Preclinical pharmacology of [2-(3-fluoro-5-methanesulfonylphenoxy)ethyl](propyl)amine (IRL790), a novel dopamine transmission modulator for the treatment of motor and psychiatric complications in Parkinson's disease.

IRL790, ([2-(3-fluoro-5-methanesulfonylphenoxy)ethyl](propyl)amine), is a novel compound, in development for the clinical management of motor and psychiatric disabilities in Parkinson's disease. The discovery of IRL790 was made applying a systems pharmacology approach, based on in vivo response profiling. The chemical design idea was to develop a new type of DA D3/D2 receptor type antagonist built on agonist rather than antagonist structural motifs. We hypothesized that such a dopamine antagonist with physicochemical properties similar to agonists would exert antidyskinetic and antipsychotic effects in states of dysregulated dopaminergic signaling, while having little negative impact on physiological dopamine transmission and, hence, minimal liability for side effects related to dopamine dependent functions. At the level of in vivo pharmacology, IRL790 displays balancing effects on aberrant motor phenotypes, reducing LIDs in the rodent 6-OHDA lesion model, and reducing psychostimulant induced locomotor hyperactivity elicited by pretreatment with either d-amphetamine or dizocilpine, without negatively impacting normal motor performance. Thus, IRL790 has the ability to normalize the behavioural phenotype in hyperdopaminergic as well as hypoglutamatergic states. Neurochemical and immediate early gene (IEG) response profiles suggest modulation of DA neurotransmission, with some features such as increased DA metabolites and extracellular DA shared by atypical antipsychotics, and others such as increased frontal cortex IEGs, unique to IRL790. IRL790 also increases extracellular levels of Ach in the prefrontal cortex and ventral hippocampus. At the receptor level, IRL790 appears to act as a preferential DA D3 receptor antagonist. Computational docking studies support preferential affinity at D3 receptors, with an agonist like binding mode. SIGNIFICANCE STATEMENT: This paper reports preclinical pharmacology, along with molecular modelling results on IRL790, a novel compound in clinical development for the treatment of motor and psychiatric complications in advanced PD. IRL790 is active in models of perturbed dopaminergic and glutamatergic signaling, including rodent 6-OHDA LIDs, and psychostimulant induced hyperactivity, in a dose range that does not impair normal behavior. This effect profile is attributed to interactions at DA D2/D3 receptors, with a 6-8 fold preference for the D3 subtype.

The key pathophysiology of PD involves loss of dopaminergic and noradrenergic neurons, in the substantia nigra and locus coeruleus, respectively, associated with severe motor symptoms and progressive autonomic and neurocognitive dysfunctions (Kalia and Lang, 2015;Vermeiren and De Deyn, 2017). Other subcortical, cortical, and autonomic pathologies contribute to non-motor symptoms in PD. It has been suggested that cortico-striatal dysconnectivity and plasticity are key drivers for both core symptoms of PD and adverse effects emerging with long term dopaminergic treatment (Villalba and Smith, 2018).
Involuntary movements occurring on levodopa treatment (LIDs) remain an unmet clinical need since the introduction of levodopa in the 1970s. An estimated 40% of PD patients develop LIDs within 4-6 years of L-DOPA treatment, and over time it afflicts almost all patients (Ahlskog and Muenter, 2001). The pathogenetic mechanisms underlying the development of motor complications emerging with long term L-DOPA treatment are not fully clarified. One major hypothesis is the impact of non-physiological fluctuations in striatal dopamine release, leading to maladaptive changes in dopaminergic and glutamatergic transmission pre-and postsynaptically (Cenci, 2014). Much focus has been on the impact of chronic L-DOPA treatment, but it has also been suggested that dopamine denervation per se is the central cause of LIDs (Borgkvist et al., 2018). Both D1 and D2/D3 receptor signaling have been implied in this context.
The development of LIDs is strongly associated with augmented DA D1 receptor mediated neurotransmission, in medium spiny neurons of the direct striatonigral pathway (Feyder et al., 2011). Upregulation of DA D3 receptors has been pointed out as a key factor contributing to dysregulated D1 receptor mediated signaling, through D1-D3 receptor-receptor interactions including heteromerization interfering with D1 receptor internalization and intracellular signaling (Marcellino et al., 2008).
Furthermore, increased DA D3 receptor binding in PD patients with LIDS has been demonstrated using positron emission tomography (PET) (Payer et al., 2016). D3 receptor knock-out reduces LIDs in mice (Solis et al., 2017), while L-DOPA treatment is reported to induce involuntary movements in non-parkinsonian rodents overexpressing D3 receptors (Cote et al., 2014).
In clinical practice, the main strategy to reduce L-DOPA induced motor complications in PD is to reduce, and adjust dopaminergic treatment, to minimize fluctuations in plasma concentrations of the dopamine agonist used (Rascol et al., 2015). This can improve motor fluctuations but does not specifically address dyskinesias. Amantadine was recently introduced in an extended release formulation for treatment of LIDs (Sharma et al., 2018).
Among the side effects, psychotic symptoms are likely related to the NMDA antagonist properties of amantadine, and psychotic symptoms are frequently observed in clinical studies on NMDA antagonists (Muir and Lees, 1995). An increased incidence of visual hallucinations was reported in a meta-analysis assessing mGluR5 antagonists in LIDs (Wang et al., 2018b). There are no other pharmacological treatments approved for LIDs.
Surgical procedures, including deep brain stimulation, are used in some cases to alleviate dyskinesias and improve overall motor function in PD (Krack et al., 2017).
IRL790 (Sonesson et al., 2012) is a novel compound in development for the treatment of mental and motor complications in PD. The discovery of IRL790 was made applying an in vivo systems pharmacology approach (Waters et al., 2017). The strategy was to find compounds that could normalize aberrant phenotypes linked to cortico-striatal dysconnectivity, through interactions with DA D2/D3 receptors. The original chemical design idea was to develop a new type of DA D2/D3 antagonist build on agonist rather than antagonist structural motifs. This would lead to compounds which mimic the specific receptor This article has not been copyedited and formatted. The final version may differ from this version.
JPET Fast Forward. Published on May 1, 2020 as DOI: 10.1124/jpet.119.264226 at ASPET Journals on July 9, 2020 jpet.aspetjournals.org Downloaded from JPET # 264226 9 interactions of DA better, however, without displaying any intrinsic activity. A new series of compounds were discovered, among which IRL790 was selected for further development.
Preclinical assessment of IRL790 indicated a pharmacokinetic profile enabling oral administration, and no safety concerns, which was corroborated in a Phase 1a trial (IRLAB data on file). IRL790 is currently in clinical development, focusing on LIDs, and PD psychosis. Results from a Phase 1b study in PD were recently published, confirming a favorable safety profile and suggesting improvement of LIDs (Svenningsson et al., 2018).
This paper describes the preclinical pharmacology of IRL790, as investigated in normal and perturbed states including rodent LIDs. The findings are discussed in terms of tentative receptor level mode-of-action, based on in vitro assays, along with molecular modelling of GPCR-ligand interactions, focused on the dopamine D3 receptor.

Animals & drugs
Male Sprague-Dawley rats were purchased from B&K Scanbur (Sollentuna, Sweden; locomotor recordings and tissue analysis in normal, non-pretreated rats), Charles River (Köln, Germany; microdialysis and MK801 interaction study) or Taconic (Ejby, Denmark; AIMs study and d-amphetamine interaction). Rats weighed 160-180 g at the time of arrival, and 220-260 g at the time of the in vivo pharmacology studies. Animals were housed five per cage with lights on between 06:00 and 18:00, at 22°C, with free access to food and water.
All experiments were carried out in accordance with Swedish animal protection legislation and with the approval of the local Animal Ethics Committee in Gothenburg (EC no 1/2011 (AIMs study), 325/08 (locomotor recordings and tissue analysis), 287/10 (microdialysis).
Locomotor recordings and ex vivo neurochemistry analysis were performed essentially as described in (Waters et al., 2014). IRL790, synthesized in-house as HCl salt, was dissolved in physiological saline (0.9% w/v NaCl) and injected subcutaneously in a volume of 5 ml/kg,

Microdialysis
In vivo brain microdialysis experiments were performed as described in (Waters et al., 1993;Ponten et al., 2010), with some minor modifications. Microdialysis probes were implanted by stereotaxic surgery 40-48 hours before the experiments, during isoflurane inhalation anaesthesia. Co-ordinates were calculated relative to bregma; dorsal striatum: AP +1.0, ML ± 2.6, DV -6.2, prefrontal cortex: AP +3.2, ML ± 1.2, DV -4.0) according to (Paxinos, 1986 levels of each analyte. After establishment of baseline IRL790 was administrated s.c. in a volume of 5 ml/kg, with 0.9% NaCl (saline) as vehicle. Vehicle controls, run in a separate experiment, received saline. The monoamine transmitters (NA, DA, 5-HT) as well as their amine (NM, 3-MT) and acid (DOPAC, 5-HIAA, HVA) metabolites were followed for 180 min and quantified by HPLC/EC. After the experiment the rats were uncoupled from the perfusion pump and decapitated. Their brains were rapidly taken out and kept in Accustain one or two days before carefully slicing the brain to verify correct probe position. Only results from rats with correctly positioned dialysis probes were included in the subsequent analysis.
Microdialysis data are expressed as mean ± SEM in percentage of baseline levels. Baseline was defined as the mean level of the three fractions collected immediately before administration of test compound.
In a separate in vivo microdialysis experiment, levels of Ach were assessed in extracellular fluid collected from the prefrontal cortex and ventral hippocampus (co-ordinates AP -5.2, ML 4.8, DV -7.5 (Paxinos, 1986), by means of liquid chromatography/tandem mass spectrometry, as described previously (Jerlhag et al., 2012).
Dialysis data were analyzed by a 2-way ANOVA with treatment as independent variable and time as repeated measure. If significant, the 2-way ANOVA was followed by a Fisher's Least Significant Difference Test. Significance between vehicle and IRL790 treatment at specific time-points is indicated in the graphs as follows: * p<0.05, ** p<0.01, *** p<0.001.

AIMs
IRL790 was tested in two independent studies, in-house and by an external laboratory, in the 6-OHDA model of AIMs (Lundblad et al., 2005). In this model, the repeated administration of L-DOPA to rats subjected to unilateral 6-OHDA lesions of the nigrostriatal dopaminergic system, elicits adverse involuntary movements, affecting the orofacial region, the limbs and the trunk. The sum of AIMs scored in orofacial region, limbs and trunk is denoted total AIMs, or alternatively, composite AIMs score. Both studies were performed using rats subjected to 13 unilateral injections of 6-OHDA (Sigma-Aldrich) into the nigrostriatal fiber bundle, as described previously (Lundblad et al., 2005). In the in-house study, 48 male Sprague-Dawley rats were stereotactically injected with 6-OHDA (10 μg in 2 μl, saline with 0.1% ascorbic acid) into the MFB, in order to achieve complete unilateral dopaminergic denervation. Half of the rats were injected in the right side and the other half were injected in the left side of MFB.
Three weeks after lesion animals were subjected to an apomorphine-induced rotation test (about two hours). Limb, axial and orolingual dyskinesias were evaluated individually and the composite score was then calculated. IRL790 was initially tested at 1, 3 and 10 mg/kg s.c., given 20 min before L-DOPA (n=8 rats/group). Subsequently, the effects of 20 mg/kg s.c were tested in a separate experimental session (n=7 rats/group, controls received L-DOPA only).
Graphs display means of composite dyskinesia scores, with errors bars representing SEM.
Graphs with medians and quartiles are provided as supplementary data. In the external study, a repeated measures ANOVA was performed on the composite scores, applying Mauchly's sphericity test and Greenhouse-Geisser and Huyhn-Feldt adjusted significance tests, followed by post-hoc Dunnett's test comparing IRL790 at different doses vs. controls.
The results from the test of 20 mg/kg + L-DOPA vs L-DOPA treated controls were analysed separately. The results were confirmed with non-parametric tests; Kruskal-Wallis test for the initial dose response study, and Mann-Whitney U-test comparing 20 mg/kg vs L-DOPA controls, based on total composite scores. An integrated dose response analysis using total composite scores collected from both experimental sessions was undertaken, fitting a sigmoid curve to the data, using the TIBCO Spotfire software. In the in-house study, statistical comparison between control and IRL790 treatment were performed using ANOVA with time as repeated measure, confirmed with Mann-Whitney U-test, based on total composite scores.

Analysis of mRNA
For ex vivo mRNA analysis, brains were removed immediately following the completion of locomotor recordings and were dissected into four regions: Limbic system (containing nucleus accumbens, most parts of the olfactory tubercle, ventral pallidum and amygdala), striatum, frontal cortex and hippocampus. Tissue samples were stored at -80°C until further processing. Total RNA was prepared using the guanidine isothiocyanate method (Chomczynski and Sacchi, 1987). RNA pellets were dissolved in RNase-free water and This article has not been copyedited and formatted. The final version may differ from this version.
JPET Fast Forward. Published on May 1, 2020 as DOI: 10.1124/jpet.119.264226 at ASPET Journals on July 9, 2020 jpet.aspetjournals.org Downloaded from stored at -80°C. The RNA concentration was determined spectrophotometrically using a NanoDrop ND-1000 (Saveen Werner, Limhamn, Sweden) and the quality and integrity of random samples were checked using an Experion automated electrophoresis system (Bio-Rad, Sundbyberg, Sweden). Reverse transcription was performing using a SuperScript III kit (Invitrogen, Groningen, Netherlands) as follows: 1 µg of total RNA was mixed with 5 µl 2 x RT Reaction Mix and 1 µl of RT Enzyme Mix. The reaction volume was adjusted to 10 µl with RNase-free water. The mixture was incubated at 25°C for 10 min, 50°C for 30 min then 85°C for 5 min. Escherichia coli RNase H (1U) was added and the reaction mixture was incubated at 37°C for 20 min then 85°C for 5 min. The final cDNA was diluted 40 times in 10 mM Tris-HCl, 0.1 mM EDTA pH 7.8 and stored at -20°C. Results are presented as means ± SEM, expressed as percentages of control group means.
For the gene mapping on multiple compounds, multivariate analysis was performed as described in (Waters et al., 2017), applying PLS regression analysis using a discriminant

In vivo pharmacology
The pharmacological properties of IRL790 were investigated in a series of in vivo studies in rats, including models of psychosis and AIMS, based on disrupted dopaminergic or glutamatergic neurotransmission. Neurochemical biomarkers collected include monoaminergic indices assessed in brain tissue ex vivo, and by in vivo brain microdialysis, as well as IEGs.

Effects on behavior
In normal, non-pretreated rats, there was no significant effect on locomotor activity at 3.7-100 µmol/kg (significant effect of time, F(11,165)=61,1, adjusted p<0.00001, but not of treatment or time*treatment; Figure 1). IRL790 was further investigated in two pharmacological rat models of psychosis, i.e. in rats displaying aberrant, hyperactive motor behaviour due to pre-treatment with the catecholamine releaser d-amphetamine In the external study, IRL790 was initially tested at 1, 3 and 10 mg/kg s.c., corresponding to 3.2, 9.6 and 32 µmol/kg s.c, administered 20 min before L-DOPA (n=8 rats/group). As IRL790 did not appear to reduce the L-DOPA-induced motor activation in the test cages, a fourth dose of 20 mg/kg (64 µmol/kg) was tested in a separate session (n=7 rats/group). A dose-dependent anti-dyskinetic effect was observed with a reduction by approximately 40% in the total composite AIMs score as compared to the control group at 10 mg/kg, and a 45% reduction at 20 mg/kg ( Figure 5) controls, and at 20 mg/kg, p=0.002 vs controls. At the highest dose, L-DOPA-induced rotation was slightly increased in the L-DOPA + IRL790 treated group suggesting that the anti-dyskinetic effect of IRL790 did not reduce the beneficial L-DOPA induced motor activation ( Figure 6). Taken together, combining the two experimental sessions covering the dose range 1-20 mg/kg (3.2-64 µmol/kg) s.c, a dose dependent reduction of LIDs was observed, with an ED50 of 4 mg/kg (13 µmol/kg; Figure 7), with a reduction by 40 to 50% in the total composite AIMs score as compared to the control group at 10 and 20 mg/kg.

Effects on neurochemistry and IEGs
Ex vivo neurochemical analysis of brain tissue revealed dose dependent effects on monoaminergic indices, in particular DOPAC and HVA, reaching approximately 250-300% relative to control group means at the highest dose (100 µmol/kg) in all three brain structures sampled, suggesting increased dopamine metabolism across brain regions (Figure 8). There were also decreases in cortical NA and DA, at the top dose (reduced to 80% and 85% of control mean, respectively, p<0.05), and in striatal DA at 33 and 100 µmol/kg (83%, p<0.05 and 78%, p<0.001, respectively, relative to controls).
Neurochemical effects were also assessed by in vivo brain microdialysis, capturing monoamine analytes and Ach in extracellular fluid in conscious, freely moving rats.
Dialysates were collected from the striatum, and the prefrontal cortex, for 3 hours following the administration of IRL790, at 16.7 or 50 µmol/kg s.c., and assessed with respect to monoaminergic neurochemical indices. In a separate microdialysis study, Ach levels were Effects on IEG mRNA are shown in figure 11. IRL790 dose dependently and potently increased Arc in the striatum, reaching 241% (p<0.01), 329% (p<0.001), and 384% (p<0.001) vs. vehicle controls at 11, 33, 100 µmol/kg, respectively. Arc was also significantly increased at 11-100 µmol/kg in the frontal cortex and in the limbic region, reaching 153-162% of control in the frontal cortex and 166-186% of control in the limbic region ( Figure 10).
Cfos, homer and egr were increased in the striatal and limbic regions. NPAS4 was increased in the striatum. As with Arc, the effects on cfos, homer, egr, and NPAS4 were generally potent, reaching statistical significance even at the lowest dose tested (11 µmol/kg), with particularly large magnitudes for striatal cfos, homer and NPAS4 (300-800% of control means at the top dose). There were also minor effects on Nptx2, however of small magnitude and with no consistent dose response pattern: small increases were observed in the striatum at 33 and 100 µmol/kg (30-50% increase vs. controls), and a slight decrease in the frontal cortex (around 20% decrease vs. controls at 11 and 33 µmol/kg). For the gene map, IEG expression data on 13 compounds were analysed using PLS regression, as described previously (Waters et al., 2017). A five-component model was obtained, describing 72% of the variance in the X-block (R 2 X), and 26% of the variance in the Y-block (R 2 Y), Q 2 cum= 0.184. W*c loadings for the two first components are shown in

In vitro pharmacology
In vitro binding characteristics of IRL790 were assessed in a broad radioligand binding screen, followed by determination of IC50/Ki values at selected targets, at which at least 20% inhibition of control specific binding was observed (Table 1).
IRL790 was also tested for antagonist properties in in vitro cellular assays with human recombinant receptors. Antagonist properties at DA D2S, 5-HT1A and 5-HT2A receptors were observed, see Table 3.
Computational docking studies Figure 13 shows the best scoring binding pose for IRL790 in the crystal structure of the dopamine D3 receptor (Chien et al., 2010). IRL790 binds in a horizontal orientation with reference to the vertically oriented transmembrane helices. The methyl-sulfone moiety of IRL790 points towards the serine residues Ser192 and Ser193 (S 5.42 and S 5.43 using Ballesteros−Weinstein numbering; (Ballesteros and Weinstein, 1995) in the transmembrane helix 5 (TM5) and the basic amine interacts with the aspartic acid residue Asp110 (D 3.32 ) in TM3. These conserved residues among monoaminergic G-protein coupled receptors are known to be key interaction residues for receptor agonists (Malo et al., 2012). Thus, the modelling results indicate that IRL790 preferably binds in a typical agonist-like binding mode.
The best scoring binding pose for IRL790 in the structure model of the dopamine D2 receptor is in essence similar to the best D3 pose, but the internal conformational energy of IRL790 is higher and the docking score slightly worse for the D2 pose. The only sequence position in the binding site region which differ between the D2 and D3 receptors is in the second extracellular loop (ECL2) at the C+1 (Conner et al., 2007) position, i.e., the position after the conserved cysteine residue making a disulfide bridge to TM3, which is close to the binding pocket entrance (See Figure 14). The D3 receptor has a Serine pointing away from the ligand (IRL790) in the crystal structure and the D2 receptor has a bulkier isoleucine amino acid which is pointing towards the ligand and causing the entrance to be narrower in the D2 structure model.

Discussion
IRL790 was developed with the aim to create an antidyskinetic and antipsychotic compound, exerting its pharmacological effects through interactions with DA D3/D2 type receptors. The underlying design strategy was to use full or partial dopamine receptor agonists and modify their pharmacological properties away from agonism towards antagonism without following the conventional route of increasing size and lipophilicity. Given that agonists of DA D3/D2 receptors tend to be smaller, more hydrophilic molecules whereas antagonists are usually larger and more lipophilic (Malo et al., 2010;Li et al., 2016), we speculated that this approach could yield DA D3/D2 antagonists which mimic the specific interactions of DA (i.e., active state of the receptor) better than large, lipophilic antagonists, which binds to and stabilize the inactive state of the receptor (Goddard and Abrol, 2007), with slow receptor dissociation (Tresadern et al., 2011). We hypothesized that such a dopamine antagonist with physicochemical properties similar to agonists would exert antidyskinetic and antipsychotic effects in states of dysregulated dopaminergic signaling, while having little negative impact on physiological dopamine transmission and, hence, minimal liability for motor side effects.
This prediction was to a large extent manifested in IRL790s in vivo pharmacological profile.
The starting point for discovery and development of IRL790 was a series of 2-(aminomethyl)chromans (Mewshaw et al., 1997)  antipsychotics, attributable to dopamine receptor antagonism, known to produce increases in DOPAC and HVA (Carlsson and Lindqvist, 1963;Magnusson et al., 1986), in combination with interactions at 5HT1a, 5HT2a, 5HT7, or alpha2 receptors, proposed to result in enhanced release of dopamine (Alex and Pehek, 2007;Wesolowska and Kowalska, 2008;Marcus et al., 2010). IRL790 also increased extracellular levels of ACh in the pfc and hippocampus, an effect observed with DA D3 antagonists, and some atypical, but not typical antipsychotics (Lacroix et al., 2006).The gene expression profile also shares some features with antipsychotics. Arc is an immediate early gene triggered by, e.g., synaptic NMDA receptor activation (Bramham et al., 2010). It is involved in consolidation of memory and related processes, such as long-term potentiation (LTP) and is a key regulator of synaptic plasticity. Cfos, encoding a transcription factor, is a more non-specific marker of neuronal activation (de Bartolomeis et al., 2017). The dose dependent increase of striatal Arc, and cfos, is a common feature of dopamine D2 antagonists (Robbins et al., 2008;Fumagalli et al., 2009;Waters et al., 2014;de Bartolomeis et al., 2017), proposedly linked to antagonism at DA D2 heteroceptors at glutamatergic cortico-striatal terminals, leading to enhanced synaptic NMDA mediated signaling onto striatal neurons (Waters et al., 2014). With IRL790, we also see dose dependent increases of Nptx, NPAS, egr, and homer in the striatum, likely reflecting downstream effects of the modulation of synaptic activity in striatal neurons. The increase in frontal cortex Arc, on the other hand, is not shared by dopamine antagonists, which generally decrease this biomarker upon acute administration (Waters et al., 2014;de Bartolomeis et al., 2017). Enhanced cortical ACh may contribute to this cortical Arc increase (Teber et al., 2004). The gene map provides a comparative overview of IEG expression patterns for a set of compounds relevant for the PD population. The IEG profile of IRL790 is reflected in a position in the map representing both features shared with antipsychotics; as This article has not been copyedited and formatted. The final version may differ from this version.
JPET Fast Forward. Published on May 1, 2020 as DOI: 10.1124/jpet.119.264226 at ASPET Journals on July 9, 2020 jpet.aspetjournals.org Downloaded from discussed above, and features unique to IRL790 e.g., increases in frontal and limbic IEGs (Arc,cfos,NPAS4). This profile is distinct from the other classes represented in the model, including cariprazine, a novel antipsychotic described as a DA D3-vs. D2-receptor-preferring partial agonist (Garnock-Jones, 2017) that is more akin to other antipsychotics in its IEG expression profile. Moreover, IRL790 displays antagonist properties at the D3 receptor, and thus differs from cariprazine which is a high affinity partial agonist at these sites. This notwithstanding, IRL790 paradoxically displays an, agonist-like receptor binding mode (see docking studies below). Taken together, the binding properties imply a different interplay with the endogenous ligand at D3 receptors, as compared to cariprazine, in turn likely influencing down-stream effects measured in vivo, including IEGs. IRL790 is also distinct from compounds used in dementia, which generally show their most prominent IEG increases in frontal cortex and hippocampus,with less impact on striatal IEGs.
The effects of IRL790 on locomotor activity in rodents were studied in several models. There was no effect on spontaneous locomotor activity over a dose range eliciting significant effects on neurochemical and gene biomarkers. In contrast, IRL790 reduced locomotor hyperactivity elicited by pretreatment with either d-amphetamine or MK-801, demonstrating the ability to normalize the behavioural phenotype in hyperdopaminergic as well as hypoglutamatergic states.
Balancing effects on aberrant motor phenotypes were also evident in the investigations made using the rodent 6-OHDA AIMs model. This model is considered to have predictive validity for evaluation of anti-dyskinetic efficacy of new therapies (Iderberg et al., 2012). In vitro, sub-micromolar affinities were observed at DA D3, D2 and σ1receptors, and micromolar affinities at 5-HT1a, 5-HT2a, and 5-HT7 receptors. Functional in vitro studies showed no intrinsic activity at the receptors studied, except at 5HT1A receptors where a partial agonist effect at high concentrations, EC50 ≈100 µM, was detected. Additional functional studies confirmed antagonist actions of IRL790 at DA D3, D2, 5HT1a and 5HT2a receptors. The highest affinity was found at DA D3, followed by D2 (6-8 fold less) receptors.
Dopamine D3  D3/D2 receptors are also implicated in psychosis treatment, with most therapeutic agents being D2-preferring receptor antagonists or partial agonists. Interestingly, the D3/D2 preference ratio displayed by IRL790 is similar to that reported for the recently launched partial DA receptor agonist cariprazine, which has shown broad antipsychotic effectiveness (Garnock-Jones, 2017;Nemeth et al., 2017;Fleischhacker et al., 2019). IRL790 is less potent at D3 receptors than cariprazine, but is nonetheless within the same nanomolar affinity range as the endogenous transmitter DA itself for this site (Kiss et al., 2010;Stahl, 2017). IRL790 would therefore be expected to effectively compete with DA for the D3      Effects of IRL790 on rotations in AIMs model, external study.
Rats were subjected to unilateral 6-OHDA lesion followed by four weeks treatment with L-DOPA, 6.5 mg/kg dose s.c. (plus benserazide 12 mg/kg), in order to establish full expression of dyskinesias. On the test day IRL790 was administered at 1, 3 and 10 mg/kg µmol/kg s.c.
Controls are drawn at log (dose) = -2. N= 7-8/dose. Shown are means ± SEM. ED50 = 4 mg/kg (black circle) was estimated by means of fitting a sigmoidal curve (Hill equation), assuming Emin=50 (no reduction), Emax 25 (50% reduction of AIMs).  Effects of IRL790 on rat brain regional dialysate levels of monoamines. Effects of IRL790 on rat brain regional dialysate levels of ACh.       Figure 1 This article has not been copyedited and formatted. The final version may differ from this version.