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OBJECTIVE: To increase the potential of membrane-acting peptides as possible novel drug-like compounds by increasing lipophilicity and thereby enhancing membrane affinity. DESIGN: An Fmoc-protected enantiomerically pure lipophilic amino acid (Fmoc-Lad-OH), which contains a nine carbon atom hydrophobic side chain, was designed. Fmoc-Lad-OH can be introduced into any peptide sequence using standard solid phase peptide synthesis to increase the lipophilicity of a peptide without sacrificing important polar segments of a peptide like for instance the N and C-termini. The antimicrobial decapeptide anoplin was chosen as a model peptide to test the hypothesis. METHODS: Fmoc-Lad-OH was prepared via organic synthesis and incorporated into the anoplin peptide sequence using solid-phase peptide synthesis followed by reversed-phase HPLC purification. Biological activity was evaluated using microtiter dilution bacterial growth assays, haemolytic assays and membrane vesicle leakage experiments. RESULTS: All three lipophilic analogues show a dramatic increase in antimicrobial activity: up to 4-8 times better for Escherichia coli (Gram-negative] and over one order of magnitude for Staphylococcus aureus (Gram-positive) compared to anoplin. Although the haemolytic activity was increased for the lipophilic analogues, the concentration at which 50% lysis will occur (EC50) was still one order of magnitude higher than the determined MICs. In the membrane vesicle leakage experiments the lipophilic analogues showed a higher lytic activity than anoplin, in agreement with the observed MIC values. CONCLUSION: Introduction of Lad into anoplin clearly showed a positive effect, which suggests that Fmoc-Lad-OH could be used as a general approach to increase membrane affinity of membrane-acting peptides.

OBJECTIVE To investigate the relationship between ajmaline serum levels (pharmacokinetics, PK) and changes in the ST segment on the electrocardiogram (pharmacodynamics, PD) in patients with suspected Brugada syndrome (BS). DESIGN AND METHODS We determined the PK profile of ajmaline in 34 patients referred to the cardiology clinic for diagnosis of Brugada syndrome. The PD profile was derived from the ST elevations on the electrocardiograms of 26 patients during the diagnostics of BS. The PK and PD data were examined and fitted into non-linear mixed effect modelling (NONMEM 7). The final PK model was evaluated with data from 8 newly included patients. RESULTS Pharmacokinetics of ajmaline was best described with a two compartment model and the PK/PD data as a direct effect with a linear relationship between ajmaline serum levels and ST segment elevation. In BS-positive patients the baseline ST segment was 0.06 mV (rsd 35%) with a slope of 0.03 mV•mL/mg (rsd 94%). In BS-negative patients no association was found between ajmaline serum levels and ST segment elevation. CONCLUSION Pharmacokinetics of ajmaline in patients with suspected BS is best described with a two compartment model with linear elimination. A direct association between ajmaline serum levels and ST segment elevation in BS-positive patients was found. No association was found between ajmaline serum levels and ST segment elevation in BSnegative patients. In future studies this model can be used to examine the effects of co-variates in order to optimize the use of ajmaline in the diagnosis of BS

OBJECTIVE: To increase the potential of membrane-acting peptides as possible novel drug-like compounds by increasing lipophilicity and thereby enhancing membrane affinity. DESIGN: An Fmoc-protected enantiomerically pure lipophilic amino acid (Fmoc-Lad-OH), which contains a nine carbon atom hydrophobic side chain, was designed. Fmoc-Lad-OH can be introduced into any peptide sequence using standard solid phase peptide synthesis to increase the lipophilicity of a peptide without sacrificing important polar segments of a peptide like for instance the N and C-termini. The antimicrobial decapeptide anoplin was chosen as a model peptide to test the hypothesis. METHODS: Fmoc-Lad-OH was prepared via organic synthesis and incorporated into the anoplin peptide sequence using solid-phase peptide synthesis followed by reversed-phase HPLC purification. Biological activity was evaluated using microtiter dilution bacterial growth assays, haemolytic assays and membrane vesicle leakage experiments. RESULTS: All three lipophilic analogues show a dramatic increase in antimicrobial activity: up to 4-8 times better for Escherichia coli (Gram-negative] and over one order of magnitude for Staphylococcus aureus (Gram-positive) compared to anoplin. Although the haemolytic activity was increased for the lipophilic analogues, the concentration at which 50% lysis will occur (EC50) was still one order of magnitude higher than the determined MICs. In the membrane vesicle leakage experiments the lipophilic analogues showed a higher lytic activity than anoplin, in agreement with the observed MIC values. CONCLUSION: Introduction of Lad into anoplin clearly showed a positive effect, which suggests that Fmoc-Lad-OH could be used as a general approach to increase membrane affinity of membrane-acting peptides.

OBJECTIVE To investigate the relationship between ajmaline serum levels (pharmacokinetics, PK) and changes in the ST segment on the electrocardiogram (pharmacodynamics, PD) in patients with suspected Brugada syndrome (BS). DESIGN AND METHODS We determined the PK profile of ajmaline in 34 patients referred to the cardiology clinic for diagnosis of Brugada syndrome. The PD profile was derived from the ST elevations on the electrocardiograms of 26 patients during the diagnostics of BS. The PK and PD data were examined and fitted into non-linear mixed effect modelling (NONMEM 7). The final PK model was evaluated with data from 8 newly included patients. RESULTS Pharmacokinetics of ajmaline was best described with a two compartment model and the PK/PD data as a direct effect with a linear relationship between ajmaline serum levels and ST segment elevation. In BS-positive patients the baseline ST segment was 0.06 mV (rsd 35%) with a slope of 0.03 mV•mL/mg (rsd 94%). In BS-negative patients no association was found between ajmaline serum levels and ST segment elevation. CONCLUSION Pharmacokinetics of ajmaline in patients with suspected BS is best described with a two compartment model with linear elimination. A direct association between ajmaline serum levels and ST segment elevation in BS-positive patients was found. No association was found between ajmaline serum levels and ST segment elevation in BSnegative patients. In future studies this model can be used to examine the effects of co-variates in order to optimize the use of ajmaline in the diagnosis of BS

The development ofa new drug often takes more than ten years. Until recently, researchers were obliged to carry out experimental research on the efficacy of variants of those molecules of which the effect was known. It took a long time before a structure-activity relationship could be established. Currently, the full effect of the stormy developments in the Life Sciences, including the sequencing of the human genome, has not yet been fully felt in clinical practice. However, in laboratories researching medicinal products, the technology and the strategies being applied are changing drastically. Understanding disease processes and pharmaconprotein interactions at molecular level make it possible to develop biologically active pharmaceuticals using efficient developmental techniques involving crystallography and computer models. At the same time it is becoming clear that more research is necessary in order to understand the pharmacokinetic characteristics and toxicological activity of medicines under development. This second phase in the development of medicinal products too, is being strongly stimulated by the recent changes.

Background: We investigated the combination of selective serotonin reuptake inhibitors (SSRIs) with the beta -adrenoceptor/serotonin 1A (5-HT1A) antagonist pindolol, based on the concept that 5-HT1A receptor blockade would eliminate the need for desensitization of presynaptic 5-HT1A receptors and therefore hasten the onset of action and improve the efficacy of SSRIs. However, since pindolol plasma levels after 2.5 mg three times a day are about 60 nmol/L, and the K-i for the 5-HT1A receptor is 30 nmol/L, it is questionable whether pindolol levels in the brain would be sufficient to antagonize 5-HT1A receptors. Using microdialysis in the guinea pig, we correlated brain and plasma levels of pindolol with its capability of augmenting paroxetine-induced increases in brain 5-HT levels. In addition, central beta -receptor antagonism of pindolol was studied by investigating blockade of beta -agonist-induced increases in brain cyclic adenosine monophosphate (cAMP) formation. Methods: Using microdialysis and jugular vein catheterization, we studied the ability of systemically administered pindolol to antagonize central 5-HT1A and beta -adrenoceptors, while simultaneously monitoring pindolol plasma and brain concentrations. Results: Augmentation of paroxetine-induced increases in extracellular 5-HT levels in the ventral hippocampus was only observed at steady stare plasma levels exceeding 7000 nmol/L (concurrent brain levels 600 nmol/L). In contrast, antagonism of beta -agonist-induced increases of brain cAMP levels was already observed at pindolol plasma levels of 70 nmol/L (concurrent brain levels

A series of new analogues of N-[4-methoxy-3-(4-methylpiperazin-1-yl)phenyl] 2'-methyl-4'-(5-methyl-1,2,4-oxadiazol-3-yl)biphenyl-4-carboxamide (1; GR127935) as potent and selective 5-HT1B/1D antagonists were synthesized and evaluated pharmacologically. Their receptor binding profiles were comparable to that of 1. The 1,3,4-oxadiazole isomer 2 and the 4'-aminocarbonyl and 4'-amidinyl analogues (9 and 10) of 1 had higher affinities at the rat 5-HT1B receptor (IC50 = 0.93, 1.3, and 0.5 nM, respectively) and calf 5-HT1D receptor (IC50 = 37, 10, and 3 nM, respectively) than did 1 (1.6 and 52 nM for rat 5-HT1B and calf 5-HT1D receptors, respectively). In the functional in vitro testing of 5-HT1B/1D antagonistic properties, 2, 9, 18, 11b (O-demethylated derivative of 2), 13a (O-methylsulfonyl analogue of 2), and 16 (which differs from 2 with a sulfonamide linker) showed more pronounced effects in the K+-induced 5-HT release in the cortex of guinea pig than did 1 and 3 (SB224289). Compounds 2, 9, and 10 were equally potent as 1 in rabbit saphenous vein model (pA(2) > 9). A biochemical study of 2 with in vivo microdialysis in the rat brain showed that it is capable of augmenting citalopram (a selective serotonin reuptake inhibitor, SSRI) induced 5-HT release in rat ventral hippocampus, while preventing the decrease in acetylcholine release elicited by citalopram administration. The molecular structure of 2 was determined by single-crystal X-ray analysis. The log P and log D values of these compounds were calculated. This study contributes to the SAR study of N-piperazinylphenyl biphenylcarboxamides as selective and potent 5-HT1B/1D antagonists.