dorsal raphe nucleus reversed the anxiogenic effects of 8-OH-DPAT in the X-maze model. The implication of these results for the understanding of the pharmacology of 5-HT in anxiety is discussed.

The effects of some neurotoxins on tetrahydrobiopterin metabolism

Previous studies in man have shown that following dosing with L--3,4-dihydroxyphenylalanine (L-DOPA) and cotrimoxazole, plasma biopterins were raised. By analogy with dihydropteridine reductase deficient children in whom plasma biopterins are greatly elevated and the observations that these preparations were dihydropteridine reductase inhibitors, it was assumed that these raised plasma levels were due to increased efflux from tissues which resulted in tissue depletion of biopterins. In some human disease states such as senile dementia of the Alzheimer type lowered plasma biopterins were observed; by analogy with tetrahydrobiopterin synthesis deficient children these reduced plasma biopterins were attributed to lowered tetrahydrobiopterin synthesis and concomitant low tissue biopterin levels. Because of ethical considerations it was not possible to measure directly the tissue biopterins changes in either case. The Wistar rat was used as a model for human tetrahydrobiopterin metabolism, since tissues not normally accessible for study in humans, such as the brain and liver, could be examined for their effects on tetrahydrobiopterin metabolism after administration of the various agents. Plasma total biopterins in normal conditions were found to be much higher than in healthy humans. The elevation of plasma total biopterins concentration following the administration of dihydropteridine reductase inhibitors to humans, such as L-DOPA and cotrimoxazole was not observed in the rat. However, the administration of inhibitors of de novo tetrahydrobiopterin biosynthesis, such as diaminohydroxypyrimidine (DAHP) and bromocriptine was shown to decrease plasma biopterins concentration. In general, hepatic biopterins were decreased after administration of both dihydropteridine reductase inhibitors and de novo biosynthesis inhibitors. Drugs which are direct (bromocriptine) or indirect (L-DOPA and Sinemet Plus) agonists at dopamine receptors were investigated and were shown to decrease hepatic total biopterins concentration, but had no effect on brain biopterins. Bromocriptine was demonstrated as a potent inhibitor of de novo tetrahydrobiopterin biosynthesis in vivo and in vitro. Cotrimoxazole decreased brain tetrahydrobiopterin concentration. DAHP was effective in causing hyperphenylalaninaemia due to tetrahydrobiopterin deficiency in the rat. p-hydroxyphenylacetate was shown to be an effective inhibitor of dihydropteridine reductase in vivo. Phenylacetate administration had no observable effect on tetrahydrobiopterin metabolism, but did cause tyrosinaemia. It is proposed that scopolamine reduces tetrahydrobiopterin turnover. Lead and aluminium exposure caused deranged tetrahydrobiopterin metabolism. Aluminium, but not lead decreased brain choline acetyltransferase activity. Phenylalanine loading in normal human subjects was followed by an elevation in plasma biopterins which was not observed after tyrosine loading. Plasma N : B ratios correlated well with VEP latencies after tyrosine loading, but not after phenylalanine loading in healthy subjects. The use of derived pterin measurements as an indicator of tetrahydrobiopterin turnover or tetrahydrofolate status is discussed in the text.

Drug repurposing screen identifies Foxo1-dependent angiopoietin-2 regulation in sepsis

Objective: The recent withdrawal of a targeted sepsis therapy has diminished pharmaceutical enthusiasm for developing novel drugs for the treatment of sepsis. Angiopoietin-2 is an endothelial-derived protein that potentiates vascular inflammation and leakage and may be involved in sepsis pathogenesis. We screened approved compounds for putative inhibitors of angiopoietin-2 production and investigated underlying molecular mechanisms. Design: Laboratory and animal research plus prospective placebo-controlled randomized controlled trial (NCT00529139) and retrospective analysis (NCT00676897). Setting: Research laboratories of Hannover Medical School and Harvard Medical School. Patients: Septic patients/C57Bl/6 mice and human endothelial cells. Interventions: Food and Drug Administration-approved library screening. Measurements and Main Results: In a cell-based screen of more than 650 Food and Drug Administration-approved compounds, we identified multiple members of the 3-hydroxy-3-methyl-glutaryl-CoA reductase inhibitor drug class (referred to as statins) that suppressed angiopoietin-2. Simvastatin inhibited 3-hydroxy-3-methyl-glutaryl-CoA reductase, which in turn activated PI3K-kinase. Downstream of this signaling, PI3K-dependent phosphorylation of the transcription factor Foxo1 at key amino acids inhibited its ability to shuttle to the nucleus and bind cis-elements in the angiopoietin-2 promoter. In septic mice, transient inhibition of angiopoietin-2 expression by liposomal siRNA in vivo improved absolute survival by 50%. Simvastatin had a similar effect, but the combination of angiopoietin-2 siRNA and simvastatin showed no additive benefit. To verify the link between statins and angiopoietin-2 in humans, we performed a pilot matched case-control study and a small randomized placebo-controlled trial demonstrating beneficial effects on angiopoietin-2. Conclusions: 3-hydroxy-3-methyl-glutaryl-CoA reductase inhibitors may operate through a novel Foxo1-angiopoietin-2 mechanism to suppress de novo production of angiopoietin-2 and thereby ameliorate manifestations of sepsis. Given angiopoietin-2's dual role as a biomarker and candidate disease mediator, early serum angiopoietin-2 measurement may serve as a stratification tool for future trials of drugs targeting vascular leakage.

Non-classical inhibitors of dihydrofolate reductase

This thesis comprises two main objectives. The first objective involved the stereochemical studies of chiral 4,6-diamino-1-aryl-1,2-dihydro-s-triazines and an investigation on how the different conformations of these stereoisomers may affect their binding affinity to the enzyme dihydrofolate reductase (DHFR). The ortho-substituted 1-aryl-1,2-dihydro-s-triazines were synthesised by the three component method. An ortho-substitution at the C6' position was observed when meta-azidocycloguanil was decomposed in acid. The ortho-substituent restricts free rotation and this gives rise to atropisomerism. Ortho-substituted 4,6-diamino-1-aryl-2-ethyl-1,2-dihydro-2-methyl-s-triazine contains two elements of chirality and therefore exists as four stereoisomers: (S,aR), (R,aS), (R,aR) and (S,aS). The energy barriers to rotation of these compounds were calculated by a semi-empirical molecular orbital program called MOPAC and they were found to be in excess of 23 kcal/mol. The diastereoisomers were resolved and enriched by C18 reversed phase h.p.l.c. Nuclear overhauser effect experiments revealed that (S,aR) and (R,aS) were the more stable pair of stereoisomers and therefore existed as the major component. The minor diastereoisomers showed greater binding affinity for the rat liver DHFR in in vitro assay. The second objective entailed the investigation into the possibility of retaining DHFR inhibitory activity by replacing the classical diamino heterocyclic moiety with an amidinyl group. 4-Benzylamino-3-nitro-N,N-dimethyl-phenylamidine was synthesised in two steps. One of the two phenylamidines indicated weak inhibition against the rat liver DHFR. This weak activity may be due to the failure of the inhibitor molecule to form strong hydrogen bonds with residue Glu-30 at the active site of the enzyme.

Design and synthesis of inhibitors of dihydrofolate reductase

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