A Phase I, First-In-Human, Healthy Volunteer Study to Investigate the Safety, Tolerability, and Pharmacokinetics of CVN424, a Novel GPR6 Inverse Agonist for Parkinson's Disease

CVN424 is a novel small molecule and first-in-class candidate therapeutic to selectively modulate GPR6, an orphan G-protein coupled receptor. Expression of GPR6 is largely confined to the subset of striatal projection neurons that give rise to the indirect (striatopallidal) pathway, important in the control of movement. CVN424 improves motor function in preclinical animal models of Parkinson's disease. Here we report results of a phase 1, first-in-human study investigating the safety, tolerability, and pharmacokinetics of CVN424 in healthy volunteers. The study (NCT03657030) was randomized, double-blind, and placebo controlled. CVN424 was orally administered in ascending doses to successive cohorts as inpatients in a clinical research unit. Single doses ranged from 1 mg to 225 mg, and repeated (7 day) daily doses were 25 mg, 75 mg, or 150 mg. CVN424 peak plasma concentrations were reached within 2h post-dose in the fasted state and increased with increasing dose. Dosing after a standardized high-fat meal reduced and delayed the peak plasma concentration but total plasma exposure was similar. Mean terminal half-life ranged from 30h to 41h. CVN424 was generally well tolerated: no serious or severe adverse effects were observed, and there were no clinically significant changes in vital signs or laboratory parameters. We conclude that CVN424, a nondopaminergic compound that modulates a novel therapeutic target, was safe and well tolerated. A phase 2 study in patients with Parkinson's disease is underway. Significance Statement This is the first-in-human clinical study of a first-in-class candidate therapeutic. CVN424 modulates a novel drug target, GPR6, which is selectively expressed in a pathway in the brain that has been implicated in the motor dysfunction of patients with Parkinson's disease. This study paves the way for investigating this novel mechanism of action in patients with Parkinson's disease.


SAD
Single ascending dose

Introduction
The ability to pharmacologically modulate specific cell types and circuits offers great promise for development of new therapies to improve the management of Parkinson's disease and other neurological disorders (Fishell and Heintz, 2013). Parkinson's disease patients commonly experience lapses in symptom relief (e.g., motor fluctuations) despite treatment with standard-of-care dopaminergic medications, and dosage increases intended to overcome those episodes are often limited by side effects, notably drug-induced dyskinesia.
Parkinson's disease motor symptoms result from degeneration of dopamine-producing neurons of the nigrostriatal pathway and the consequent impact on dopamine-receptive striatal neurons and their efferent circuits. There are two major types of dopamine-receptive neurons in the striatum, which differ in the type of dopamine receptor they express and that give rise to separate efferent pathways. Medium spiny neurons that express dopamine D1 receptors give rise to the striatonigral "direct" pathway, whose activity normally facilitates movement but can also drive the involuntary movements of levodopa-induced dyskinesia 6 (Ryan et al., 2018). Medium spiny neurons that express D2 receptors give rise to the striatopallidal "indirect" pathway, whose activity inhibits movement. Normally, dopamine acting via these D2 receptors lowers the neuronal level of 3'-5'-cyclic adenosine monophosphate (cAMP), facilitating movement by reducing neuronal activity in these striatal neurons and thus lessening the motor-inhibitory influence of the indirect pathway.
Conversely, under the pathological condition of dopamine depletion in Parkinson's disease, the indirect pathway becomes hyperactive (DeLong & Wichmann, 2015) and contributes to bradykinesia and "freezing" of gait.
CVN424 is a potent and selective inverse agonist of GPR6, an orphan G-protein coupled receptor that is selectively expressed by the D2 receptor-positive neurons of the indirect pathway (Heiman et al., 2008). Expression of GPR6 is very low or absent in D1 receptorpositive neurons of the direct pathway and in other central nervous system regions and peripheral tissues (Morales et al., 2018)  . Since GPR6 is a Gs-coupled receptor with high constitutive activity (Uhlenbrock et al., 2002), it normally increases cAMP and thereby activates the indirect pathway, in opposition to the inhibitory effect of the Gicoupled D2 receptors. CVN424 suppresses this constitutive activity, reducing cellular cAMP levels   and potentially attenuating the pathological hyperactivity of the indirect pathway seen in Parkinson's patients ( Figure 1). Thus, CVN424 is predicted to mimic the salutary effect of dopaminergic medications on the indirect pathway but without concurrent activation of the direct pathway. CVN424 should thereby facilitate voluntary movement without exacerbating levodopa-induced dyskinesia, and thus has therapeutic potential in Parkinson's disease as a monotherapy or as an adjunct to levodopa. Preclinical testing of CVN424 included an extensive panel of pharmacology studies conducted in vitro and in vivo in rodents . These findings established that CVN424 was orally bioavailable and central nervous system penetrant. Importantly, CVN424 was effective at enhancing motor function in Parkinson's disease preclinical models, such as the 6hydroxydopamine lesion model in rodents , supporting the GPR6 inverse agonist therapeutic hypothesis.
Based on these data, a first-in-human trial was conducted to investigate the safety, tolerability, and pharmacokinetics of CVN424 in healthy subjects.

Study Drug
Aqueous suspensions of CVN424 contained hydroxypropyl methylcellulose 200 mg, Tween-80 50 mg, and 10 mL water. Placebo contained the same ingredients but omitted active drug.
Applying a 20-fold safety margin below the human-equivalent dose corresponding to the NOAEL from nonclinical toxicology studies, the maximum recommended starting dose for this first-in-human study per FDA guidance was 0.8 mg/kg, or 48 mg for a 60-kg subject. However, based on preclinical data, that dosage was predicted to yield up to 98% receptor occupancy of GPR6 sites in the brain. To be conservative, the starting dosage for the SAD was set at 1 mg, with an expected peak receptor occupancy of 58%.

PK assessments
In the SAD cohorts, blood samples were obtained for PK analyses on day 1 pre-dose and serially through 72 hours post-dose. In the MAD cohorts, blood samples were collected for PK analyses on day 1 pre-dose and serially through 24 hours post-dose (i.e., day 2 pre-dose); predose on days 3, 4, 5, and 6; and on day 7 pre-dose and serially through 72 hours post dose.
Plasma obtained after centrifugation was stored frozen at -70 o C until analysis.
Plasma concentrations of CVN424 were quantitated using a validated assay based on liquid chromatography coupled with tandem mass spectrometry (Frontage Laboratories, Inc.; Exton, PA). The assay has a dynamic range of 0.100 -100 ng/mL; to extend the range, high concentration samples were retested after dilution.
The following PK parameters were calculated for CVN424 from plasma concentration and actual time data for each subject by noncompartmental analysis using Phoenix® WinNonlin®  Accumulation ratios based on AUC (Rac(AUC)) and C max (R ac(Cmax ) and dose-normalized AUCs, C max , and C max,ss were also calculated.
The PK parameters were summarized by treatment using summary statistics. Approximate attainment of steady state was visually assessed by plotting mean trough concentrations.
To evaluate dose proportionality, a power model was fitted to describe the relationship between Y (C max , AUC 24 , AUC t , and AUC ∞ for single-dose cohorts and C max,ss , AUC 24 , AUC t , and AUC tau for multiple-dose cohorts) and X (dose) using the least-squares linear regression model . Dose proportionality was concluded if the 90% CI of the slope β lies entirely within [1 + ln(0.8) / ln(r), 1 + ln(1.25) / ln(r)], where r is a ratio that describes the dose range and was defined as the ratio of highest dose/lowest dose (Smith et al., 2000).
To evaluate the effect of food on PK of CVN424 in suspension formulation, a linear mixed-effect model (SAS PROC MIXED) with treatment as a fixed effect and measurements within subject as repeated measures was fitted to the natural log-transformed PK parameters C max , AUC 24 , AUC t , AUC ∞ , and for use in estimation of effects and construction of CIs for SAD cohort S2 Fed compared with SAD cohort S2 Fasted. Point estimates and 90% CIs for differences on the log scale were exponentiated to obtain estimates for the ratios of geometric means and respective 90% CIs on the original scale.
This article has not been copyedited and formatted. The final version may differ from this version.

Statistical analyses
The safety set included all subjects who were enrolled and received study drug. The safety set was used for demographic, baseline characteristics, and safety summaries. The PK set included all subjects who received study drug and had at least 1 measurable plasma concentration.
Medical history and adverse events were coded using the Medical Dictionary for Regulatory

Subjects
A total of 64 subjects were enrolled, 48 received CVN424 (30 in the SAD cohorts and 18 in the MAD cohorts), and 16 received placebo (10 in the SAD cohorts and 6 in the MAD cohorts). One MAD subject who received 75 mg dose of CVN424 discontinued early because of an adverse event of dysphagia; all other subjects completed the study as per protocol. The demographic characteristics of the enrolled subjects are summarized in Table 1.

Pharmacokinetics
The mean plasma concentration-time profiles of CVN424 in the SAD and MAD cohorts are presented in Error! Reference source not found..

Safety and tolerability
A summary of treatment emergent adverse events (TEAEs) is presented in

Discussion
The current standard of care for Parkinson's disease is symptomatic treatment by dopamine replacement, dopamine agonists, or analogous mechanisms. After several years of treatment, many patients experience motor fluctuations that limit the effectiveness of those drugs and may also develop dyskinesias that are exacerbated by dopaminergic agents (e.g., levodopainduced dyskinesias). Novel, non-dopaminergic therapies like CVN424 have potential to improve treatment of such patients.
This first-in-human study investigated the safety, tolerability, and PK of single and multiple escalating doses of CVN424 in healthy volunteers. CVN424 mean peak (C max ) and total (AUC 24 , AUC t , and AUC ∞ ) plasma exposure increased with increase in CVN424 dose across the evaluated dose range (1-225 mg once daily, or 25-150 mg once daily for 7 days), although the increase appeared to be less than dose proportional. The food effect comparison showed a 33% reduction in C max and a delay in t max with food. A small increase in the overall exposure was noted when given with food (comparison of AUC ∞ , 19%), although AUC 24 and AUC t values met equivalence criteria. Moderate plasma accumulation of CVN424 was observed following 7 day once daily dosing of CVN424 oral suspension, as expected given the drug's half-life, with apparent steady-state concentrations of CVN424 being achieved following 4 or 5 days of dosing.
The administration of CVN424 was safe and well tolerated and resulted in no serious adverse events. The only early discontinuation was for dysphagia (75mg group), an adverse event not reported by any other subject.
This article has not been copyedited and formatted. The final version may differ from this version. Transient increases in temperature and heart rate, observed as first-dose effects, were not clinically significant in the study subjects. It is uncertain whether similar vital sign changes will be observed in Parkinson's disease patients, who may be elderly with various cardiac and autonomic co-morbidities and using concomitant medications. Curiously, the rise in heart rate averaged across all CVN424 cohorts was about 8 beats per minute per degree Celsius rise in temperature (data not shown), which is close to the 8.5 beats per minute physiological rise in heart rate linked to each degree Celsius rise in febrile patients in previous studies (Karjalainen J, 1986). If that is not coincidental, perhaps CVN424 induces a temperature rise that indirectly leads to the increase in heart rate. An in vitro selectivity screen of CVN424 against 110 receptors, channels, and enzymes showed inhibition greater than 50% at 10 µM for adrenergic α1 receptors (maximum inhibition seen was 70% for α1D) and the sigma receptor , although it was not determined if this inhibition in binding translates to a functional effect. The maximum total plasma concentration achieved in this trail was 1097 ng/ml (2.3 µM) at the top dose of the SAD cohort. Although adrenergic receptors are known to effect heart rate it is unlikely that the free drug concentration was sufficient to modulate these receptors. Therefore, the mechanism by which CVN424 or modulation of GPR6 might cause the observed changes in vital signs is uncertain.
The data presented here detail the PK profile of a suspension formulation, while use of solid form in future clinical studies may require bridging PK analysis to assess bioequivalence.
While GPR6 expression is most prominent in striatal indirect pathway neurons, GPR6 expression has been observed in a subset of hypothalamic neurons in mice and humans (Henry et al., 2015; Cerevance unpublished data], which suggests a possible mechanism to account for our       (a) 1.500 (1.00, 1.52)   Abbreviations: AE, adverse event; SAD, single ascending dose; TEAE, treatment-emergent AE. Note: A TEAE was defined as any AE with onset occurring within 30 days after study drug administration.
N represents the number of subjects within each treatment and overall; n represents the number of subjects at each level of summarization.