There is a clear need to develop novel pharmacological tools to improve our understanding of Smoothened (Smo) function in normal and pathological states. Here, we report the discovery, the mechanism of action, and the in vivo activity of N- (2-methyl-5-(3-(3,4,5-trimethoxybenzoyl)

4 Histamine derivatives substituted with methyl groups in a, b or N a position of the side chain retained a nanomolar potency at the H 3 R, but their affinity was dramatically decreased at the H 4 R. With relative potencies to histamine of 282 and 0.13% at the H 3 R and H 4 R, respectively, (7)-a,bdimethylhistamine is a potent and selective H 3 R agonist. 5 Chiral a-branched analogues exhibited a marked stereoselectivity at the H 3 R and H 4 R, the enantiomers with a configuration equivalent to L-histidine being preferred at both receptors. 6 The methylsubstitution of the imidazole ring was also studied. The relative potency to histamine of 4-methylhistamine (4-MeHA) at the H 4 R (67%) was similar to that reported at H 2 receptors but, owing to its high affinity at the H 4 R (K i ¼ 7.071.2 nM) and very low potency at H 1 -and H 3 -receptors, it can be considered as a potent and selective H 4 R agonist. 7 On inhibition of forskolin-induced cAMP formation, all the compounds tested, including 4-MeHA, behaved as full agonists at both receptors. However, the maximal inhibition achieved at the H 4 R (BÀ30%) was much lower than at the H 3 R (BÀ80%). Thioperamide behaved as an inverse agonist at both receptors and increased cAMP formation with the same maximal effect (B þ 25%). 8 In conclusion, although the pharmacological profiles of the human H 3 R and H 4 R overlap, the structure-activity relationships of histamine derivatives at both receptors strongly differ and lead to the identification of selective compounds.

5-Methoxycarbonylamino-N-acetyltryptamine (MCA-NAT) has been initially described as a ligand at non MT(1), non MT(2) melatonin binding site (MT3) selective versus MT(1) and MT(2), two membrane melatonin receptors. MCA-NAT activity has been reported by others in different models, in vivo, particularly in the intra-ocular pressure (IOP) models in rabbits and monkeys. Its activity was systematically linked to either MT3 or to a new, yet unknown, melatonin receptor. In this article, the melatonin receptor pharmacology of MCA-NAT is described. MCA-NAT has micromolar range affinities at the melatonin receptors MT(1) and MT(2), while in functional studies, MCA-NAT proved to be a powerful MT(1)/MT(2) partial agonist in the sub-micromolar range. These data strongly suggest that MCA-NAT actions might be mediated by these receptors in vivo. Finally, as described by others, we show that MCA-NAT is unable to elicit any type of receptor-like functional responses from Chinese hamster ovary cells over-expressing quinone reductase 2, the MT3.

Clozapine and its active metabolite NDMC interact with the four human histamine receptors at clinically relevant concentrations. This interaction may substantiate, at least in part, the atypical antipsychotic profile of clozapine, as well as its central and peripheral side effects such as sedation and weight gain.

Clinical assessment of drug-drug interactions (DDIs) in children is not a common practice in drug development. Therefore, physiologically-based pharmacokinetic (PBPK) modeling can be beneficial for informing drug labeling. Using ivabradine and its metabolite (both cytochrome P450 3A4 enzyme (CYP3A4) substrates), the objectives were (i) to scale ivabradinemetabolite adult PBPK/PD to pediatrics, (ii) to predict the DDIs with a strong CYP3A4 inhibitor, and (iii) to compare the sensitivity of children to DDIs using two CYP3A4 hepatic ontogeny functions: Salem and Upreti. A scaled parent-metabolite PBPK/PD model from adults to children satisfactorily predicted pharmacokinetics (PK) and pharmacodynamics (PD) in 74 children (0.5-18 years) regardless of CYP3A4 hepatic ontogeny function applied. However, using the Salem ontogeny, mean predicted parent and metabolite area under the concentration-time curve over 12 hours (AUC 12h ) and heart rate change from baseline were 2-fold, 1.5-fold, and 1.4-fold higher in young children (0.5-3 years old) compared with Upreti ontogeny, respectively. Despite these differences, choice of appropriate hepatic CYP3A4 ontogeny was challenging due to sparse PK and PD data. Different sensitivity to ivabradine-ketoconazole DDIs was simulated in young children relative to adults depending on the choice of hepatic CYP3A4 ontogeny. Predicted ivabradine and metabolite AUC DDI /AUC control were 2-fold lower in the youngest children (0.5-1 year old) compared with adults (Salem function). In contrast, the Upreti function predicted comparable ivabradine DDIs across all age groups, although predicted metabolite AUC DDI/ AUC control was 1.3-fold higher between the youngest children and adults. In the case of PD, differences in predicted DDIs were minor across age groups and between both functions. Current work highlights the importance of careful consideration of hepatic CYP3A4 ontogeny function and implications on labeling recommendations in the pediatric population.

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1. We have applied the concept of using MBIs to produce CYP-Silensomes to quantify the contribution of the major CYPs to drug metabolism (fmCYP). 2. The target CYPs were extensively and selectivity inhibited by the selected MBIs, while non-target CYPs were inhibited by less than 20% of the homologous control activities. Only CYP2D6-Silensomes exhibited a CYP2B6 inhibition that could be easily and efficiently encountered by subtracting the fm measured using CYP2B6-Silensomes to adjust the fm. 3. To validate the use of a panel of 6 CYP-Silensomes, we showed that the fmCYP values of mono- and multi-CYP metabolised drugs were well predicted, with 70% within ± 15% accuracy. Moreover, the correlation with observed fmCYP values was higher than that for rhCYPs, which were run in parallel using the same drugs (<45% within ±15% accuracy). Moreover, the choice of the RAF substrate in rhCYP predictions was shown to affect the accuracy of the fmCYP measurement. 4. These results support the use of CYP1A2-, CYP2B6-, CYP2C8-, CYP2C9-, CYP2D6 and CYP3A4-Silensomes to accurately predict fmCYP values during the in vitro enzyme phenotyping assays in early, as well as in development, phases of drug development.

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