Renzo Carletti

Neurokinin 1 receptor and relative abundance of the short and long isoforms in the human brain

Substance P exerts its various biochemical effects mainly via interactions through neurokinin‐1 receptors (NK1). Recently, the NK1 receptor has attracted considerable interest for its possible role in a variety of psychiatric disorders including depression and anxiety. However, little is known regarding the anatomical distribution of NK1 in the human central nervous system (CNS). Riboprobe in situ hybridization, quantitative PCR and in vitro autoradiography were performed. Highest NK1 mRNA levels were localized in the locus coeruleus and ventral striatum, while moderate hybridization signals were observed in the cerebral cortex (most abundant in the visual cortex), hippocampus and different amygdaloid nuclei. Very low levels of the NK1 mRNA were detected in the cerebellum and thalamus. In view of the existence of a long and short isoform of the NK1 receptor, it was of interest to assess whether there was a differential distribution of the two splice variants in the human CNS and peripheral tissues. A quantitative TaqMan PCR analysis showed that the long NK1 isoform was the most prevalent throughout the human brain, while in peripheral tissues the truncated form was the most represented. 3H‐Substance P autoradiography revealed a good correlation between receptor binding sites and NK1 mRNA expression throughout the brain, with the highest levels of binding in the locus coeruleus. These results provide the anatomical evidence that the NK1 receptors have a strong association with neuronal systems relevant to mood regulation and stress in the human brain, but do not suggest a region‐specific role of the two isoforms in the CNS.

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.

[3H]GR205171 displays similar NK1 receptor binding profile in gerbil and human brain

1 In this study, [3H]GR205171 (3(S)‐(2‐methoxy‐5‐(5‐trifluoromethyltetrazol‐1‐yl)‐phenylmethylamino)‐2(S)‐phenylpiperidine), a potent and selective NK1 receptor antagonist, was characterised in autoradiographic studies in gerbil brain and in binding experiments on homogenates from gerbil and human brain cortex and striatum. 2 In autoradiographic studies in gerbil brain, highest levels of [3H]GR205171 binding sites were observed in caudate putamen, nucleus accumbens, medial and cortical nuclei of the amygdala and intermediate levels were detected in the hypothalamus, basolateral amygdala, septum, and cortex. 3 Saturation experiments in homogenates of brain striatum from gerbil showed that [3H]GR205171 binds to a single receptor population with a pKd value of 10.8±0.2 and a Bmax value of 607±40 fmol mg−1. A lower number of NK1 receptor sites was found in cortex, where a Bmax of 94±6 fmol mg−1 protein was obtained. Saturation experiments performed on homogenates from brain striatum of two human subjects and brain cortex of three human subjects showed that [3H]GR205171 binds with pKd values not different from gerbil and Bmax values ranging from 318±51 to 432±27 fmol mg−1 protein in striatum and from 59±1 to 74±21 fmol mg−1 protein in cortex. The natural ligand [3H]Substance P (SP) bound with sub‐nanomolar affinity to 15 and 6% sites compared to [3H]GR205171 in gerbil and human striatum, respectively. 4 In competition binding experiments, GR205171 and the NK1 receptor antagonists aprepitant (MK‐869), L‐733,060 and NKP‐608 bound with similar pKi values in gerbil and human striatum, irrespective of the use of [3H]GR205171 or [3H]SP as radioligand. The following rank order was found in terms of pKi values: GR205171>aprepitantL‐733,060>NKP‐608. In homologous displacement experiments in gerbil and human striatum, SP showed nanomolar affinity, whereas in [3H]GR205171 competition experiments SP bound with pIC50 values in the micromolar range and Hill slopes significantly lower than one. 5 It is concluded that the similarities of [3H]GR205171 binding characteristics and pharmacology between gerbil and human in cortex and striatum support the use of gerbil in preclinical models to study the effects of NK1 receptor antagonists in the central nervous system.

Down-regulation of amygdala preprotachykinin A mRNA but not 3H-SP receptor binding sites in subjects affected by mood disorders and schizophrenia.

Increasing evidence suggests that substance P (SP) neurokinin‐1 (NK1) receptors are involved in stress and emotional responses, representing a potential target for the treatment of anxiety and depression in humans. Given the important role of the amygdaloid complex in the regulation of emotional behavior, we examined the mRNA levels of preprotachykinin A [PPT‐A, a precursor of both SP and neurokinin A (NKA)] and 3H‐SP binding sites in the amygdala of patients affected by bipolar disorder, major depression or schizophrenia as compared with matched control individuals. By means of in situ hybridization, a significant reduction of PPT‐A mRNA expression levels was detected in the three diagnostic groups, mainly in the basal, lateral and accessory basal amygdaloid nuclei, but not in the temporal cortical area proximal to the amygdala. Receptor autoradiography performed on adjacent sections showed no change in 3H‐SP binding sites as compared with controls. No significant correlation was found between levels of PPT‐A mRNA expression or binding sites and subject age, gender, hemisphere side, cause of death or history of substance misuse (marijuana, alcohol, cocaine/amphetamine). An inverse relationship between PPT‐A mRNA expression levels and lifetime antipsychotic treatment (Fluphenazine) in the schizophrenic and bipolar disorder groups was found. Post‐mortem delay was also negatively correlated with NK1 binding sites. The results confirm an involvement of the tachykinins in psychiatric disorders, suggesting there is a generalized impairment of the SP system in the amygdala in mood disorders and schizophrenia rather than this being a disease‐related phenomenon.

Identification, biological characterization and pharmacophoric analysis of a new potent and selective NK1 receptor antagonist clinical candidate

The last two decades have provided a large weight of preclinical data implicating the neurokinin-1 receptor (NK1) and its cognate ligand substance P (SP) in a broad range of both central and peripheral disease conditions. However, to date, only the NK1 receptor antagonist aprepitant has been approved as a therapeutic and this is to prevent chemotherapy-induced nausea & vomiting (CINV). The belief remained that the full therapeutic potential of NK1 receptor antagonists had yet to be realized; therefore clinical evidence that NK1 receptor antagonists may be effective in major depression disorder, resulted in a significant further investment in discovering novel CNS penetrant druggable NK1 receptor antagonists to address this condition. At GlaxoSmithKline after the discovery of casopitant, that went on to demonstrate efficacy as a novel antidepressant in the clinic, additional novel analogues of this NK1 receptor antagonist were designed to further enhance its drug developability characteristics. Herein, we therefore describe the discovery process and the vivo pharmacological and pharmacokinetic profile of the new NK1 receptor antagonist 3a (also called orvepitant), selected as clinical candidate and further progressed into clinical studies for major depressive disorder. Moreover, molecular modeling studies enabled us to improve the pharmacophore model of the NK1 receptor antagonists with the identification of a region able to accommodate a variety of heterocycle moieties.

Receptor distribution studies

HIGHLIGHTSDistribution studies may contribute to the understanding of novel receptor systems.GABAB, NMDA and NK1 receptor studies in Translational Medicine strategies are shown.PET ligands to effectively bridge the preclinical/clinical interface are needed. &NA; Receptor distribution studies have played a key role in the characterization of receptor systems (e.g. GABAB, NMDA (GluNRs), and Neurokinin 1) and in generating hypotheses to exploit these systems as potential therapeutic targets. Distribution studies can provide important information on the potential role of candidate receptors in normal physiology/disease and alert for possible adverse effects of targeting the receptors. Moreover, they can provide valuable information relating to quantitative target engagement (e.g. % receptor occupancy) to drive mechanistic pharmacokinetic/pharmacodynamic (PK/PD) hypotheses for compounds in the Drug Discovery process. Finally, receptor distribution and quantitative target engagement studies can be used to validate truly translational technologies such as PET ligands and pharmacoEEG paradigms to facilitate bridging of the preclinical/clinical interface and thus increase probability of success.