Main Research Findings
1) Administration of ASP2905 was shown to alleviate the majority of both positive and negative symptoms of schizophrenia and the related cognitive impairments.

2) ASP2905 is found to be an effective selective inhibitor of KCNH3 that has the potential for treating disorders related to the regulation of neurotransmitters, dopamine and acetylcholine, that assist with various aspects of cognitive functioning.

 

Selected Data

1) The research team of Takahashi et al examined the antipsychotic profile of ASP2905 in animal models of schizophrenia, as well as its potential to treat cognitive dysfunctioning and symptoms related to the disorder. For the purpose of this experiment 4-5 weeks old male ddY mice were used to investigate locomotion, while 3 week old ddY mice were used to assess chronic phencyclidine hydrochloride-forced swimming test (PSP-FST). ASP2905 was synthesized and suspended in 0.5 % methylcellulose while both methamphetamine hydrochloride and phencyclidine hydrochloride (PCP) were dissolved in saline. ASP2905 was administered in doses of 10 ml/kg to the mice and 1 ml/kg of the rats, while PCP was administered using an osmotic minipump that infused 15 ml/kg of the compound on postnatal days 7, 9, and 11. The animals were maintained under standard laboratory conditions and allowed to acclimatize to the laboratory for 1 hour before each experiment initiated [1].

Several cognition trials were conducted in order to assess the effect of the drugs on symptoms of schizophrenia. First was the chronic PCP-FST test that began by dividing the mice into two different groups according to their recorded mobility time during a pre-swimming trial. The pre-swimming trial included placing the mice in an experiment room to acclimatize 1 hour prior to the study, followed by placing the mice in a cylindrical pool with a diameter of 14 cm and a depth of 13 cm. The mice were allowed to swim around while their activity was measured for 3 minutes. Two days after the pre-swimming trial the mice were anesthetized with pentobarbital and received subcutaneous implants of osmotic minipumps. These mice were further split into 4 groups based on their mobility time in the pre-swimming trial and the measured body weight on the day of the test [1].

The following cognitive test that was performed was the neonatal PCP-water-finding-task (WFT). 101 mice were utilized in this portion of the study and divided into 7 groups according to body weight prior to the training trial. 18 mice were excluded from the training trail including those assigned to the vehicle-saline-treated untrained group and the vehicle-PCP-treated untrained group. An open field apparatus measuring 30 cm x 50 cm and 15 cm high was used for the water finding test; a 10 cm x 10 cm x 10 cm alcove was placed in the middle of one wall, as well as a drinking tube with its tip suspended 5.5 cm and 8 cm above the floor for the training trial and test trial, respectively [1].

On the first day of the training trial the proper dose of ASP2905 or clozapine was orally administered and the mice were returned to the cages. After 60 minutes the animals were placed in the apparatus and allowed to explore for 3 minutes. Over the 3 minute training trial the researcher recorded the subjects’ latency to move, ambulation measured by line crossing, the number of water-tube touched, and the time it took to enter the alcove and make contact with the water tube.

Following the 3 minute training tail the mice were returned to their cages and deprived of water until the initiation of the test trial where water was only delivered through the drinking tube. Day 2 marked the beginning of the test trial where each mouse was placed into the apparatus while the researchers recorded the time taken for the mouse to move from their starting position, the time taken to enter the alcover and the time to start drinking water. The time it took to enter the alcove and drink the water was calculated and used as an index of latent learning [1]. The results were further compared to the compound-treated trained groups and the vehicle-PCP-treated trained groups.

In order to assess hyperactivity induced by methamphetamine, the mice were administered 4 oral doses of ASP2905 and placed in individual cages, followed by a 1 mg/kg dose of methamphetamine 30 minutes later. The mice were monitored for 90 minutes after administration of the nootropic; the first 30 minutes was defined as spontaneous activity while the following 60 minutes was defined as hyperactivity. To assess hyperactivity induced by PCP the mice were administered 4 oral doses of ASP2905 and placed in individual cages, followed by a 2 mg/kg dose of PCP 30 minutes later [1]. The mice were monitored for 60 minutes after administration of the nootropic; the first 30 minutes was defined as spontaneous activity while the last 30 minutes was defined as hyperactivity. Spontaneous locomotor activity was measured in the same manner and measured using a Supermex sensor paired with an infrared pyroelectric detector used to measure body heat radiation [1].

2) The research team of Takahashi et al assessed the efficacy of ASP2905 to treat spontaneously hypertensive rats as they serve as a validated model of ADHD, inattention, impulsivity, and slow habituation. In order to prepare for experimentation ASP2905 hydrochloride and ASSP2905 hemifumarate were prepared by suspending the compound in 0.5% methylcellulose. Atomoxetine hydrochloride was dissolved in distilled water while amphetamine sulfate, guanfacine hydrochloride, and methylphenidate were dissolved in saline. ASP2905 was administered in doses of 10 mL/kg to ddY juvenile, stroke-prone spontaneously hypertensive rats (SPSHR), 2 mL/kg doses to Wistar-Kyoto rats, and 1 mL/kg doses to Wistar/ST and Sprague-Dawley rats [2]. Additionally, methylphenidate was administered via subcutaneous injection to juvenile SHRSP and WKY rats in a 2 mL/kg dose and in a 1 mL/kg dose to Sprague Dawley rats; while amphetamine and guanfacine were administered in a 1 mL/kg dose via subcutaneous injection into Sprague Dawley rats.

On day one of the training trial ASP2905 was given to the test subjects in doses ranging from 0.0078, 0.0156, 0.0313, and 0.0625 mg/kg. The mice were placed back into their individual home cages and after 30 minutes they were placed into an open field apparatus and allowed to explore for 3 minutes. Prior to the training trial the mice were not motivated to drink water; the researchers placed a water tube in the alcove of the apparatus, however no water was delivered. Immediately after the training trial the mice were returned to their cages and deprived of water until the initiation of the test trial. Test subjects were excluded from the test trial if they approached the drinking tube less than two times or if they failed to explore the apparatus entirely; the cutoff time was 600 seconds.

The next test the animals underwent was the five-trail passive avoidance task (5T-PAT) that included a step-through passive avoidance apparatus and a light-dark box. 4 week old male SHRSP and WKY rats were initially administered ASP2905 in doses of 0.03, 0.1, or 0.3 mg/kg, or methylphenidate in doses of 0.03, 0.1, or 0.3 mg/kg, followed by the return to their home cage. After 30 minutes they were placed into the illuminated room and when they moved into the dark room an electric shock of 0.15 mA was delivered through the metal grid flooring. Testing took place for 180 seconds and the time it took for the rat to enter the dark room was defined as step-through latency. After the first initial treatment dose was administered this test took place 5 different times at one minute intervals in order to determine the cumulative latency [2].

In order to perform microdialysis experiments, 9-10 week old male Wistar and ST rats were anesthetized with sodium pentobarbital while a stereotactic apparatus implanted a microdialysis guided cannula into the medial prefrontal cortex. The rats recovered from surgery for at least 4 days prior to the day of the microdialysis experiment and on the first day each animal was placed in a plastic cage while a dialysis probe was inserted into the cannula and perfused with modified Ringer’s solution containing physostigmine at a rate of 1.0 uL/min and 2.0 uL/min for dopamine and acetylcholine, respectively. Dialysates for dopamine and acetylcholine were collected every 30 minutes and every 20 minutes, respectively. After 3 samples were collected ASP2905 was orally administered in doses of 0.01, 0.03, and 0.1 mg/kg for dopamine and doses of 0.1 and 1 mg/kg for acetylcholine. Dialysates were then collected every 180 minutes for dopamine and every 100 minutes for acetylcholine [2].

The levels of extracellular dopamine in the medial prefrontal cortex were assessed through the use of an Eicom HTEC-500 high pressure liquid chromatography system and separated using a reverse-phase PP-ODS column eluted with 500 mg/mL sodium 1-decanesulfonate, 1% methanol, and 50 mg/mL of EDTA.The levels of extracellular acetylcholine in the medial prefrontal cortex were measured using a combination of an ECD, an enzymatic assay, and a combination of HPLC. Additionally, isopropylhomocholine served as an internal standard for the purpose of this study while a solution comprised of 100 mM KHCO3 contain 300 mg/L sodium 1-decanesulfonate and 50 mg/L EDTA was delivered at a rate of 500 uL/min. Samples were collected and separated using a column packed with a polystyrene resin; acetylcholine was converted to H2O2 through the use of a postcolumn enzyme reactor that detected H2O2 with an ECD-300 [2].

For the purpose of an electroencephalogram (EEG) analysis, 8 week old male Sprague Dawley rats were anesthetized with isoflurane and placed into a stereotactic apparatus. An incision was made to expose the skull and the researchers identified the bregma in order to epidurally implant stainless steel screw electrodes with wire leads into the vertex, cerebellum, and frontal sinus. The assembly was secured to the skull and recording took place over 10 days after allowing the subjects to heal from surgery and acclimate to the recording chamber and electrically shielded field. At this time the rats were orally administered 1, 3, and 10 mg/kg of ASP2905 or 3, 10, and 30 mg/kg of atomoxetine, and EEG was performed 60 minutes later. Alternatively, amphetamine was subcutaneously administered in doses 0.3, 1, and 3 mg/kg, methylphenidate in doses of 0.3, 1, and 3 mg/kg, or guanfacine in doses of 0.03, 0.1, and 0.3 mg/kg, and EEG was performed 30 minutes later [2].

 

Discussion

1) The effects of mobility in the test subjects was assessed through the administration of PCP followed by treatment with ASP2905. The results of the Chronic PCP-FST found that when PCP was administered to the animals daily for 14 days they experienced prolonged immobility time while in the FST apparatus. Administration of ASP2905 at doses of 0.03 mg/kg and 0.1 mg/kg were found to significantly reverse the effects elicited by PCP. Similar results were seen when the animals’ were administered Clozapine in doses of 3 mg/kg, however the effect was not as great. Alternatively, when assessing spontaneous locomotor activity, ASP2905 did not elicit any significant effects when given to the animals at concentrations up to 3 mg/kg. On the other hand, the nootropic administered at doses of 0.1 and 0.3 mg/kg drastically decreased hyperactivity induced by PCP and methamphetamine [1].

Figure 1: Changes in the test subjects’ total mobility time in response to treatment with ASP2905.

Latent learning was measured through the finding latency in the WFT. The mice treated with salines as neonates experienced a shortened finding latency compared to the mice treated with PCP as neonates that did not experience any changes in finding latency. These findings suggest that neonatal treatment with PCP is related to the impairment of latent learning. That being said, when the mice treated with PCP as neonates were administered ASP2905 in doses of 0.03 and 0.1 mg/kg resulting in a significantly shortened finding latency in the WFT. In contrast to the nootropic, injection of clozapine in doses of 0.3, 1, and 3 mg/kg did not lead to any remarkable changes in the finding latency [1].

Figure 2: Changes in the finding latency of the test subjects’ during the WFT in response to treatment with ASP2905.

2) The first portion of the experiment conducted by the research team of Takahashi et al assessed how finding latency during a water finding test was affected when ASP2905 was administered to the test subjects in varying doses. The results of the WFT revealed that the control mice were able to find the water novel with a finding latency of 153 +/- 15 seconds. When ASP2905 was administered to the test subjects in doses of 0.0078, 0.0156, 0.0313, and 0.0625 mg/kg the finding latency of each animal was found to be significantly shorter. These findings were confirmed with one-way ANOVA statistical analysis and Dunnett’s multiple comparison test [2].

Figure 3: Changes in the finding latency of the test subject’s during the water finding task in response to treatment with ASP2905.

The next portion of the study examined the potential of both methylphenidate and ASP2905 to prolong the cumulative latencies of SHRSP rats recorded throughout the 5-trial passive avoidance task. The results initially reported that the cumulative latencies of the juvenile SHRSP were far shorter than the cumulative latencies of the WKY rats. When ASP2905 was administered in doses of 0.1 and 0.3 mg/kg, the cumulative latencies of the SHRSP mice was found to significantly increase when compared to the control group of test subjects. Additionally, when methylphenidate was administered in doses of 0.1 and 0.3 mg/kg the cumulative latencies of the SHRSP mice was found to significantly increase in comparison to the control group of animals. Both of these findings were confirmed through the use of one-way ANOVA statistical analysis and Dunnett’s multiple comparison test [2].

Figure 4: Changes in the cumulative latency of juvenile SHRSP mice during the 5-trial passive avoidance task, in response to A) ASP2905 and B) methylphenidate.

ASP2905 was also administered orally to the test subjects in order to determine the effects that the nootropic has on extracellular levels of dopamine and acetylcholine in the medial prefrontal cortex. The results reported that oral administration of ASP2905 in doses of 0.03 and 0.1 mg/kg significantly increased the extracellular levels of dopamine when compared to the animals that only received saline. Furthermore, oral administration of ASP2905 in doses of 0.1 and 1.0 mg/kg was found to significantly increase extracellular levels of acetylcholine in comparison to the animals that were only administered saline. These findings were confirmed through the use of one-way ANOVA statistical analysis and Dunnett’s multiple comparison test [2].

Figure 5: Changes in the extracellular levels of dopamine (A, B) and acetylcholine (C, D) in the medial prefrontal cortex of the test subjects.

In the last portion of the study EEG analysis was used to reveal changes in alpha power in rats administered varying doses of ASP2905, amphetamine, atomoxetine guanfacine, or methylphenidate. In the test subjects administered ASP2905 and methylphenidate all experienced a significant increase in alpha power. Amphetamine was also found to not only increase alpha power but cause a decrease in theta power, as well, while administration of atomoxetine and guanfacine did not lead to any significant changes in alpha power. These findings were confirmed through the use of one-way ANOVA statistical analysis and Dunnett’s multiple comparison test [2].

Figure 6: Changes in alpha power in the test subjects in response to treatment with A) ASP2905, B) amphetamine, C) methylphenidate, D) atomoxetine, and E) guanfacine.

 

Disclaimer

**LAB USE ONLY**
*This information is for educational purposes only and does not constitute medical advice. THE PRODUCTS DESCRIBED HEREIN ARE FOR RESEARCH USE ONLY. All clinical research must be conducted with oversight from the appropriate Institutional Review Board (IRB). All preclinical research must be conducted with oversight from the appropriate Institutional Animal Care and Use Committee (IACUC) following the guidelines of the Animal Welfare Act (AWA).

 

Citations

[1] Takahashi S, Okamura A, Yamazaki M, Ni K. ASP2905, a specific inhibitor of the potassium channel Kv12.2 encoded by the Kcnh3 gene, is psychoactive in mice. Behav Brain Res. 2020 Jan 27;378:112315. doi: 10.1016/j.bbr.2019.112315. Epub 2019 Oct 22. PMID: 31654662.

[2] Takahashi S, Ohmiya M, Honda S, Ni K. The KCNH3 inhibitor ASP2905 shows potential in the treatment of attention deficit/hyperactivity disorder. PLoS One. 2018 Nov 21;13(11):e0207750. doi: 10.1371/journal.pone.0207750. PMID: 30462746; PMCID: PMC6248980.

 

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