Pridopidine: Overview of Pharmacology and Rationale for its Use in Huntington’s Disease

The dopamine system in Huntington’s disease

Dopamine is a monoamine neurotransmitter modulating several aspects of brain function, including motor control [20], and disrupted dopaminergic signaling has been implicated in a number of neurological and psychiatric conditions [21–24]. Motor control is exerted by dopamine released from the nigrostriatal pathway, modulating the activity of MSNs involved in the facilitation of movement, and inhibition of unwanted movement [25]. MSNs are GABAergic neurons, expressing high densities of dopamine receptors, and a progressive decline in striatal dopamine receptor density is one of the earliest findings in patients with HD [26, 27]. Such changes have been well described in post mortem studies, and corroborated in vivo by positron emission tomographic (PET) studies [28–31]. In comparison to these post-synaptic changes, the integrity of the pre-synaptic dopaminergic system in HD has been less extensively studied. While the dopamine neuron population in the substantia nigra appears preserved [32], a loss of dopamine terminals has been reported [33]. The latter finding has been confirmed in a PET study including a smaller number of patients with HD [28]. Studies in transgenic animal models suggest that a change in dopaminergic function, such as compromised dopamine release, is an early sign of neuropathology in HD [34–36]. Clinical studies have demonstrated that HD is characterized by pre-synaptic as well as post-synaptic dopamine-related dysfunctions with reduction in striatal dopamine synthesis, dopamine storage, dopamine transporter binding, and both dopamine D1 and D2 receptor binding [37]. Loss of both pre- and post-synaptic markers of dopamine neurotransmission is positively correlated with cognitive performance in both asymptomatic and symptomatic HD patients [38], but the integrity of extrastriatal dopamine D2 receptors has been reported to appear relatively well preserved in patients with HD [39]. A recent review on dopamine changes in Huntington’s disease outlined an integrated view, in essence suggesting a continuous decline in dopamine receptors over the patient’s life-span, and biphasic changes in striatal dopamine levels, initially increased but thereafter decreasing, in parallel with the transition from hyper- to hypokinetic motor disturbances [40].

It is well established that pharmacological treatments that modify dopaminergic function impact on the motor symptoms of HD. Levodopa challenges were used to provoke chorea as a clinical diagnostic test more than a decade before genetic testing was available [41]. Conversely, tetrabenazine, a monoamine-depleting drug, and dopamine D2 receptor antagonist drugs (antipsychotics) are used to alleviate chorea [42, 43]. On the other hand, Parkinsonian symptoms such as bradykinesia and hypokinesia in HD are hypothesized to be linked to dopaminergic impairment as these symptoms are aggravated by the use of antipsychotic medication [44, 45]. Furthermore, HD patients treated with antidopaminergic drugs have been reported to display a more rapid disease progression [46] and a more severe phenotype [47]. Thus, motor symptoms in HD are sensitive to drugs that alter dopaminergic transmission, where enhancement of dopaminergic activity is associated with increased chorea, and attenuation is conceivably associated with worsening of negative motor symptoms such as bradykinesia.

Pridopidine – discovery and overview of pharmacology

Pridopidine was discovered using an in vivo systems pharmacology approach, focusing on measuring biomarkers of neurotransmission in neuronal circuits known for their involvement in the control of psychomotor functions. The theory is that systematic in vivo profiling of CNS compounds, using a comprehensive range of neurochemical, gene expression and behavioral biomarkers collected in both healthy and diseased states, would give a reliable handle on preclinical properties and allow for predictions of clinical features of the novel compounds tested in the system. Data on a wide variety of known therapeutic agents including antipsychotics, antidepressants, anxiolytics and psychostimulants, as well as experimental reference compounds, are collected, and then used to define a multidimensional compound “map” serving as a guide towards the sought after in vivo profile [69]. This methodology was developed to enable a rational and predictive drug discovery process in a field where target-based discovery approaches have hitherto shown to be largely unsatisfactory in terms of determining clinically promising molecules and molecular action mechanisms [70]. In the specific program that led to the discovery and subsequent development of pridopidine in HD, the vital impact of central monoaminergic and specifically dopaminergic pathways in the cortico-subcortical circuitry regulating motor function was considered. The complex, progressive symptomatology in HD, combining hypo- and hyperkinetic motor disturbances, cognitive and psychiatric symptoms, suggest any pharmacological intervention should be delicately balanced not to disturb remaining functions or worsen aspects of the heterogeneous symptoms. The combined basic research efforts and considerations described above led to the formulation of a preclinical target profile. In short, a compound of interest should: 1) show no interference with spontaneous locomotor patterns over a wide dose range; 2) have the ability to normalize states of hypoactivity; 3) have the ability to normalize states of hyperactivity; and 4) act primarily through the dopamine system. The initial screening was performed in vivo as described in the following (see section Overall in vivo phenotypic response profile).

From the medicinal chemistry perspective, the fourth criterion above suggested the conceptual starting point of the program to focus on design and synthesis of compounds with dopamine D2 receptor agonist-like chemical motifs. The key chemical strategy in the program was to modify the agonist motif so that the pharmacophore elements essential for intrinsic activity were lost, to obtain compounds that primarily interact with the dopamine D2 receptor in a similar way as agonists, but without the ability to stabilize the active and catalytic conformation(s) of the receptor G-protein complex. The working hypothesis was that this should result in a portfolio of compounds with agonist-like receptor interactions but with antagonist-like pharmacological features. Further, the hydrophilic characteristics of the agonist motif were to be retained to ensure similar binding mode and adherence to druggability features important for favorable drug metabolism and pharmacokinetics, and reduced safety and toxicological risks.

Starting from a series of compounds with partial dopamine D2 agonist properties reported by Mewshaw et al. [71] including 3-(1-benzylpiperidin-4-yl)phenol, the key elements for its intrinsic activity were identified to be the phenol group, the anilinic nitrogen and the large N-alkyl group. A series of modifications of these elements led to a range of phenylpiperidines, including 4-[3-(methylsulfonyl)phenyl]-1-propylpiperidine (ACR16, pridopidine) [72], displaying the desired profile.

Overall in vivo phenotypic response profile

The initial in vivo pharmacological assessment of pridopidine was performed by acute dose response studies in intact rats, collecting behavioral data and ex vivo monoaminergic neurochemical indices according to a systematic, standardized model, followed by multivariate analysis of the response profile, including comparisons to a wide range of CNS compounds and a tentative therapeutic classification [69, 73]. This “screening” suggested that three major criteria were met; i.e., no behavioral inhibition, subtle behavioral activation in the hypoactive phase, and significant effects on dopaminergic indices reflecting dopamine D2 receptor modulation. Furthermore, the multivariate comparison to reference compounds indicated clear similarities with the cluster of anti-psychotic compounds tested, as well as some effects in cortical brain areas overlapping with effects of pro-cognitive compounds. This prompted further preclinical investigations in specific models of dopaminergic hyperactivity as well as glutamatergic hypoactivity, and assays of cortical neurotransmission.

Behavioral pharmacology

Pridopidine reduces both hyperactivity and the behavioral abnormalities pharmacologically induced in animal models of elevated dopamine or decreased glutamate neurotransmission, while the locomotor activity of intact animals is unaffected over the same dose range. Hence, pridopidine counteracts hyperactivity induced by psychotomimetics including d-amphetamine and N-methyl-d-aspartate (NMDA) antagonist MK-801 [72, 74]. In addition, pridopidine enhances locomotor activity in animals with a low baseline psychomotor activity, as seen in animals habituated to their environment [74], as well as in rats co-treated with the VMAT inhibitor tetrabenazine [75]. Pridopidine is unable to induce profound hypoactivity or catalepsy, indicating that it has a low likelihood of displaying the adverse neurological effects associated with classical dopamine D2 receptor antagonist antipsychotics [74, 76]. Pridopidine does not induce catalepsy, even at doses producing D2 receptor occupancy reaching 80% or above [76]. Considering qualitative aspects of the behavioral effects of pridopidine, it has been shown to restore social interactions in rats treated with MK-801 [77], and ameliorate the behavioral primitivization induced by MK-801 in mice [78]. Both findings are proposed to imply beneficial effects on cognitive symptoms. Furthermore, pridopidine is efficacious in the conditioned avoidance response model of antipsychotic activity [76]. Pridopidine has also been shown to display potent and efficacious antidepressant activity in the mouse tail suspension test [74]. A chronic study in a rodent model of L-DOPA induced motor complications (sensitization to repeated L-DOPA upon unilateral 6-OH-dopamine lesion) demonstrated that pridopidine reduced L-DOPA induced rotational behavior while not impairing forward locomotion [79]. In behavioral studies using the R6/2 mouse model of HD, Squitieri et al., recently reported data indicating that pridopidine reduces and/or prevents the expression of the HD motor phenotype on the horizontal ladder task and in open-field locomotor measurements during 6 weeks’ treatment, starting in the pre-symptomatic stage. It also prolonged the life-span in R6/2 mice. Wild-type behaviors in the same assays were not affected by pridopidine [80].

Neurochemical effects

Pridopidine increases synthesis, release and metabolism of dopamine in sub-cortical areas [74], mimicking the effects of dopamine D2 antagonists in general [22]. The increases in dopamine turnover and transmission biomarkers produced by dopamine D2 antagonists in sub-cortical areas are due to inhibition of dopamine-D2-receptor-dependent negative feedback [22, 81]. These results indicate that pridopidine acts as an antagonist at dopamine D2 receptors in vivo. Furthermore, in an in vivo study designed to detect agonist properties, Natesan et al., tested pridopidine’s ability to reverse DOPA accumulation induced by reserpine (an inhibitor of the vesicular monoamine transporter) [76]. They found that pridopidine had no effect in this sensitive agonist assay. Pridopidine increases plasma levels of prolactin in rats, suggesting antagonistic effects at hypothalamic dopamine D2 receptors in vivo [82]. The interaction of pridopidine at dopamine D2 receptors is further demonstrated by in vivo binding experiments showing that pridopidine dose-dependently displaces the dopamine D2 antagonist raclopride from dopamine D2 receptors [76]. Taken together, the in vivo dopaminergic neurochemistry and in vivo binding data on pridopidine consistently indicate that pridopidine acts as a full dopamine D2 antagonist. In vivo microdialysis studies in conscious rats have demonstrated that pridopidine dose-dependently increases the efflux of dopamine and noradrenaline in the cortical and subcortical projection areas of the ascending dopamine projections [74]. At doses efficacious with respect to the key behavioral effects, prefrontal cortex and striatal levels of both dopamine and noradrenaline are increased.

Analysis of gene expression profiles has demonstrated that pridopidine increases the expression of activity-regulated cytoskeleton-associated protein [83] (Arc) mRNA in the frontal cortex and the striatum [84]. Furthermore, pridopidine has been shown to reverse the reduction in striatal Arc levels induced by MK-801 [85]. The increase of Arc mRNA expression suggests an increase in synaptic activity [86], possibly related to facilitation of NMDA receptor activation [87, 88]. In addition to the ex vivo biomarker studies demonstrating cortical effects of pridopidine, a recent study shows that pridopidine increases firing in prefrontal pyramidal cells [79]. The pridopidine-driven increase in pyramidal cell firing was antagonized by the dopamine D1 antagonist SCH23390, suggesting that the pyramidal cells are indirectly activated by pridopidine through increased levels of dopamine binding with D1 receptors. The cortical effects of pridopidine, as reflected, by increased dopamine release in the frontal cortex, increased Arc gene expression, and increased cortical neuronal activity, may contribute to its potency in behavioral assays. This is reported to be higher than the potency in increasing striatal 3,4-dihydroxyphenylacetic acid, essentially reflecting D2 antagonism in vivo [74]. It has been shown that addition of a compound that increases cortical dopamine release improves the potency of D2 antagonists in behavioral models of antipsychotic activity [89, 90]. Also, dopamine D1 agonists per se have been shown to exert anti-psychotic like behavioral effects [91].

In vitro pharmacology

In vitro binding studies demonstrate affinity in the micromolar range at dopamine D2 receptors. [72]. Assessments on functional responses in different settings in vitro show that pridopidine displays competitive antagonism with a fast dissociation rate from the dopamine D2 receptor [92], and that pridopidine, just as in the in vivo assays, lacks intrinsic activity at dopamine D2 receptors [92–94].

The affinity of pridopidine measured at dopamine D2 receptors is slightly higher using agonist vs antagonist counter ligands, k_{i antagonist}/k_i agonist  = 2.3 [72]. Dopamine D2 receptors exists in two states (i) the resting and low-affinity state (D2R^Low) and (ii) the active, catalytic, high-affinity state (D2R^High). Dopamine D2 receptors agonists bind with preference to receptors in D2R^High and induce a full catalytic reaction, i.e. they have affinity and intrinsic activity. Pridopidine has been proposed to preferentially bind to the high-affinity state, but without intrinsic activity [72, 95, 96]. This would differentiate pridopidine from classical D2 receptor antagonists, which, in contrast, stabilize the D2R^Low state and do not show preference for either receptor state. In summary, in vitro as well as in vivo studies addressing dopamine receptor interactions consistently indicate that pridopidine acts as a competitive, low-affinity dopamine D2 antagonist with fast-off receptor-dissociation kinetics and with a slight preference for the agonist binding site.

Pridopidine has also been reported to display in vitro affinity in the micromolar range at adrenergic alpha 2A/C receptors (k_i 20/2.3 μM), serotonergic 5HT1A (k_i 3.1 μM) and 5HT2A (k_i 15 μM) receptors, and histamine H3 (k_i 7.2 μM) receptors [79]. Interactions at these receptors, each of which has been shown to modulate extracellular levels of monoamines [97–100], as well as glutamatergic transmission in the cortex [101–104], may contribute to the in vivo effects of pridopidine. Apart from the monoaminergic receptors, pridopidine has been reported to display moderate affinity at the sigma-1 receptor in vitro [105]. The significance of this is unclear, as sigma-receptor binding is displayed by a wide range of CNS-active compounds from different therapeutic classes, but it has been proposed to contribute to neuroprotective properties of pridopidine [80]. In support of this, a recent ex vivo study on striatal gene expression in rats showed upregulation of several neuroprotective pathways by pridopidine, including brain derived neurotrophic factor (BDNF), glucocorticoid receptor, and AKT/phosphoinositide 3-kinase (PI3K), and dopamine D1 pathways, and further suggested that sigma1-receptor activation is necessary for the effect of pridopidine on the effect on the BDNF pathway, based on assessment of in vitro BDNF secretion in neuroblastoma cells, which was antagonized by co-incubation with a sigma-1 antagonist [106]. Recently published additional gene expression analyses from the same study demonstrated upregulation of calcium regulating proteins including calbindin and homer1a upon chronic treatment with pridopidine. Furthermore, it was shown that pridopidine normalized calcium homeostasis and prevented spine loss in aging corticostriatal co-cultures from YAC128 mice. Spine rescue and normalized Ca2 + levels were prevented by deleting sigma-1 receptors, suggesting pridopidine exerts synapto-protective effects, through sigma-1 receptors and effects on calcium signaling [107].

Summary of preclinical pharmacology

The in vivo pharmacological profile of pridopidine can be summarized as state-dependent inhibition or activation of dopamine-dependent psychomotor functions (Fig. 1). This is achieved through low-affinity/fast-off antagonism of dopamine D2 receptors, combined with an increased cortical and sub-cortical monoamine release resulting in enhanced activity of prefrontal cortex neurons. It thus appears as if pridopidine exerts its behavioral stabilizing effects by acting on several key nodes of the cortico-striatal network regulating psychomotor functions. In addition to these acute in vivo effects as described in terms of major neuronal pathways, emerging data suggest sigma-1 receptor mediated effects, putatively involving regulation of intraneuronal calcium signaling, which may be of relevance in particular with regard to neuroprotective effects of pridopidine.

Fig.1

Pridopidine is able to enhance or inhibit dopamine-dependent functions. Graphic illustration of psycho-motor stabilization: In vivo pharmacological studies have consistently demonstrated state dependent behavioral effects of pridopidine; reducing psychomotor activity in hyperactive states, and enhancing activity in hypoactive states. This is proposed to translate to stabilization of both hyper- and hypokinetic motor disturbances in HD; to some extent overlapping with observations of disturbed dopamine transmission over the course of the disease [40].

Pridopidine strengthens the indirect pathway via antagonism of dopamine D2 receptors

The mechanism of action for pridopidine involves dopamine D2 receptor antagonism [72, 92, 95, 96]. MSNs projecting to the indirect pathway are negatively modulated by dopamine through dopamine D2 receptors. Hence, dopamine attenuates the GABAergic output from these neurons. Diminished activity in this pathway results in a reduced capacity to suppress involuntary movements. By blocking the dopamine D2 receptors on the MSNs of the indirect pathway, the inhibitory influence of dopamine is reduced, and the output via the indirect pathway is strengthened. Therefore, by antagonizing dopamine D2 receptors in the striatum, pridopidine normalizes the aberrant function in the indirect pathway, which results in attenuation of involuntary movements (Fig. 2, right panel). This is supported by clinical results suggesting reduced involuntary motor symptoms in HD patients treated with pridopidine, reported from large randomized clinical trials [63, 64].

Pridopidine strengthens the direct pathway by stimulating dopamine D1 receptors

MSNs projecting to the direct pathway are positively modulated by dopamine through dopamine D1 receptors. Hence, dopamine enhances the GABAergic output from these neurons. Administration of pridopidine increases the release of striatal dopamine [74]. This implies that pridopidine, by increasing synaptic availability of dopamine, thus indirectly stimulating the dopamine D1 receptors, could strengthen the striatal output in the direct pathway (Fig. 2). This would result in improvements of voluntary movement control in HD. In our view, this hypothesis seems to be supported by clinical outcomes of the MermaiHD (Multinational European Multicentre ACR16 study in Huntington’s Disease) and HART (Huntington’s disease ACR16 Randomized Trial) trials indicating improvement in voluntary motor control such as oculomotor and postural function, and hand movements [63, 64].

Consistent with the notion that pridopidine activates striatal D1 receptors, and antagonizes D2 receptors, pridopidine has been demonstrated to dose-dependently increase expression of striatal Arc mRNA [84]. To the best of our knowledge, pridopidine displays no affinity or direct activity at any glutamate receptors investigated. Therefore, such NMDA receptor activation is not likely to occur as a direct effect. Rather, given the functional association between D1 and NMDA receptors in MSNs [108] the increase in striatal Arc mRNA levels could arise indirectly as a consequence of synaptic NMDA receptor modulation, related to activation of dopamine D1 receptors. This is in line with the findings that dopamine D1 receptor agonists increase, and dopamine D1 antagonists decrease striatal Arc expression [109, 110]. Activation of DA D1 receptor mediated pathways by pridopidine was corroborated in a recent gene profiling study indicating upregulation of D1 mediated gene expression upon repeated administration of pridopidine [106]. The effect of pridopidine on striatal Arc levels may also be associated with the direct antagonism of striatal D2 receptors, leading to reduced inhibitory tone on cortico-striatal glutamate release, and therefore to increased glutamate transmission. Induction of striatal Arc gene expression has been reported for several D2 receptor antagonists [111, 112]. Further studies, e.g. investigating the localization of Arc induced in D1 vs D2 receptor expressing MSNs, would be needed to determine more precisely how MSNs of the direct and indirect pathways are affected by pridopidine. Furthermore, animal studies have suggested a biphasic pattern of changes in cortico-striatal communication in HD, with increased signaling in early stages, before reaching the late stage represented in Fig. 2 [53, 55]. Thus, it would be of interest to assess the impact of pridopidine on cortico-striatal communication at different stages of the disease.

Pridopidine strengthens cortical activity

In manifest HD, progressive thinning of the cortex is observed [113], and preclinical studies suggest decreased communication in the cortico-striatal glutamatergic projections, in addition to the degeneration of striatal MSNs [53]. These alterations are associated with cognitive impairments [114] and are proposed to result in reduced activation of the direct and indirect pathway [53] (Fig. 2, left panel), hampering motor control [53]. The pathogenetic impact of disrupted cortico-striatal connectivity for the HD phenotype has further been demonstrated in pre-clinical HD models, showing that expression of mutant htt in both the cortex and the striatum is required to develop the full pathological phenotype [115].

Pridopidine has been demonstrated to dose-dependently increase dopamine in the prefrontal cortex [74]. The strengthening of frontal cortex dopamine transmission is further hypothesized to drive down-stream effects in the cortico-striatal circuitry, regulating motor functions.

As a more direct read-out of neuronal activity in the frontal cortex, administration of pridopidine has been demonstrated to dose-dependently increase Arc gene expression in rat frontal cortex, interpreted as increased activation of synaptic NMDA receptors [84]. Preliminary observations that the extra-synaptically acting NMDA receptor antagonists memantine and ifenprodil display similar effects as pridopidine, with respect to frontal cortex Arc gene expression, provide indirect support for the notion that pridopidine shifts the balance towards synaptic NMDA receptor signaling [85]. Given the synergistic interaction between dopamine D1 and NMDA receptor signaling in cortical pyramidal cells, it is proposed that such enhancement of synaptic NMDA receptor signaling by pridopidine arises indirectly due to increased cortical dopamine transmission followed by activation of dopamine D1 receptors. This is supported by recently recorded observations that pridopidine increases the firing frequency of rat pyramidal neurons in the frontal cortex, and that this effect could be blocked by administration of the D1 antagonist SCH23390 [79]. Increased activity of dopamine D1-expressing glutamatergic cells in the cortex would promote cortico-striatal communication, and indirectly drive the indirect and direct pathways (Fig. 2). The effects of pridopidine to reduce hypoactivity in partially monoamine-depleted rats, concurring with increased frontal cortex Arc gene expression, provide some support for a cortically driven improvement of voluntary motor function by pridopidine.

Pridopidine displays clear-cut effects on cortical dopamine transmission, which are likely to contribute to effects such as those on Arc gene expression and pyramidal cell firing activity, and to the overall behavioral profile. However, pridopidine may also influence cortical neurons by other mechanisms. In vivo microdialysis studies have demonstrated increased levels of not only dopamine, but also noradrenaline, which modulates cortical activity through alpha 1 and alpha 2 receptors [74, 79]. α2-adrenoceptor blockade has been shown to induce cortical Arc gene expression, likely by increased NA release [116]. Furthermore, the affinity of pridopidine at adrenergic alpha2c, 5HT1a and histamine H3 receptors [79], as well as sigma-1 receptors [105], may be of relevance. Regardless of the exact mechanism for the Arc upregulation in vivo, it provides evidence of unique cortical effects of the dopidines, as compared to other DA modulating compounds in general, and antipsychotics in particular [84]. Such effects could contribute to the characteristic behavioral pharmacology of the dopidines, and further support the notion of potential cognitive enhancing properties.

The proposed in vivo mode of action of pridopidine is based on the state of manifest HD, a symptomatic stage in which impairment of both the indirect and direct pathways has developed (Fig. 2). This would suggest the use of pridopidine for motor symptom alleviation in advanced HD, targeting both voluntary and nonvoluntary motor impairment. However, emerging evidence suggesting potential beneficial effects on disease progression could motivate earlier interventions.