Characterization of PF-6142, a Novel, Non-Catecholamine Dopamine Receptor D1 Agonist, in Murine and Nonhuman Primate Models of Dopaminergic Activation

Abstract

Selective activation of dopamine D1 receptors remains a promising pro-cognitive therapeutic strategy awaiting robust clinical investigation. PF-6142 is a key example from a recently disclosed novel series of non-catechol agonists and partial agonists of the dopamine D1/5 receptors (D1R) that exhibit pharmacokinetic (PK) properties suitable for oral delivery. Given their reported potential for functionally biased signaling compared to known catechol-based selective agonists, and the promising rodent PK profile of PF-6142, we utilized relevant in vivo assays in male rodents and male and female non-human primates (NHP) to evaluate the pharmacology of this new series. Studies in rodents showed that PF-6142 increased locomotor activity and prefrontal cortex acetylcholine release, increased time spent in wakefulness, and desynchronized the EEG, like known D1R agonists. D1R selectivity of PF-6142 was supported by lack of effect in D1R knock-out mice and blocked response in the presence of the D1R antagonist SCH-23390. Further, PF-6142 improved performance in rodent models of NMDA receptor antagonist-induced cognitive dysfunction, such as MK-801-disrupted paired-pulse facilitation, and ketamine-disrupted working memory performance in the radial arm maze. Similarly, PF-6142 reversed ketamine-induced deficits in NHP performing the spatial delayed recognition task. Of importance, PF-6142 did not alter the efficacy of risperidone in assays predictive of antipsychotic-like effect in rodents including pre-pulse inhibition and conditioned avoidance responding. These data support the continued development of non-catechol based D1R agonists for the treatment of cognitive impairment associated with brain disorders including schizophrenia.

Keywords: Parkinson’s disease; prefrontal cortex; pro-cognitive therapeutics; schizophrenia; working memory.

Figure 1

Acetylcholine following administration of PF-6142 in rat and mouse. Acetylcholine levels in the rat prefrontal cortex (PFC) following subchronic dosing with PF-6142. (A) Time course data comparing the effect of vehicle or PF-6142 (10 mg/kg, SC) on ACh levels in the rat PFC after repeated dosing for 5 d. (B) 75–180-min total area under the curve of time course data. Points represent the mean + SEM, One-way ANOVA with Dunnett’s post-test adjusted *p < 0.05, **p < 0.01 vs. vehicle. N = 7–8. (C) wild-type (WT) and D1 KO mouse acetylcholine levels in the PFC. 75–180-min total area under the curve (AUC) data for WT and D1 KO mice treated with PF-6142 (10 mg/kg, SC) and SCH-23390 (0.32 mg/kg, SC). Two-way ANOVA with Tukey’s post-test adjusted *p < 0.05, **p < 0.01 vs. vehicle + vehicle within genotype. N = 6–7.

Figure 2

Mouse locomotor activity (LMA) following administration of PF-6142. (A) Treatment with PF-6142 increases the number of beam breaks and pretreatment with the D1 antagonist, SCH-23390, effectively and dose dependently blocks the hyperactivity induced by PF-6142. (B) Daily administration of D1 agonists. (C) The hyperactive response is greatly diminished in the D1 KO mice compared to WT mice. Data are shown as the mean beam breaks + SEM. N = 8. One-way ANOVA with Dunnett’s post-test adjusted *p < 0.05, **p < 0.01 vehicle + PF-6142 (10 mg/kg) (A). Two-way ANOVA with Dunnett’s (B) or Tukey’s (C) post-test adjusted *p < 0.05, **p < 0.01 vs vehicle (B) or vehicle + vehicle (C).

Figure 3

Quantitative electroencephalography and polysomnography analysis of treatment with PF-6142. (A) Systemic PF-6142 treatment significantly and dose dependently modulates oscillatory power in freely moving rats. Vehicle or PF-6142 (low: 1.0 mg/kg, high: 5.6 mg/kg, SC) was administered in the morning during the inactive period of the animals in their home cage. Delta (0.5–4 Hz), theta (4–9 Hz), alpha (9–13 Hz), beta (13–28 Hz), and gamma (28–80 Hz) oscillation power was analyzed during the first 4 h following treatment together with the total power contained in the signal. Statistical analyses revealed that treatment only with the high dose resulted in significant (*p < 0.05 vs. vehicle, #p < 0.05 vs. low) decrease of delta and total powers and increase of beta and gamma powers. Symbols (x for vehicle, Δ for low, and + for high dose) show values for individual animals. All animals are shown, outliers are not indicated separately, the upper and lower hinges on the boxplots show the 25^th and the 75^th percentiles, respectively, horizontal bar in the boxplot shows median value, whiskers extend to the minimum and the maximum values. (B) Systemic PF-6142 treatment significantly and dose dependently increases the time spent in wakefulness in freely moving rats. The fraction of time rats spent awake (Wake), in slow-wave sleep (SWS), or in REM sleep during the first 4 h following treatment were analyzed. Statistical analyses revealed that treatment with the high dose (5.6 mg/kg, SC) resulted in a significant (*p < 0.05 vs. vehicle, ^#p < 0.05 vs. low) decrease of the time spent in SWS and REM sleep, and an increase of the time spent awake. Boxplots are set up as described in (A).

Figure 4

PF-6142 Does not alter the antipsychotic-like activity of risperidone in rodents. (A) As expected, risperidone produced increases % PPI in adult male C57BL/6J mice consistent with an antipsychotic-like profile in this assay. Administration of PF-6142 alone or in combination with risperidone does not alter PPI responses (n=8 per treatment group). (B) Statistically significant reductions in startle responses were observed with the highest dose of risperidone (0.56 mg/kg) which was not altered in the presence of PF-6142. (C) PF-6142 alone produced modest non-significant reductions in % PPI in adult C57BL/6J mice. (D) Risperidone produces significant reductions in avoidance responding consistent with antipsychotic-like activity in adult male rats (n=8–9 per treatment) in the conditioned avoidance responding assay. There is no effect of PF-6142 alone or in combination with risperidone in avoidance responding. One-way ANOVA with Tukey’s post-test adjusted *p < 0.05, **p < 0.01 vs. vehicle + Risperidone (0.56 mg/kg) ***p < 0.001 vs. vehicle + vehicle. Note:2-way RM ANOVA (A). One-way ANOVA with Dunnett’s post-hoc test ****p < 0.0001 vs. vehicle + vehicle (D).

Figure 5

PF-6142 effects on ketamine-induced working memory deficits. (A) Pre-treatment with PF-6142 dose-dependently prevents ketamine-induced deficits in the rat radial arm maze assay. Data are presented as mean errors + SEM. Kruskal-Wallis test with Dunn’s multiple comparison post-test *p < 0.05, **p < 0.01 vs. vehicle + ketamine. N=10–21. (B) Treatment with PF-6142 prevents ketamine-induced deficits in the non-human primate spatial delayed response task. One-way ANOVA with Dunnett’s post-test adjusted *p < 0.05 vs. vehicle + Ketamine and N = 8 NHP.

Figure 6

Effects of PF-6142 on NMDA antagonist disrupted paired-pulse facilitation (PPF) and delta oscillations. (A) PF-6142 significantly reverses NMDA blockade-induced changes in PPF. PPF (calculated as P2/P1) values as a function of drug treatment. Note that PF-6142 reverses MK-801-evoked decrease of PPF with an ED₅₀ of 0.35 mg/kg (*: p<0.05 vs. Control, #: p<0.001 vs. MK-801; n=5). The upper and lower hinges on the boxplots show the 25^th and the 75^th percentiles, respectively, horizontal bar in the boxplot shows median value, whiskers extend to the minimum and the maximum values, “o” indicates data points outside of the 1.5*inter-quartile range of the hinges. (B) PF-6142 significantly reverses NMDA blockade-induced mPFC low frequency delta activity increase. Power contained in the low frequency delta (0–1.8 Hz) band expressed as a percentage of the total delta (0–4 Hz) power. Note that PF-6142 completely reverses MK-801 induced increase of low frequency delta oscillation (*: p<0.02 vs. Control; #: p<0.02 vs. MK-801; n=5). (C) Antagonism of D1Rs blocks effects of PF-6142 on PPF. Figure shows PPF values as a function of drug treatment. Note that administration of SCH-23390 alone had no effect on either the P1 or P2 components or PPF, while this pretreatment completely blocked PF-6142 effects even at high doses. (D) Antagonism of D1Rs blocks effects of PF-6142 on mPFC low frequency delta activity. Power contained in the low frequency delta (0–1.8 Hz) band expressed as a percentage of the total delta (0–4 Hz) power. Note that as with PPF SCH-23390 completely blocks effects of PF-6142 on reversing MK-801-induced changes (*p<0.05 vs. Control, ^#p<0.03 vs. SCH-23390; n=5).