Efects Of DPTQ, A Novel Positive Allosteric Modulator Of The Dopamine D1 Receptor, On Spontaneous Eye Blink Rate And Spatial Working Memory in The Nonhuman Primate Part 1

Abstract

Rationale

Dopamine (DA) signaling through the D1 receptor is integral to multiple aspects of cognition, including the core process of working memory. The discovery of positive allosteric modulators (PAMs) of the D1 receptor has enabled treatment modalities that may have alternative benefits to orthosteric D1 agonists arising from a synergism of action with functional D1 receptor signaling.

Dopamine is a neurotransmitter that has important effects on how the brain works. It plays an important role in regulating emotions, eating, emotion, learning, memory, and many other aspects. Recent studies have found a strong relationship between dopamine and memory.

Studies have shown that dopamine can enhance an individual's ability to remember situations and events. This is because dopamine regulates the interactions between neurons in the brain, making relevant information easier to store and retrieve. At the same time, dopamine can strengthen the connection between neurons, improve the transmission efficiency between neurons, and further promote the formation and maintenance of memory.

In real life, we can increase dopamine secretion in various ways to improve our memory. For example, exercising can promote the secretion of dopamine, so regular physical exercise is an effective way to improve memory. In addition, some foods such as beans, nuts, whole wheat bread, etc. can also increase the secretion of dopamine and help us improve memory.

In daily study and work, we can also improve memory by stimulating the brain's dopamine system. For example, we can activate the dopamine system by setting goals, rewarding ourselves, and providing regular feedback, thereby improving our memory and understanding of the knowledge we have learned. In addition, a positive attitude and good mood can also promote the secretion of dopamine and improve memory.

To sum up, there is a close relationship between dopamine and memory. Through scientific methods and the correct attitude, we can promote the secretion of dopamine, improve our memory, and better cope with challenges in study and work. It can be seen that we need to improve memory, and Cistanche deserticola can significantly improve memory, because Cistanche deserticola can also regulate the balance of neurotransmitters, such as increasing the levels of acetylcholine and growth factors. These substances are very important for memory and learning. In addition, Meat can also improve blood flow and promote oxygen delivery, which can ensure that the brain receives sufficient nutrients and energy, thereby improving brain vitality and endurance.

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Objectives

To investigate this potential, we have studied the effects of the novel D1 PAM DPTQ on a spatial delayed response working memory task in the rhesus monkey. Initial studies indicated that DPTQ binds to primate D1R with high affinity and selectivity and elevates spontaneous eye blink rate in rhesus monkeys in a dose-dependent manner consistent with plasma ligand exposures and central D1activation.

Results

Based on those results, DPTQ was tested at 2.5 mg/kg IM in the working memory task. No acute effect was observed 1 h after dosing, but performance was impaired 48 h later. Remarkably, this defect was immediately followed by a significant enhancement in cognition over the next 3 days. 

In a second experiment in which DPTQ was administered on days 1 and 5, the early impairment was smaller and did not reach statistical significance, but a statistically significant enhancement of performance was observed over the following week. Lower doses of 0.1 and 1.0 mg/kg were also capable of producing this protracted enhancement without inducing any transient impairment.

Conclusions

DPTQ exemplifies a class of D1PAMs that may be capable of providing long-term improvements in working memory.

Keywords

Positive allosteric modulator · Dopamine · D1 · Eye blink · Working memory · Spatial delayed response · Cyclic AMP · Plasticity.

Introduction
Dopamine (DA) is critically involved in many aspects of brain function including motor activity, wakefulness, and cognition. The D1 receptor in particular is crucial to maintaining high-order cognitive functions in the primate, including attention, executive function, and working memory, and this receptor is abundant in the primate prefrontal cortex where its signaling is integral to the core cognitive process of working memory (Sawaguchi and GoldmanRakic 1991; Goldman-Rakic et al. 2004). 

Working memory is a key indicator of outcome in patients with schizophrenia and its function becomes diminished in aging and neurodegenerative disorders (Bodnar et al. 2008; Rhode and Katz 2017; Zokaei and Husain 2019). These are areas of high unmet medical need. In particular, depletion of dopamine transmission (including D1 receptor signaling) in aging is known to contribute to age-related cognitive decline (Roth and Joseph 1994; Volkow et al. 1998; Karrer et al. 2017). 

Consequently, D1 receptor function has been the subject of extensive study at the behavioral, cellular, and molecular levels (Muly et al. 1998; Castner et al. 2000; Goldman-Rakic et al. 2000; Dunah and Standaert 2001; Castner and Williams 2007; Liu et al. 2017).

The development of D1 agonists to enhance cognition has presented a difficult challenge due to inverted U-shaped dose-response (Williams and Goldman-Rakic 1995; Vijayraghavan et al. 2007). In addition, prolonged D1 stimulation by high affinity/long-acting agonists can lead to tolerance development (Asin and Wirtshafter 1993; Lewis et al. 1998; Gulwadi et al. 2001; Smith et al. 2002, Ryman-Rasmussen et al. 2007). 

Hence, clinical studies with full D1 agonists have had limited success (Blanchet et al. 1996; Giardina and Williams 2001; Zang et al. 2009). More recent approaches for D1 activation have been introduced including partial dopamine D1 agonists (Roberts et al. 2010; Balice-Gordon et al. 2020; Kozak et al. 2020; Riesenberg et al. 2020). 

An alternative approach would be to develop a positive allosteric modulator (PAM) for the D1 receptor, based on the hypothesis that the activity of a PAM would be dependent on endogenous dopaminergic tone, potentiating dopamine when and where it is released. This would offer a more physiological approach that could provide a better therapeutic margin, possibly avoiding the inverted U-shaped dose-response and tolerance development seen with some D1 agonists. Recently, several selective D1PAMs from the tetrahydroisoquinoline structural class have been reported and characterized and pharmacological differentiation from D1 agonists was established in a series of in vitro and in vivo studies (Svensson et al. 2017; Bruns et al. 2018; Hao et al. 2019; Meltzer et al. 2019 and Svensson et al. 2019). 

Due to a species difference in D1 binding for the D1PAMs, these neurochemical and behavioral studies were performed in a transgenic mouse in which the murine D1 receptor was replaced with its human equivalent (hD1 mouse). Overall, the data supported a pro-cognitive potential for this approach (Bruns et al. 2018 and Meltzer et al. 2019). For the present study, we selected the D1PAM DPTQ (Hao et al. 2019), a close structural analog of the previously reported pharmacological tool DETQ and the clinical candidate LY3154207 (medaled; Hao et al. 2019; Wilbraham et al. 2021, Biglan et al. 2022 and McCarthy et al. 2022). 

DPTQ has an EC50 of 76 nM for potentiation of the dopamine-induced increase in cAMP in stably transfected human D1 cloned cells and possesses suitable physicochemical properties with acceptable brain penetration after systemic dosing (Hao et al. 2019).

Our main objectives with this study were to establish central pharmacodynamic activity and explore potential efficacy on cognition with a D1PAM in a higher species, the rhesus monkey. Members of the tetrahydroisoquinoline D1PAM series have been previously shown to have a high affinity for this species, similar to humans (Svensson et al. 2017, Wang et al. 2018 Hao et al. 2019). 

For initial pharmacodynamic testing, we selected the spontaneous eye blink rate model in the rhesus monkey to establish doses needed to achieve a central D1 response for DPTQ in a higher species. D1 agonists are known to increase the eye blink rate in this model (Elsworth et al. 1991; Jutkiewicz and Bergman 2004). In addition, Parkinson’s disease patients have reduced spontaneous eye blink rate (Deuschl and Goddemeier 1998; Karson et al 1982). 

Although there are many interactive neurotransmitter systems involved in spontaneous eye blink rate, this model can be used as a marker for central dopamine D1 receptor activation (Jutkiewicz and Bergman 2004) and might also reflect effects on cognition (Taylor et al. 1999; Jongkees and Colzato 2016). A previous study with DETQ showed enhanced spontaneous eye blink rate, although with a smaller response than that which was observed with the D1 agonist SKF82958 (Bruns et al., 2018). 

For cognitive testing in the rhesus monkey, we selected the spatial delayed response task (Roberts et al., 2010), a highly established translational measure of spatial working memory that has been shown to engage the same neural circuitry in human and nonhuman primates (Jonides et al., 1993; GoldmanRakic, 1996; McCarthy et al., 1996; Postle et al., 2000).

Methods

In vitro testing in the cAMP assay

The human, rhesus monkey, dog, or mouse D1 receptor was cloned into the Jump-In vector system (Life Technologies Corp, CA) and transiently transfected into HEK293 cells. Increases in cAMP by DPTQ (free base, Eli Lilly) were measured in the presence of an EC20 concentration of dopamine. The EC50 values for each species along with % max stimulation (where dopamine max stimulation equals 100%) are presented in Table 1. These in vitro studies were performed at Eli Lilly and Company, Indianapolis, in the USA. For further details on the measurement of effects on cAMP accumulation in these transiently transfected cells, see Hao et al. (2019).

Spontaneous eye blink rate in the rhesus monkey

Animals

Eleven 5–6-year-old, male rhesus monkeys (Chengdu Ping, An Animal Breeding and Research Base, Sichuan Province, China) with an average weight of 4.83 kg were included in the study. The animals were randomly divided into three groups, DPTQ higher cumulative dose group (n=4); DPTQ lower cumulative dose group (n=3); and vehicle (20% Captisol (CyDex Pharmaceuticals, Inc., KS) in NaPO4 buffer) group (n=4). 

The D1 agonist SKF82958 was included as a positive control; the results for this compound (not shown here) were recently published together with data for a different D1PAM (DETQ, see Bruns et al. 2018). The D1 selective antagonist SCH39166 hydrobromide (Cat No.: 2299, Batch No.: 3A/129719) was purchased from Tocris, MN, and was prepared in Millipore pure water with an injection volume of 0.2 ml/kg. The D1 antagonist was dosed 15 min before vehicle or DPTQ (n=4 animals per group).

The monkeys were housed individually in a climate-controlled (22–24℃) and humidity-controlled (40–70%) vivarium. A 12 h light/12 h dark cycle was in effect (light on from 7 am–7 pm). Monkeys had unlimited access to water and received a daily allotment of high-protein monkey chow, supplemented with fruit every day. Before compound administration and during test hours, monkeys were not fed with fruits or food.

The Spontaneous Eye Blink Rate study was performed at ChemPartner, Shanghai, China. The monkey housing in the Shanghai ChemPartner large animal facility is fully accredited by AAALAC. The animals were socially pair-housed in 2 interconnected cages, enabling visual and verbal communication with neighboring monkeys as well as self-viewing from a hanging mirror on each cage. The cage dimension for each monkey was 0.9 (L)×0.9 (W)×2 (H) m, with a gourd, foraging ball, or ball chew provided inside. 

The enrichment program included television shows 2–3 times per week and background music for several hours every other day. Animal health status was checked periodically by the attending veterinarian and daily by the animal care staf for their (1) appearance, hair, and tail; (2) feces and urination; and (3) gross behavior, food consumption, and signs of illness. All training and testing protocols were approved by the Institutional Animal Welfare IACUC guidelines and policy committee at ChemPartner (Shanghai, China).

Apparatus and experimental protocol

The spontaneous eye blinking rate was measured during observational experiments conducted in a specially constructed monkey chair (Mason et al. 2019). A U-shaped neck plate that helped to fixate the orientation of the head was used to facilitate observation of eye blink rate response. A compact video camera (Panasonic/wv-cp480/CH) on a tripod was positioned in front of the seated monkey. Two camera images were combined by a video collector (Color QUAD system) and transferred to the EthoVision software (Noldus, Leesburg, VA) on the computer for online viewing and recording.

Two weeks before testing, monkeys were trained daily to sit in the monkey chair for two 2-hour periods with a food reward. During post-training test sessions conducted between 9:00 a.m. and 4:30 p.m. every day, each monkey was studied for nine consecutive 15-minute components. Each 15-minute block consisted of a 10-minute habituation period followed by a 5-minute period during which eye blinking was counted. 

The video was displayed on a computer screen to allow continual observation of the subject. Observers blind to the treatment conditions later scored each 5-minute videotaped session. An eye blink was defined as a visible, rapid opening and closing of the eyelid. Two observers independently confirmed the measurement with reliability>90%.

Pharmacological testing

The effects of different D1 ligands on spontaneous eye blink rate were studied by a cumulative dosing procedure (Jutikiewicz and Bergman 2004). Active compound or vehicle (Captisol 20% w/v NaPO4 buffer) was administered after obtaining two baseline eye blink recordings. The ligand was given in random order to different monkeys of each group as described above. To study dose-response and time course, incremental doses of DPTQ were administrated at the end of 15-minute components of each test session.

Chemicals

DPTQ (free base, see Hao et al. 2019) was supplied by Eli Lilly Company. Captisol (Lot No.: NC04A-05034) was prepared as follows: Captisol 20% w/v NaPO4 buffer 25 mM, pH 8. The injection volume for DPTQ was 0.5 ml/kg IM. DPTQ at 0.1 mg/kg, 0.5 mg/kg, and 1 mg/ kg were dosed as clear solution, while the 5 mg/kg and 10 mg/kg doses were formulated as fine suspensions. 

The above vehicle solution was stored at 4℃ in a refrigerator and restored to room temperature before use. When preparing DPTQ fresh daily, a portion of the vehicle (20% Captisol) was added to the compound and stirred to wet, followed by the addition of the remainder of the vehicle and mixing until the solution became homogeneous. The suspension was then sonicated on an ice bath for 40 min to reduce particle size. Animals were dosed while in the chair.

Data analysis for eye blink rate

Results for each monkey were expressed as the rate of eye blinking (blinks per minute) averaged over each 5-minute testing epoch. The effects of vehicle and D1 PAM on eye blinking were averaged for each group of monkeys and expressed as mean±SE. The effects of DPTQ and vehicle were analyzed with two-way ANOVA (D1 PAM and time) followed by the Bonferroni post hoc test (GraphPad Prism, San Diego CA).

Spatial working memory task

Animals

Sixteen rhesus and one stumptail macaque participated in the study (details provided below) that was performed at Yale University, New Haven CT, USA. Animals were used and cared for in full accordance with Yale University’s IACUC guidelines and policies in addition to all U.S. Federal policies and regulations. The monkeys were fed their normal diet each day, including appropriately nutritional biscuits and multiple fruits and vegetables for enrichment. Testing and any training occurred just before their main food course.
This food consisted of biscuits, delivered each day, and all animals had full 24-hour access to ad libitum water. Participation in cognitive testing was ensured by the enrichment of the environmental stimulation and the provision of the animals’ favorite food treats as rewards in the task, including yogurt, raisins, almonds, grapes, fruit loops, and multiple other food items (Roberts et al 2010).

Cognitive testing

Spatial delayed response is an established translational measure of spatial working memory that has been shown to engage the same neural circuitry in human and non-human primates (McCarthy et al. 1996). The circuitry involved in this response is almost exclusively the “dorsal stream” involving the dorsolateral prefrontal cortex, particularly Area 46, and the lateral intraparietal sulcus (GoldmanRakic 1996). For a full description of the task used here, see Roberts et al. (2010). Animals were tested in a sound-attenuated room incorporating a testing chamber. In this task one of several well locations is baited with food given to the animal and the wells are then covered with identical plaques. 

An opaque shutter is then lowered for one of 5 variable delays and then raised to allow the animal to make a response to one of the well locations to retrieve the food. Each delay length was repeated 4 times in a semi-random distribution across the 20 trials in each testing session. Each animal was stabilized before commencing any administration by gradually incrementing the number of wells (starting at just 2) and the lengths of the delay. The 5 variable delay lengths were set at 0, 1, 2, 3, and 4 s multiplied by a factor “N.” Thus, N was incremented by 1 up to a maximum of 10 until the subject performed at 80% or more across 3 consecutive test sessions whereupon the number of wells was incremented by 1. 

This process was repeated for each individual until they reached stable performance (65–75% ± ≤2.5% correct). This normalized level of performance allows for the sensitive detection of any impairment or improvement produced by experimental conditions. For baseline data for this study, we took 7 or more consecutive data points that had been collected in previous weeks which fell within the stable limits for the test. For the total group of 17 animals participating in this study, the median well number was 4 (range 2–7) and the median N value was 3 (range 1–7). Animals were typically tested 2 to 3 days a week.

Compound administration

DPTQ (free base, Eli Lilly & Co.) was prepared as described above for the spontaneous eye blink test. Animals (4 males and 6 females, mean age=22.1±1.7 years) were originally assigned to receive either vehicle or DPTQ 2.5 mg/kg IM in a semi-random design. One animal received a vehicle but was not dosed with DPTQ. This same group of animals was later used in a repeated dosing study with DPTQ 2.5 mg/kg vs. vehicle. After a protracted washout period of several months, we tested the lower doses of 0.1 mg/kg and 1.0 mg/kg in ascending order. 

This group of 10 animals included 3 from the previous study (3 males, and 7 females (including one aged stumptail macaque); average age=22.6±1.7 years). The youngest animal in this study was aged 17.3 years. None of the animals were naïve and had all been used in previous studies with other compounds. They were given an extensive washout of weeks or months before this study and were established to have normal baseline performance on the task. 

During the study, animals were washed out based on known PK properties (half-life) of this drug as well as the baseline performance of each animal. That is, performance was required to return to baseline before commencing any other condition and a washout period of at least two weeks was typically employed. Cognitive testing was conducted 1 h post-injection to match the time of maximal effect observed in the original eye blink study.

Cognitive data analysis

The effect of the administration of the vehicle and each dose of DPTQ was analyzed using a 1-way analysis of variance (ANOVA) followed by post hoc comparisons based on false discovery rate (FDR), using the two-stage step-up method of Benjamini et al. (2006; GraphPad Prism 9.1). This methodology was chosen considering the high number of groups (days) and the limited number of tests (animals). 

An α level of 0.05 was used to determine statistical significance. After initial testing to ensure no significant effects of the vehicle (see “Results” below) comparisons were performed with baseline (averaged over several sessions) as the control.

Plasma ligand exposure analyses

Plasma samples were collected in EDTA tubes at 0, 0.25, 0.50, 1, 2, 4, 8, 12, and 24 h after a bolus IM injection and stored at−70C until further analysis by LC/MS (for further details see Hao et al. 2019). For the lower dose of 0.1 mg/kg, IM, plasma samples were collected from three of the animals participating in the spatial working memory task at Yale University, at approximately 1.5 h after dosing (~ 30 min after completion of behavioral test session). 

Animals were acclimated to sitting freely in a customized chair and having one leg held for hair trimming (if necessary), wiping with 70% alcohol, and saphenous venipuncture using a BD Vacutainer tube inside a safety holder. For the 5 mg/kg, IM dose, plasma samples were collected in a separate study in three male adult macaque monkeys at Eli Lilly and Company, Indianapolis, IN. The collection and storage of samples followed the procedure described above, and analysis of D1 ligand levels was carried out at Eli Lilly and Company, Indianapolis, IN, USA, using liquid chromatography and tandem mass spectrometry.

Results

Effects of DPTQ on cAMP accumulation in four species

DPTQ was tested in HEK293 cells transiently expressing human, dog, rhesus monkey, and mouse dopamine D1 receptors. Potentiator EC50 and Emax values for the dog and rhesus monkey D1 receptors were similar to those for the human D1 receptor (Table 1). However, the potentiator EC50 for the mouse D1 receptor was shifted rightward about 14- to 23-fold compared to the rhesus monkey and human respectively. In addition, the Emax for the mouse D1 receptor was only 41%, compared with 77–88% for the other three species.

Spontaneous eye blink rate

The mean baseline eye blink rates before dosing did not exceed 10 blinks/min, and the eye blink rates were relatively stable across test sessions within individual subjects after vehicle injection. DPTQ was tested in two cumulative dose regimens (Fig. 1). The higher cumulative dose of DPTQ (0.5 mg/kg, 5 mg/kg, and 10 mg/kg, IM) resulted in the highest rates of eye blinking. The time course effect following these higher doses showed that significant increases in the post-DPTQ eye blink rate occurred at 30 min (p<0.01), 45 min (p<0.001), 60 min (p<0.01), and 75 min (p<0.05) (Fig. 1). The lower cumulative dose of DPTQ (0.1 mg/ kg, 1 mg/kg and 5 mg/kg, resulted in small but significant (p < 0.05) increases in rates of eye blinking only at the 45-min and 60-min time points (Fig. 1). During recording and observation, some vehicle group monkeys fell asleep or became drowsy in the monkey chair as time went by. In contrast, all of the DPTQ-treated monkeys remained alert throughout the test period of close to 2.5 hours.

Both high and lower cumulative doses of DPTQ resulted in some degree of oral movement such as tongue protrusion (data not shown). However, only the higher cumulative dose showed a clear effect at 30 min after DPTQ administration when compared to the vehicle group. This is consistent with an activation of tongue movement following D1 receptor stimulation reported in monkeys (Bédard and Boucher 1989).

Administration of the D1 selective antagonist SCH39166 (0.03 mg/kg, IM) alone induced a brief reduction in eye blink rate, and visual observations indicated that the animals were slightly sedated. This effect lasted for about 15 min (Fig. 2). Animals pretreated with SCH39166 showed only a small, non-significant (p<0.05) increase in eye blink rate induced by higher accumulated doses (0.5, 5, 10 mg/kg) of DPTQ (Fig. 2; compare to Fig. 1).

Analyses of plasma levels of DPTQ in the monkey

In the first study, plasma samples were collected at various time points from three animals for up to 24 h after a dose of 5 mg/kg IM (Fig. 7). High total plasma levels of DPTQ were measured for the initial 2 h ranging from about 9000 to 1800 nM with estimated unbound plasma concentrations of 674 to 125 nM. Based on data that DPTQ has an unbound brain vs. unbound plasma concentration ratio of about 0.3 and unbound ligand fraction of 0.068 (J. Cramer, unpublished data), the projected unbound brain concentrations for DPTQ after 5 mg/kg injection would thus range from 202 down to 38 nM over the first 2 h post-dosing. These concentrations are within the range of the hD1 EC50 value for DPTQ in the cAMP assay reported to be 76 nM (Hao et al. 2019) and at or above the monkey D1 EC50 of 38 nM reported here using transiently expressed cells (Table 1). 

In the second study, samples were collected after the low dose of 0.1 mg/kg, IM from three animals at ~1.5 h after dosing in the cognition study. As shown in Fig. 3, the low dose of 0.1 mg/kg IM resulted in about 50×lower plasma levels of DPTQ. The total D1 PAM plasma concentrations ranged from 56 to 91 nM (Fig. 7). Using the same assumptions as above for unbound ligand fraction and unbound brain vs. unbound plasma ratio, the estimated unbound ligand concentrations in the brain would be 1–2 nM at 1.5 h after the 0.1 mg/kg, IM dose. These levels are far below both the monkey and human EC50 values for potentiation of dopamine-induced activation of cAMP in the vitro assay.

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