Simone Braggio

Assessing brain free fraction in early drug discovery.

Importance of the field: The incorporation of brain tissue binding routinely in CNS drug discovery screening strategies has markedly changed the way CNS drug discovery is performed and is proving to be a valuable tool in identifying new therapies for CNS diseases. For many years emphasis has been placed on the magnitude of the brain to blood ratio, the bigger the better, even though, in many cases, brain total concentration (Cbrain) has no or, at best, poor correlation with receptor occupancy/pharmacodynamic readout. Today, Cbrain values measured during in vivo experiments are corrected for the fraction unbound measured through in vitro experiments using brain tissue homogenate or brain tissue slice to obtain an estimate of the brain unbound concentration (Cu,brain), and this has been demonstrated across a range of CNS targets to give a much better correlation with receptor occupancy/pharmacodynamic readout. This apparently simple change in CNS lead optimisation strategy has de facto revolutionised the vision of the brain penetration concepts. Areas covered in this review: This review will provide an overview of the use and applications of assessing brain free fraction to determine Cu,brain. Take home message: Assessing brain free fraction to determine Cu,brain in CNS lead optimisation strategies is the surrogate of choice for rapidly assessing biophase concentration for the majority of CNS targets.

6-(3,4-Dichlorophenyl)-1-[(Methyloxy)methyl]-3-azabicyclo[4.1.0]heptane: A New Potent and Selective Triple Reuptake Inhibitor

A pharmacophore model for triple reuptake inhibitors and the new class of 1-(aryl)-6-[alkoxyalkyl]-3-azabicyclo[3.1.0]hexanes were recently reported. Further investigation in this area led to the identification of a new series of potent and selective triple reuptake inhibitors endowed with good developability characteristics. Excellent bioavailability and brain penetration are associated with this series of 6-(3,4-dichlorophenyl)-1-[(methyloxy)methyl]-3-azabicyclo[4.1.0]heptanes together with high in vitro potency and selectivity at SERT, NET, and DAT. In vivo microdialysis experiments in different animal models and receptor occupancy studies in rat confirmed that derivative 17 showed an appropriate profile to guarantee further progression of the compound.

Discovery process and pharmacological characterization of a novel dual orexin 1 and orexin 2 receptor antagonist useful for treatment of sleep disorders.

The hypothalamic peptides orexin-A and orexin-B are potent agonists of two G-protein coupled receptors, namely the OX(1) and the OX(2) receptor. These receptors are widely distributed, though differentially, in the rat brain. In particular, the OX(1) receptor is highly expressed throughout the hypothalamus, whilst the OX(2) receptor is mainly located in the ventral posterior nucleus. A large body of compelling evidence, both pre-clinical and clinical, suggests that the orexin system is profoundly implicated in sleep disorders. In particular, modulation of the orexin receptors activation by appropriate antagonists was proven to be an efficacious strategy for the treatment of insomnia in man. A novel, drug-like bis-amido piperidine derivative was identified as potent dual OX(1) and OX(2) receptor antagonists, highly effective in a pre-clinical model of sleep.

1,2,4-Triazolyl Azabicyclo[3.1.0]hexanes: A New Series of Potent and Selective Dopamine D3 Receptor Antagonists

The discovery of new highly potent and selective dopamine (DA) D(3) receptor antagonists has recently allowed the characterization of the DA D(3) receptor in a range of preclinical animal models of drug addiction. A novel series of 1,2,4-triazol-3-yl-azabicyclo[3.1.0]hexanes, members of which showed a high affinity and selectivity for the DA D(3) receptor and excellent pharmacokinetic profiles, is reported here. Members of a group of derivatives from this series showed good oral bioavailability and brain penetration and very high in vitro affinity and selectivity for the DA D(3) receptor, as well as high in vitro potency for antagonism at this receptor. Several members of this series also significantly attenuate the expression of conditioned place preference (CPP) to nicotine and cocaine.

Drug efficiency: a new concept to guide lead optimization programs towards the selection of better clinical candidates

As a result of their wide acceptance and conceptual simplicity, drug-like concepts are having a major influence on the drug discovery process, particularly in the selection of the ‘optimal’ absorption, distribution, metabolism, excretion and toxicity and physicochemical parameters space. While they have an undisputable value when assessing the potential of lead series or in evaluating inherent risk of a portfolio of drug candidates, they result much less useful in weighing up compounds for the selection of the best potential clinical candidate. We introduce the concept of drug efficiency as a new tool both to guide the drug discovery program teams during the lead optimization phase and to better assess the developability potential of a drug candidate.

Synthesis and pharmacological characterization of novel druglike corticotropin-releasing factor 1 antagonists.

To identify new CRF(1) receptor antagonists, an attempt to modify the bis-heterocycle moiety present in the top region of the dihydropyrrole[2,3]pyridine template was made following new pharmacophoric hypothesis on the CRF(1) receptor antagonists binding pocket. In particular, the 2-thiazole ring, present in the previous series of compounds, was replaced by more hydrophilic non aromatic heterocycles able to make appropriate H-bond interactions with amino acid residues Thr192 and Tyr195. This exploration, followed by an accurate analysis of the substitution of the pendant aryl ring, enabled to identify in vitro potent compounds showing excellent pharmacokinetics and outstanding in vivo activity in animal models of anxiety, both in rodents and primates.

1-(Aryl)-6-[alkoxyalkyl]-3-azabicyclo[3.1.0]hexanes and 6-(aryl)-6-[alkoxyalkyl]-3-azabicyclo[3.1.0]hexanes: a new series of potent and selective triple reuptake inhibitors.

The discovery of new highly potent and selective triple reuptake inhibitors is reported. The new classes of 1-(aryl)-6-[alkoxyalkyl]-3-azabicyclo[3.1.0]hexanes and 6-(aryl)-6-[alkoxyalkyl]-3-azabicyclo[3.1.0]hexanes are described together with detailed SAR. Appropriate decoration of the scaffolds was achieved with the help of a triple reuptake inhibitor pharmacophore model detailed here. Selected derivatives showed good oral bioavailability (>30%) and brain penetration (B/B > 4) in rats associated with high in vitro potency and selectivity at SERT, NET, and DAT. Among these compounds, microdialysis and in vivo experiments confirm that derivative 15 has an appropriate developability profile to be considered for further progression.

Exploration of the amine terminus in a novel series of 1,2,4-triazolo-3-yl-azabicyclo[3.1.0]hexanes as selective dopamine D3 receptor antagonists.

A novel series of 1,2,4-triazol-3-yl-azabicyclo[3.1.0]hexanes with high affinity and selectivity for the DA D(3) receptor and excellent pharmacokinetic profiles was recently reported. We also recently discussed the role of the linker associated with the triazole moiety. In this manuscript, we are reporting a detailed exploration of the region of the receptor interacting with the amine terminus of the scaffold wherein SAR and developability data associated with these novel templates was undertaken.

New fused benzazepine as selective D3 receptor antagonists. Synthesis and biological evaluation. Part one : [h]-fused tricyclic systems

The synthesis and SAR of a new series of potent and selective dopamine D(3) receptor antagonists is reported. The introduction of a tricyclic [h]-fused benzazepine moiety on the recently disclosed scaffold of 1,2,4-triazol-3-yl-thiopropyl-tetrahydrobenzazepines is reported. A full rat pharmacokinetic characterization is also reported.

Development and validation of a high-throughput method for the quantitative analysis of d-amphetamine in rat blood using liquid chromatography/MS3 on a hybrid triple quadrupole-linear ion trap mass spectrometer and its application to a pharmacokinetic study

Amphetamines are a group of sympathomimetic drugs that exhibit strong central nervous system stimulant effects. D-Amphetamine ((+)-alpha-methylphenetylamine) is the parent drug in this class to which all others are structurally related. In drug discovery, d-amphetamine is extensively used either for the exploration of novel mechanisms involving the catecholaminergic system, or for the validation of new behavioural animal models. Due to this extensive use of D-amphetamine in drug research and its interest in toxicologic-forensic investigation, a specific and high-throughput method, with minimal sample preparation, is necessary for routine analysis of D-amphetamine in biological samples. We propose here a sensitive, specific and high-throughput bioanalytical method for the quantitative determination of D-amphetamine in rat blood using MS(3) scan mode on a hybrid triple quadrupole-linear ion trap mass spectrometer (LC-MS/MS/MS). Blood samples, following dilution with water, were prepared by fully automated protein precipitation with acetonitrile containing an internal standard. The chromatographic separation was achieved on a Waters XTerra C18 column (2.1mm x 30mm, 3.5microm) using gradient elution at a flow rate of 1.0mL/min over a 2min run time. An Applied Biosystems API4000 QTRAP mass spectrometer equipped with turbo ion-spray ionization source was operated simultaneously in MS(3) scan mode for the d-amphetamine and in multiple reaction monitoring (MRM) for the internal standard. The MS/MS/MS ion transition monitored was m/z 136.1-->119.1-->91.1 for the quantitation of d-amphetamine and for the internal standard (rolipram) the MS/MS ion transition monitored was m/z 276.1-->208.2. The linear dynamic range was established over the concentration range 0.5-1000ng/mL (r(2)=0.9991). The method was rugged and sensitive with a lower limit of quantification (LLOQ) of 0.5ng/mL. All the validation data, such as accuracy, precision, and inter-day repeatability, were within the required limits. This method was successfully applied to evaluate the pharmacokinetics of d-amphetamine in rat. On a more general extent, this work demonstrated that the selectivity of the fragmentation pathway (MS(3)) can be used as alternative approach to significantly improve detection capability in complex situation (e.g., small molecules in complex matrices) rather than increasing time for sample preparation and chromatographic separation.