Isabelle Berrebi-Bertrand

BF2.649 [1-{3-[3-(4-Chlorophenyl)propoxy]propyl}piperidine, hydrochloride], a nonimidazole inverse agonist/antagonist at the human histamine H3 receptor: Preclinical pharmacology.

Histamine H3 receptor inverse agonists are known to enhance the activity of histaminergic neurons in brain and thereby promote vigilance and cognition. 1-{3-[3-(4-Chlorophenyl)propoxy]propyl}piperidine, hydrochloride (BF2.649) is a novel, potent, and selective nonimidazole inverse agonist at the recombinant human H3 receptor. On the stimulation of guanosine 5′-O-(3-[35S]thio)triphosphate binding to this receptor, BF2.649 behaved as a competitive antagonist with a Ki value of 0.16 nM and as an inverse agonist with an EC50 value of 1.5 nM and an intrinsic activity ∼50% higher than that of ciproxifan. Its in vitro potency was ∼6 times lower at the rodent receptor. In mice, the oral bioavailability coefficient, i.e., the ratio of plasma areas under the curve after oral and i.v. administrations, respectively, was 84%. BF2.649 dose dependently enhanced tele-methylhistamine levels in mouse brain, an index of histaminergic neuron activity, with an ED50 value of 1.6 mg/kg p.o., a response that persisted after repeated administrations for 17 days. In rats, the drug enhanced dopamine and acetylcholine levels in microdialysates of the prefrontal cortex. In cats, it markedly enhanced wakefulness at the expense of sleep states and also enhanced fast cortical rhythms of the electroencephalogram, known to be associated with improved vigilance. On the two-trial object recognition test in mice, a promnesiant effect was shown regarding either scopolamine-induced or natural forgetting. These preclinical data suggest that BF2.649 is a valuable drug candidate to be developed in wakefulness or memory deficits and other cognitive disorders.

BF2.649, A NON-IMIDAZOLE INVERSE AGONIST/ANTAGONIST AT THE HUMAN HISTAMINE H3 RECEPTOR: PRECLINICAL PHARMACOLOGY

Histamine H3 receptor inverse agonists are known to enhance the activity of histaminergic neurons in brain and thereby promote vigilance and cognition. 1-{3-[3-(4-Chlorophenyl)propoxy]propyl}piperidine, hydrochloride (BF2.649) is a novel, potent, and selective nonimidazole inverse agonist at the recombinant human H3 receptor. On the stimulation of guanosine 5′-O-(3-[35S]thio)triphosphate binding to this receptor, BF2.649 behaved as a competitive antagonist with a Ki value of 0.16 nM and as an inverse agonist with an EC50 value of 1.5 nM and an intrinsic activity ∼50% higher than that of ciproxifan. Its in vitro potency was ∼6 times lower at the rodent receptor. In mice, the oral bioavailability coefficient, i.e., the ratio of plasma areas under the curve after oral and i.v. administrations, respectively, was 84%. BF2.649 dose dependently enhanced tele-methylhistamine levels in mouse brain, an index of histaminergic neuron activity, with an ED50 value of 1.6 mg/kg p.o., a response that persisted after repeated administrations for 17 days. In rats, the drug enhanced dopamine and acetylcholine levels in microdialysates of the prefrontal cortex. In cats, it markedly enhanced wakefulness at the expense of sleep states and also enhanced fast cortical rhythms of the electroencephalogram, known to be associated with improved vigilance. On the two-trial object recognition test in mice, a promnesiant effect was shown regarding either scopolamine-induced or natural forgetting. These preclinical data suggest that BF2.649 is a valuable drug candidate to be developed in wakefulness or memory deficits and other cognitive disorders.

Matrix metalloproteinase expression in cardiac myocytes following myocardial infarction in the rabbit

Myocardial infarction (MI), leads to cardiac remodeling, thinning of the ventricle wall, ventricular dilation, and heart failure, and is a leading cause of death. Interactions between the contractile elements of the cardiac myocytes and the extracellular matrix (ECM) help maintain myocyte alignment required for the structural and functional integrity of the heart. Following MI, reorganization of the ECM and the myocytes occurs, contributing to loss of heart function. In certain pathological circumstances, the ECM is modulated such that the structure of the tissue becomes damaged. The matrix metalloproteinases (MMPs) are a family of enzymes that degrade molecules of the ECM. The present experiments were performed to define the time-course, isozyme subtypes, and cellular source of increased MMP expression that occurs following MI in an experimental rabbit model. Heart tissue samples from infarcted and sham animals were analyzed over a time-course of 1-14 days. By zymography, it was demonstrated that, unlike the sham controls, MMP-9 expression was induced within 24 hours following MI. MMP-3 expression, also absent in sham controls, was induced 2 days after MI. MMP-2 expression was detected in both the sham and infarcted samples and was modestly up-regulated following MI. Tissue inhibitor of metalloproteinase-1 (TIMP-1) expression was evaluated and shown to be down-regulated following MI, inverse of MMP-9 and MMP-3 expression. Further, MMP-9 and MMP-3 expression was detected by immunohistochemistry in myocytes within the infarct. Additional studies were conducted in which cultured rat cardiac myocytes were exposed to a hypoxic environment (2% O2) for 24 hours and the media analyzed for MMP expression. MMP-9 and MMP-3 were induced following exposure to hypoxia. It is speculated that the net increase in proteolytic activity by myocytes is a contributing factor leading to myocyte misalignment and slippage. Additional studies with a MMP inhibitor would elucidate this hypothesis.

Cell Type-specific Localization of Human Cardiac S1P Receptors

Sphingosine 1-phosphate (S1P), which derives from the metabolism of sphingomyelin, is mainly synthesized, stored, and released from platelets after activation by physiological and pathophysiological events. S1P acts in cardiovascular tissues through cell surface G-protein-coupled receptors of the endothelial differentiation gene (EDG) family, i.e., EDG1, EDG3 and EDG5. The aim of the present study was to assess the precise distribution of EDG1, EDG3, and EDG5 receptors expressed in human cardiovascular tissues to investigate their respective physiological implication. When assessed by Northern blots, EDG1, EDG3, and EDG5 displayed wide expression levels in decreasing order, respectively. In particular, EDG3 was mainly detected in the aorta. Detailed analysis by in situ hybridization (ISH) and immunohistochemistry (IHC) revealed strong EDG1 expression in cardiomyocytes and in endothelial cells of cardiac vessels. In cardiomyocytes, the EDG1 receptor is likely to be co-expressed with EDG3 and EDG5, although EDG1 exhibits the most prominent expression pattern. Unlike EDG3 and EDG5, which are expressed in the smooth muscle cell layer of the human aorta, no signal corresponding to EDG1 expression could be detected in the aorta. Moreover, only EDG3 expression was also found in smooth muscle cells of cardiac vessels. The present results provide new insight into the expression pattern of S1P receptors in human cardiovascular tissues, indicating a differential pattern of expression for these receptors in human vessels.

Inhibition of the cardiac electrogenic sodium bicarbonate cotransporter reduces ischemic injury

OBJECTIVE Although it is believed that sodium-driven acid-base transport plays a central role in the development of the reperfusion injury that follows cardiac ischemia, research to date has demonstrated only a role for Na(+)/H(+) exchange (NHE). However, Na(+)-driven HCO(-)(3) transport, which is quantitatively as important as NHE in cardiac cells, has not been examined. METHODS AND RESULTS Here the results show that a neutralizing antibody raised against the human heart electrogenic Na(+)/HCO(3)(-) cotransporter (hhNBC) blocked the recovery of pH after acidic pulse both in HEK-293 cells expressing hhNBC and in rat cardiac myocytes demonstrating the presence of an electrogenic NBC in rat cardiac myocytes similar to hhNBC. Administration of anti-NBC antibody to ischemic-reperfused rat hearts markedly protects systolic and diastolic functions of the heart during reperfusion. Furthermore, using a quantitative real-time RT-PCR (TaqMan) and Western blot analysis we demonstrated that in human cardiomyopathic hearts, mRNA and protein levels of hhNBC increase, whereas mRNA levels of the electroneutral Na(+)/HCO(3)(-) cotransporter (NBCn1) remain unchanged. CONCLUSION Our data provide evidence that inhibition of hhNBC, whose role in cardiac pathologies could be amplified by overexpression, represents a novel therapeutic approach for ischemic heart disease.

Highly Potent Fluorescence‐Tagged Nonimidazole Histamine H3 Receptor Ligands

Different (3‐phenoxypropyl)piperidine derivatives have been coupled to fluorescent moieties (5‐dimethylaminonaphthalene‐1‐sulfonyl, carbazol‐9‐ylcarbonyl, 2‐cyanoisoindol‐1‐yl, 2‐cyanobenzo[f]isoindol‐1‐yl, 2,4‐dinitrobenzen‐1‐yl, 2,4‐diaminophenyl, 7‐nitrobenzofurazan‐4‐yl, 7‐aminosulfonylbenzofurazan‐4‐yl, 4‐methylcoumarin‐6‐yl) as novel histamine H3 receptor ligands. They have been synthesised starting from piperidine in a few steps. The compounds display good to excellent histamine hH3 receptor affinities with Ki values ranging from 13.4 to 0.048 nM. Some of the new compounds belong to the most potent ligands known so far and may act as tools for identification and understanding of the binding site on the histamine H3 receptor. In vivo screening on selected derivatives of Sanger’s reagent showed antagonist potencies with ED50 values from 7.9 to 0.39 mg kg−1, p.o.

Refined Docking as a Valuable Tool for Lead Optimization : Application to Histamine H3 Receptor Antagonists

Drug‐discovery projects frequently employ structure‐based information through protein modeling and ligand docking, and there is a plethora of reports relating successful use of them in virtual screening. Hit / lead optimization, which represents the next step and the longest for the medicinal chemist, is very rarely considered. This is not surprising because lead optimization is a much more complex task. Here, a homology model of the histamine H3 receptor was built and tested for its ability to discriminate ligands above a defined threshold of affinity. In addition, drug safety is also evaluated during lead optimization, and “antitargets” are studied. So, we have used the same benchmarking procedure with the HERG channel and CYP2D6 enzyme, for which a minimal affinity is strongly desired. For targets and antitargets, we report here an accuracy as high as at least 70%, for ligands being classified above or below the chosen threshold. Such a good result is beyond what could have been predicted, especially, since our test conditions were particularly stringent. First, we measured the accuracy by means of AUC of ROC plots, i. e. considering both false positive and false negatives. Second, we used as datasets extensive chemical libraries (nearly a thousand ligands for H3). All molecules considered were true H3 receptor ligands with moderate to high affinity (from μM to nM range). Third, the database is issued from concrete SAR (Bioprojet H3 BF2.649 library) and is not simply constituted by few active ligands buried in a chemical catalogue.

Neuropeptide AF and FF Modulation of Adipocyte Metabolism PRIMARY INSIGHTS FROM FUNCTIONAL GENOMICS AND EFFECTS ON β-ADRENERGIC RESPONSIVENESS

The presence of a neuropeptide AF and FF receptor (NPFF-R2) mRNA in human adipose tissue (Elshourbagy, N. A., Ames, R. S., Fitzgerald, L. R., Foley, J. J., Chambers, J. K., Szekeres, P. G., Evans, N. A., Schmidt, D. B., Buckley, P. T., Dytko, G. M., Murdock, P. R., Tan, K. B., Shabon, U., Nuthulaganti, P., Wang, D. Y., Wilson, S., Bergsma, D. J., and Sarau, H. M. (2000) J. Biol. Chem. 275, 25965–25971) suggested these peptides, principally recognized for their pain modulating effects, may also impact on adipocyte metabolism, an aspect that has not been explored previously. Our aim was thus to obtain more insights into the actions of these peptides on adipocytes, an approach initially undertaken with a functional genomic assay. First we showed that 3T3-L1 adipocytes express both NPFF-R1 andNPFF-R2 transcripts, and that NPAF binds adipocyte membranes with a nanomolar affinity as assessed by surface plasmon resonance technology. Then, and following a 24-h treatment with NPFF or NPAF (1 μm), we have measured using real-time quantitative reverse transcriptase-PCR the mRNA steady state levels of already well characterized genes involved in key pathways of adipose metabolism. Among the 45 genes tested, few were modulated by NPFF (∼10%) and a larger number by NPAF (∼27%). Interestingly, NPAF increased the mRNA levels of β2- and β3-adrenergic receptors (AR), and to a lesser extent those of β1-ARs. These variations in catecholamine receptor mRNAs correlated with a clear induction in the density of β2- and β3-AR proteins, and in the potency of β-AR subtype-selective agonists to stimulate adenylyl cyclase activity. Altogether, these data show that NPFF-R1 and NPFF-R2 are functionally present in adipocytes and suggest that besides their well described pain modulation effects, NPAF and to a lesser extent NPFF, may have a global impact on body energy storage and utilization.

Biophysical interaction between phospholamban and protein phosphatase 1 regulatory subunit GM

Regulation of the sarco(endo)plasmic reticulum Ca2+‐ATPase (SERCA 2a) depends on the phosphorylation state of phospholamban (PLB). When PLB is phosphorylated, its inhibitory effect towards SERCA 2a is relieved, leading to an enhanced myocardial performance. This process is reversed by a sarcoplasmic reticulum (SR)‐associated type 1 protein phosphatase (PP1) composed of a catalytic subunit PP1C and a regulatory subunit GM. Human GM and PLB have been produced in an in vitro transcription/translation system and used for co‐immunoprecipitation and biosensor experiments. The detected interaction between the two partners suggests that cardiac PP1 is targeted to PLB via GM and we believe that this process occurs with the identified transmembrane domains of the two proteins. Thus, the interaction between PLB and GM may represent a specific way to modulate the SR function in human cardiac muscle.

Novel and highly potent histamine H3 receptor ligands. Part 1: withdrawing of hERG activity

Pre-clinical investigation of some aryl-piperidinyl ether histamine H3 receptor antagonists revealed a strong hERG binding. To overcome this issue, we have developed a QSAR model specially dedicated to H3 receptor ligands. This model was designed to be directly applicable in medicinal chemistry with no need of molecular modeling. The resulting recursive partitioning trees are robust (80-85% accuracy), but also simple and comprehensible. A novel promising lead emerged from our work and the structure-activity relationships are presented.