Main Research Findings

1) Metabolite 6 was found to enhance the anti-parkinsonian activity of L-DOPA while exhibiting a low risk of secondary dyskinesia.

2) The study reports the pharmacokinetics of the compound following administration to healthy subjects, noting it was safe and well-tolerated for 14 days of treatment.

 

Selected Data

1) This study performed by Ohno et al employed a comprehensive set of methods and materials to investigate the potential of Metabolite 6 (KW-6356), a novel selective adenosine A2A receptor antagonist/inverse agonist, to enhance the anti-parkinsonian activity of L-DOPA while mitigating dyskinesia. The research was conducted using common marmosets, an established animal model for Parkinson’s disease (PD), under strict ethical guidelines. Male and female marmosets, aged over two years at the study’s commencement, were housed in stainless steel cages under controlled environmental conditions including 25-29 °C, 20%-60% humidity, and a 12-hour light-dark cycle. They received ad libitum access to a specialized monkey diet and fresh water [1].

Parkinsonism was experimentally induced in the marmosets through subcutaneous administration of MPTP hydrochloride at a dose of 2.0 mg/kg once daily for 5 consecutive days, dissolved in physiological saline. To ensure consistent symptom development, some animals received additional MPTP injections approximately three weeks after the initial treatment, depending on their individual symptoms. Following MPTP administration, animals were hand-fed a specialized paste of pelleted diet, mashed fresh fruit, and liquid feed to aid their recovery from the acute effects of MPTP. A crucial selection criterion for participation in the studies was that animals exhibited clear PD-like symptoms and demonstrated a measurable response to L-DOPA at least six weeks after the initial MPTP dose.

To prepare for dyskinesia-related studies, a subset of MPTP-treated marmosets was primed for dyskinesia by orally administering L-DOPA at a dose of 10 mg/kg plus benserazide at a dose of 2.5 mg/kg twice daily for 28 days. Only those animals displaying a mean maximal dyskinesia score of ≥3.5, with individual scores ≥2, were selected for the dyskinesia studies. The investigational compound, KW-6356, was consistently administered at an optimal dose of 1 mg/kg. It was prepared as a suspension in 0.5% methyl cellulose and 10% sucrose solution. L-DOPA and benserazide, maintaining a fixed L-DOPA:benserazide ratio of 4, were prepared similarly as suspensions in 0.5% methyl cellulose and 10% sucrose. All drugs, including KW-6356, L-DOPA, and benserazide, were administered orally at a consistent volume of 2 mL/kg body weight [1].

The study design incorporated three distinct experimental studies to address different aspects of KW-6356’s therapeutic potential: Study 1aimed to evaluate the acute effects of KW-6356 monotherapy and its combination with various doses of L-DOPA/benserazide, ranging from 2.5/0.625 mg/kg to 10/2.5 mg/kg, on motor disability and locomotor activity in MPTP-treated marmosets that were not yet primed for dyskinesia (drug-naive). Study 2 investigated the acute effects of KW-6356 at a dose of 1 mg/kg, co-administered with different doses of L-DOPA/benserazide at doses of 1.25/0.312 mg/kg to 10/2.5 mg/kg, on both dyskinesia and locomotor activity in MPTP-treated marmosets that had been primed to exhibit dyskinesia. Study 3 explored the effects of chronic co-administration. Dyskinesia levels were first established using a high dose of L-DOPA at a dose of 10 mg/kg plus benserazide at a dose of 2.5 mg/kg as the “L-DOPA pre” value on Day -8. Subsequently, vehicle was administered to determine a control value (“L-DOPA post” on Day -1), followed by a low 2.5 mg/kg dose of L-DOPA plus 0.625 mg/kg of benserazide alone (“L-DOPA cont” on Day 0). Then, KW-6356 at a dose of 1 mg/kg was co-administered daily with this low-dose L-DOPA/benserazide regimen for 21 days. A one-week washout period followed this chronic treatment, and dyskinesia assessments were performed throughout [1].

Behavioral assessments were rigorous and standardized. Motor disability was continuously monitored for 6 hours post-drug administration using a one-way mirror and an established rating scale with scores ranging from 0 for minimal disability to 17 for severe. Locomotor activity was quantified by counting light beam interruptions in stainless steel cages equipped with photoelectric sensors, recording activity in 30-minute intervals for 8 or 12 hours post-drug. Dyskinesia was specifically assessed using a semi-quantitative scoring system with 0 being absent, to 4 being severe, for 3 or 6 hours post-drug, with the maximal intensity recorded during a 1-hour observation period.

Statistical analyses were performed using SAS software. For comparing mean values of total locomotor activities, paired t-tests were used for L-DOPA-only vs. L-DOPA + KW-6356 groups, and Dunnett tests for L-DOPA groups vs. vehicle. For total motor disability and maximal dyskinesia scores, the sign-Wilcoxon test was applied for paired comparisons, and the Steel test for multiple comparisons against the vehicle control. This comprehensive methodological approach allowed for a robust evaluation of KW-6356’s multifaceted effects in a preclinical model of PD [1].

2) The research presented by the research team of Tamaya et al encompassed two randomized, double-blind, placebo-controlled, Phase I studies along with an open-label component in study 6356-004, designed to thoroughly evaluate the safety, tolerability, and pharmacokinetics of KW-6356, a novel adenosine A2A receptor-selective antagonist and inverse agonist, in healthy volunteers [2].

The patient population for study 1 consisted of healthy male subjects aged 20 to less than 40 years, with a BMI between 18.5 and less than 25. Similarly, for parts A and B of study 2, inclusion criteria specified healthy male Japanese subjects aged 20 to less than 45 years, with a BMI between 18.5 and less than 30, and no mixed marriage for at least two generations. Part C of study 2 expanded the cohort to include both healthy male Japanese and White subjects, maintaining the age and BMI criteria and the requirement for at least two generations without mixed marriage for each respective ethnic group. Exclusion criteria common to both studies included significant medical conditions that could interfere with drug metabolism or pose safety risks.

Study 1 comprised two parts: a single-dose study (part 1) and a multiple-dose study (part 2). In part 1, subjects were randomized 3:1 to receive single ascending oral doses of KW-6356 ranging from 1, 3, or 10 mg, or a placebo on Day 1, following at least 10 hours of fasting. Safety data from each dose cohort were reviewed before proceeding to the next higher dose. Part 2 involved subjects receiving a 6 mg oral dose of KW-6356 once daily, or a placebo for 7 consecutive days, administered within 30 minutes after breakfast [2].

Study 2 was more complex, consisting of three parts: single-dose (part A) and multiple-dose (part B) studies in healthy Japanese subjects, and a multiple-dose open-label study (part C) in healthy Japanese and White subjects. In part A, subjects were randomized 3:1 to receive single ascending oral doses of KW-6356 ranging from 21, 42, or 60 mg, or a placebo on Day 1, after a 10-hour fast. Similar to study 1, cumulative safety data from each cohort were reviewed before dose escalation. Part B involved subjects receiving a 24 mg oral dose of KW-6356 once daily, or a placebo for 14 consecutive days, administered after breakfast. The dose for part B was determined by dividing the maximum tolerable dose from part A by the accumulation ratio. Part C was an open-label study where healthy Japanese and White subjects received a 6 mg oral dose of KW-6356 once daily, for 7 consecutive days, also after breakfast.

Safety and tolerability were paramount, assessed through various parameters throughout both studies. Subjects were monitored for treatment-emergent adverse events (AEs), which were coded using the Medical Dictionary for Regulatory Activities (MedDRA) and summarized by preferred terms and system organ classes. Any subject receiving study drug was considered safety-evaluable. Monitoring periods varied: an 8-day period with 6 days of inpatient observation for single doses in study 1 part 1, an 18-day period with 12 days inpatient for multiple doses in study 1 part 2, a 12-day period with 10 days inpatient for single doses in study 2 part A, a 27-day period with 22 days inpatient for multiple doses in study 2 part B, and an 18-day period with 12 days inpatient for multiple doses in study 2 part C. Optional early termination visits were available for subjects who discontinued prematurely [2].

Pharmacokinetic (PK) assessments involved extensive blood sampling. For single-dose studies (study 1 part 1, study 2 part A), blood was drawn at numerous time points over several days post-dose, capturing the drug’s absorption, distribution, metabolism, and elimination. For multiple-dose studies (study 1 part 2, study 2 parts B and C), samples were taken before and at various intervals after the first dose, daily before subsequent doses, and again at multiple intervals after the last dose to assess steady-state kinetics and accumulation. Plasma concentrations of KW-6356 and its main metabolite, M6, were quantified using validated bioanalytical methods involving protein precipitation and liquid chromatography-tandem mass spectrometry (LC-MS/MS). The analytical method utilized a Hypersil Gold column, a binary gradient mobile phase (ammonium acetate and acetonitrile), and atmospheric pressure chemical ionization in positive mode. Specific multiple reaction monitoring (MRM) settings were used for KW-6356 and M6, with stable isotope-labeled internal standards for accurate quantification. The validated assay ranges were 2.00-1000 ng/mL for KW-6356 and 1.00-500 ng/mL for M6, with good intra- and inter-day precision.

PK parameters such as maximum observed plasma concentration (Cmax), time to Cmax (tmax), trough concentration (Ctrough), area under the plasma drug concentration-time curve (AUC), apparent systemic clearance (CL/F), and terminal elimination half-life (t1/2) were estimated using non-compartmental analysis. Values below the limit of quantification were treated as zero for calculations. Statistical analyses for PK parameters, metabolic ratios, and accumulation ratios were performed using SAS software, with parameters derived for the first and last doses in multiple-dose parts. Subjects who received KW-6356 were considered PK-evaluable [2].

 

Discussion

1) The study conducted by the research team of Ohno et al yielded significant and nuanced results regarding the efficacy of KW-6356, an adenosine A2A receptor antagonist/inverse agonist, in improving motor symptoms and modulating dyskinesia in MPTP-treated common marmosets. The findings underscore KW-6356’s potential as a valuable therapeutic agent for PD treatment [1].

The results of the first study investigating the effects on motor disability and locomotor activity in drug-naive (not previously primed for dyskinesia) MPTP marmosets, found that oral administration of L-DOPA/benserazide alone exhibited a dose-dependent effect, leading to a reversal of motor disability and an increase in locomotor activity. Specifically, significant improvements in motor disability were observed at L-DOPA doses of 10/2.5 mg/kg. Locomotor activity was also significantly increased at L-DOPA doses of 7.5/1.875 mg/kg and 10/2.5 mg/kg. A key finding of this study was the potentiation of these anti-parkinsonian effects when 1 mg/kg of KW-6356 was co-administered with L-DOPA/benserazide. The combination significantly enhanced the L-DOPA-induced decrease in total disability score and robustly amplified the increase in total locomotor activity across a wide range of L-DOPA doses from 2.5/0.625 to 10/2.5 mg/kg. This suggests that KW-6356 can augment the therapeutic benefits of L-DOPA, potentially allowing for lower effective L-DOPA doses, which could be critical for long-term management and reducing L-DOPA-related side effects [1].

The results of the second study assessing the effects on dyskinesia and locomotor activity in L-DOPA primed MPTP marmosets, found that in the primed animals, acute administration of L-DOPA/benserazide alone resulted in a dose-dependent increase in maximal dyskinesia scores, with significant dyskinesia observed at doses of 5/1.25, 7.5/1.875, and 10/2.5 mg/kg. L-DOPA also increased locomotor activity in a dose-dependent manner in these primed animals. When KW-6356 at a dose of 1 mg/kg was co-administered acutely with L-DOPA/benserazide, it also led to increased maximal dyskinesia scores with increasing L-DOPA dosage. However, a crucial observation was that the maximal dyskinesia score induced by the combination of 1 mg/kg of KW-6356 with optimal L-DOPA at a dose of 10 mg/kg was not significantly different from that induced by optimal L-DOPA alone. This indicates that while the combination did induce dyskinesia, KW-6356 did not exacerbate the severity of dyskinesia compared to L-DOPA monotherapy in this acute setting. Furthermore, 1 mg/kg KW-6356 administered alone to primed marmosets induced only minimal dyskinesia while still significantly increasing locomotor activity, suggesting an intrinsic anti-parkinsonian effect with a low inherent dyskinesia risk.

Initial results of the third study evaluating the chronic co-administration of KW-6356 and low-dose L-DOPA on dyskinesia revealed a high “L-DOPA pre” dyskinesia level in the primed marmosets. Acute administration of a vehicle resulted in very low dyskinesia, while a low 2.5 mg/kg dose of L-DOPA alone induced mild dyskinesia. When 1 mg/kg KW-6356 was co-administered acutely with this low-dose L-DOPA, it induced mild to moderate dyskinesia. The most significant finding emerged from the chronic co-administration phase: daily administration of 1 mg/kg KW-6356 with low-dose L-DOPA at a dose of 2.5 mg/kg, for 21 days led to an increase in dyskinesia severity, reaching 2.00 ± 0.26 by Day 21, when compared to the low-dose L-DOPA alone. However, critically, the amplitude of this dyskinesia was consistently lower than that induced by an optimal, higher 10 mg/kg dose of L-DOPA in the priming phase. Following a 1-week washout period, the dyskinesia severity observed after subsequent single co-administration returned to levels comparable to those during the chronic administration, indicating a reversible effect [1].

 

Figure 1: Changes in A) total disability score and B) total locomotor activity across experimental treatment groups receiving varying doses of KW-6356 and L-DOPA.

In summary, the study demonstrates that KW-6356 effectively potentiates the anti-parkinsonian activity of L-DOPA across a range of doses. Importantly, in dyskinesia-primed marmosets, KW-6356 did not exacerbate dyskinesia when acutely co-administered with L-DOPA. Furthermore, even with chronic co-administration alongside a low dose of L-DOPA, the resulting dyskinesia severity remained significantly lower than the dyskinesia associated with optimal, high-dose L-DOPA monotherapy. These results collectively suggest that KW-6356 offers a promising therapeutic strategy to enhance L-DOPA’s efficacy while potentially mitigating the severe dyskinesia often associated with conventional high-dose L-DOPA regimens, thereby improving the overall management of PD symptoms [1].

2) The study by Tamaya et al successfully evaluated the safety, tolerability, and pharmacokinetics of KW-6356 in healthy Japanese and White subjects, demonstrating a favorable profile for the novel adenosine A2A antagonist/inverse agonist. Safety data were available for a total of 80 subjects across the two primary studies. KW-6356 was generally well tolerated, with no serious treatment-emergent adverse events reported in either study [2].

In study 1, treatment-emergent AEs were reported in a dose-dependent manner in part 1 (single ascending doses). No serious AEs were reported in study 1. Study 2 also showed dose-dependent AE incidence in part A with single ascending doses. Overall, the studies indicated that KW-6356 was well tolerated at single doses up to 60 mg and multiple doses up to 24 mg once daily for 14 days, with insomnia being the most common, dose-dependent, and drug-related AE, likely due to its wake-promoting effects.

The main metabolite, M6, also exhibited dose-proportional increases in plasma concentrations after single doses of KW-6356, with a median tmax ranging from 12 to 24 hours. M6 concentrations declined in a monophasic manner. In multiple-dose regimens where participants were administered either 6 mg or 24 mg once daily, both KW-6356 and M6 plasma concentrations reached steady state by 7 days. The mean accumulation ratios for KW-6356 were between 1.86 and 2.54, while for M6, they ranged from 1.84 to 3.20, indicating moderate accumulation upon repeated dosing. The mean metabolic ratios (unchanged drug AUC/metabolite AUC) ranged from 4.38 to 11.39, with no prominent dose-related trends, suggesting M6’s contribution to pharmacological effects was unlikely to be significant at steady-state concentrations, consistent with previous in vitro data [2].

An important finding from the multiple-dose part C of study 2 where participants were administered 6 mg once daily, was the lack of a clear difference in KW-6356 pharmacokinetics between healthy Japanese and White subjects. While M6 concentrations and exposure measured by Cmax, Ctrough, and AUC0-24, were higher in Japanese subjects compared to White subjects, the overall PK profile of the parent drug, KW-6356, remained similar across these ethnic groups. This similarity was considered to imply no clinically meaningful genetic variations in key CYP enzymes, including CYP2C8, CYP2C9, CYP2J2, CYP3A4, and CYP3A5, that would significantly alter KW-6356 exposure between the two populations, despite known differences in CYP3A5*3 allele frequencies. These results collectively support that KW-6356 has a predictable and linear pharmacokinetic profile, is well tolerated across a range of doses, and exhibits consistent PK behavior regardless of the healthy subject’s Japanese or White ethnicity [2].

 

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] Ohno Y, Okita E, Kawai-Uchida M, et al. The adenosine A2A receptor antagonist/inverse agonist, KW-6356 enhances the anti-parkinsonian activity of L-DOPA with a low risk of dyskinesia in MPTP-treated common marmosets. J Pharmacol Sci. 2023;152(3):193-199. doi:10.1016/j.jphs.2023.05.001

[2] Tayama T, Ishiuchi M, Sugiyama K, et al. Safety, Tolerability, and Pharmacokinetics of the Novel Adenosine A2A Antagonist/Inverse Agonist KW-6356 Following Single and Multiple Oral Administration in Healthy Volunteers. Clin Pharmacol Drug Dev. 2023;12(8):801-809. doi:10.1002/cpdd.1222

 

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