Safety, tolerability, and pharmacokinetics of fluoropezil (DC20), a novel AChE inhibitor: a single-center, phase I study in healthy young and elderly Chinese subjects

doi:10.21203/rs.3.rs-1744060/v1

Background

Acetylcholinesterase (AChE) inhibitors attempt to reduce the breakdown of acetylcholine levels in the brain of patients with Alzheimer’s disease (AD) by inhibiting the responsible enzyme AChE in the synaptic cleft. This study evaluated the safety, tolerability, and pharmacokinetics of fluoropezil (DC20), a novel AChE inhibitor under development for the treatment of AD in healthy young and elderly Chinese subjects.

Methods

The study on young subjects were divided into two arms: the multiple ascending-dose (MAD) arm (double-blind, randomized, placebo-controlled, multiple ascending-dose, 2 and 6 mg, N = 24), and the food effect arm (three-period, self-crossover, open-labeled, fasting/standard diet/high-fat diet administration, 4 mg, N = 12). A two-period, self-crossover, open-labeled, single ascending-dose study was designed for elderly subjects (2 and 4 mg, N = 11).

Results

For young subjects’ study: In the MAD arm, the accumulation ratios of DC20 in vivo were 2.29 and 2.15, respectively. In the food effect arm, compared with fasting administration, area under the concentration-time curve from zero to t (AUC_0-t) orally after a standard diet and high-fat diet slightly increased by about 19% and 29% and the T_max were delayed by around 1 hour. For elderly subjects’ study, T_max were 1.5 and 1.25 hour, t_1/2 were 77.1 and 74.2 hour, respectively. After oral administration of DC20 in healthy young and elderly subjects, no serious adverse events occurred, the most common adverse events associated with the study drug were gastrointestinal reactions.

Conclusion

We predicted the safety risks of DC20 in the clinical treatment of AD, which were well tolerated by the healthy young and elderly subjects. The elimination of DC20 from the body was slower in elderly subjects than in young subjects.

Trial registration numbers:

CTR20181428, CTR20190664, CTR20191878, CTR20192724 approved by the Center for Drug Evaluation, National Medical Products Administration.

Alzheimer’s disease

Fluoropezil (DC20)

Donepezil

Pharmacokinetics

Adverse Events

Acetylcholinesterase

Clinical trial

In recent years, two major pathological markers of Alzheimer's Disease (AD) have gained increasing prominence: “the neurofibrillary tangles (NFTs) and senile plaques (SP)” in the brain [1]. The neurofibrillary tangles are intracellular deposits made up of filaments of abnormally hyperphosphorylated tau [2, 3]. The senile plaques are extracellular deposits consisting of multimeric aggregates of 4-kDa polypeptides known as the amyloid protein or Aβ [4–6]. While NFTs (tau pathology) have been associated with a variety of neurodegenerative diseases including non‐Alzheimer dementias, senile plaques are unique to AD, which indicates that Aβ accumulation might be the essential step in the pathogenesis of AD and NFTs could be the secondary phenomenon in AD [6–8].

Despite the growing population of patients with AD, only five treatment options [9], which include four cholinesterase inhibitors, one N-methyl-D-aspartate receptor antagonist, are currently approved in the United States. Cholinesterase inhibitors, including tacrine (Warner-Lambert, 40 to 80 mg per day, not used anymore), donepezil (Pfizer, 5 to 10 mg per day), galantamine (Janssen, at least 16 mg per day), or rivastigmine (Novartis, 6 to 12 mg per day orally or 9.5 mg per day trans-dermally), should be considered for treatment of cognitive and functional decline in patients with mild to moderate AD.

All these drugs currently available on the market are only symptomatic in nature, trying to counterbalance the neurotransmitter disturbance of the disease. So far, no drug for disease-modifying treatment has been approved to enter the market, although a lot of funds and manpower have been invested in the research and development of disease-modifying treatment drugs.

Acetylcholinesterase (AChE) inhibitors attempt to reduce the breakdown of acetylcholine levels in the brain of patients with AD by inhibiting the responsible enzyme AChE in the synaptic cleft [10]. Thus, AChE enhance central cholinergic neurotransmission, mitigating decline in cognition at least during the first year of treatment. These drugs have been regarded as the standard and first-line treatment for AD. [11].

A further therapeutic option for moderate to severe AD is memantine (Lundbeck, Denmark), which is an uncompetitive, moderate-affinity N-methyl-D-aspartate (NMDA) antagonist believed to protect neurons from excitotoxicity. Memantine may reduce behavioral and psychological symptoms of dementia [12] and improve cognitive abilities, activities of daily living (ADL), and behaviors in people with moderate to severe AD after 6 months of use [13].

Preclinical studies showed that fluoropezil (DC20) could be used as a new generation AChE inhibitor with a clear mechanism, a high selectivity of pharmacological targets, and a wide safety window [14]. This Phase I study aimed to evaluate the safety, tolerability, and pharmacokinetics of DC20 in healthy young and elderly Chinese subjects.

Study design

The study of young subjects included two following arms: the multiple ascending-dose (MAD) arm, and the food effect arm (Figure 1). The MAD arm (Part A) was a Phase I, single-center, double-blind, randomized, placebo-controlled, multiple ascending-dose study of healthy young subjects that explored multiple doses of 2 and 6 mg DC20 q.d. Before dose-escalation, all available safety data were reviewed to determine the appropriate dose for the next cohort. The food effect arm (Part B) was a Phase I, single-center, three-period, self-crossover, fasting/standard diet/high-fat diet administration study. Subjects in this arm were administrated 4 mg DC20, and the washout period was 7-days. The high-fat diet contains high-calorie of about 800-1000 kcal which consisting of about 150 kcal of protein, about 250 kcal of carbohydrates, and about 500-600 kcal of fat.

The study of elderly subjects (Part C) was a Phase I, single-center, two-period, self-crossover, open-label, single ascending-dose study (Figure 1). The washout period was a minimum of 10-days (adjusted according to the pharmacokinetics results) between two doses (2 mg and 4 mg) of drug administrations. Before dose-escalation, all available safety data were reviewed to determine the appropriate dose for the next cohort.

The subjects received DC20 in the morning after a 12-h fast. Except for standard diet cohort and high-fat diet cohort in the food effect arm, other cohorts in all the studies administration of DC20 in the fasted state. In fast cohorts, food intake was not allowed for 4 hours after drug administration, while water was allowed ad libitum. In standard diet cohort and high-fat diet cohort, drug administrated in 30 minutes after starting of food intake, while water was not allowed ad libitum in 1 hour. All the subjects stayed at the study site from the evening one day before dosing initiation to seven days after administration. After the blood samples were collected, prescriptive inspections of the subjects were performed. The data on safety, tolerability, and pharmacokinetics were collected throughout the study.

All the studies were conducted in Shanghai, China and approved by the Center for Drug Evaluation, National Medical Products Administration as CTR20181428, CTR20190664, CTR20191878, CTR20192724. All the studies were conducted by all applicable regulatory and Good Clinical Practice guidelines and the Declaration of Helsinki. All subjects provided written informed consent before study participation.

Subjects

For young subjects’ study, the inclusion criteria for eligible subjects were following overtly healthy males and females ages from 18 to 40 years old, with a body mass index (BMI) ranging from 19 to 26 kg/m². The recruitment of subjects was based on medical history, screening evaluations (including electrocardiograms (ECG), clinical laboratory blood tests, and blood pressure assessment), and physical examination. Exclusion criteria were the following: subjects with the central nervous system, cardiovascular system, digestive system, respiratory system, urinary system, and blood system disease.

The requirements for recruitment of elderly subjects were different from that of young subjects since elderly subjects usually have a lot have complex characteristics and chronic diseases. Inclusion criteria: healthy men and women, aged ≥ 60 years old, with a BMI ranging from 19 to 30 kg/m². The recruitment of subjects was based on medical history, screening evaluations (including ECG, clinical laboratory blood tests, and blood pressure assessment), and physical examination. Those with no evidence of diseases of the major organs and no clinically significant abnormalities of chest X-ray, abdominal B-ultrasound; those with chronic diseases and abnormal test results, which did not affect the clinical trial observation items (e.g., hyperlipidemia and non-insulin-dependent diabetes are well-controlled, etc.). Exclusion criteria were the following: any medical condition that could constitute a risk when taking the study medication, history of significant allergy, alcoholic or controlled drug abuse; any surgical condition or other kinds of condition that could significantly affect drug ADME or any surgical condition or condition that could be harmful to the subjects.

Pharmacokinetics

For the study of young subjects, there were two arms: MAD arm, and food effect arm. In the MAD arm (Part A), plasma samples were taken at pre-dose (0) and 0.25, 0.5, 0.75, 1, 1.5, 2, 4, 6, 8, 10, 12, 24 and 48 hour post-dose on days 1, 9 and some additional samples were taken on days 7, 8, 11, 12. For food effect arm (Part B), plasma samples were taken at pre-dose (0) and 0.25, 0.5, 0.75, 1, 1.5, 2, 4, 6, 8, 10, 12, 24, 48, 72 and 96 hour post-dose. For the study of elderly subjects (Part C), blood samples were collected at pre-dose (0) and 0.5, 1, 1.5, 2, 3, 4, 6, 8, 10, 12, 24, 48, 72, 96, and 120 hour post-dose.

Pharmacokinetic plasma samples were analyzed using liquid chromatography-tandem mass spectrometry (LC/MS/MS) for DC20 at Center for Drug Metabolism and Pharmacokinetics, Shanghai Institute of Materia Medica. The analytical method was validated for a concentration range of 0.1 to 20.0 ng/ml in plasma for both DC20. The coefficient of variation over this range was ≤ 15%. The samples were quantified for DC20 using a validated liquid chromatography/tandem mass spectrometry (LC/MS/MS) method. Briefly, the separation was conducted on a UPLC-30A liquid chromatographic system (Shimadzu, Kyoto, Japan). The analytical column used the BEH C18 column (50*2.1 mm, 1.7 um, waters, MA, USA.).

Safety and Tolerability

All subjects were included in the safety analysis. Blood samples were taken for safety laboratory assessments (hematology and clinical chemistry) at screening, pre-dose, throughout the clinical phase of the study, and at follow-up (at least 7 days after the final dose of the study drug). Safety assessments included assessment of vital signs, physical examinations, AEs, and clinical laboratory tests including clinical chemistry, hematology, urinalysis, and 12-lead ECG analysis were performed according to predefined schedules. AEs were monitored throughout the study. All AEs were recorded and evaluated by investigators in terms of intensity (mild, moderate, or severe), duration, severity, outcome, and relationship to study drug.

Statistical analysis

Safety parameters, including safety labs, vital signs, and ECG parameters, were summarized descriptively. Pharmacokinetics parameters for DC20 was calculated by standard noncompartmental methods of analysis using WinNonlin version 5.3 and included the area under the concentration-time curve from zero to t, where t is the last time point with a measurable concentration (AUC_0-t), area under the concentration curve from zero to infinity (AUC_0-_∞), area under the concentration-time curve during one dosing interval at steady state (AUC₍_τ_{, ss)}), and maximum concentration (C_max) after single or repeated doses. Other parameters, such as time of maximal concentration (T_max), elimination half-life (t_1/2), accumulation ratio (R_AUC), volume of distribution (Vλz/F), and linearity index, were calculated, data permitting. Subjects who vomited on the day of pharmacokinetics assessment were excluded from the analysis.

Ethical approval

Ethical approvals were obtained from Shanghai Xuhui Central Hospital. Informed consent was obtained from all patients participating in the studies.

Subjects

For the study in the young subjects (Part A and Part B), the demographics of each study population were well balanced between cohorts and arms. The mean age for the MAD arm and food effect arm subjects was 29.5 (range 19–39) and 28.8 (range 19–35) years, respectively; the mean BMI was 23.31 (range 20.3–25.9) and 23.15 (range 19.4–25.7) kg/m², respectively. The majority of subjects in each study were male and Han Chinese (79.2% and 22/24 in the MAD arm; 91.7% and 11/12 in the food effect arm).

For the study of elderly subjects (Part C), all subjects were of Han Chinese, with a mean age of 67.2 (range 62-73) years, the mean BMI was 25.2 (range 22.2-28.4) kg/m². None of the subjects drank alcohol, had a history of drug use, and had blood donation/blood loss within 3 months. Only 1 subject (9.1%) had a history of allergies.

There were totally 3 subjects withdraw in these studies: 1 subject in the MAD arm (2mg cohort), 1 subject in the food effect arm (high fat cohort), and 1 subject in elderly subjects’ study (4 mg cohort). None of subjects withdraw events were related with serious adverse events (SAEs)

Pharmacokinetic Properties

For the study of young subjects, there were two arms of results: MAD arm and food effect arm.

In the MAD arm (Part A), DC20 were successive administration for 9 days. For 2 mg cohort and 6 mg cohort, the C_max of DC20 were 5.67 and 18.0 ng/ml, and AUC_{(τ, ss)} were 55.7 and 139 h*ng/ml, respectively. In terms of AUC_{(τ, ss)}, the R_AUC of were 2.29 and 2.15, respectively (Table 1). The plasma concentrations of DC20 in the MAD arm are shown in Figure 2.

In the food effect arm (Part B), the pharmacokinetics parameters of DC20 were summarized in Table 2. Compared high-fat diet administration with fasting administration, C_max of DC20 did not significantly change after T_max was delayed from 1 h to 2 h, and AUC_0-t increased by about 29%. Compared standard diet administration with fasting administration, C_max of DC20 did not significantly change, the peak time was delayed from 1 h to 1.75 h, and AUC_0-t increased by about 19%. The plasma concentrations of DC20 in the food effect arm are shown in Figure 3. The ratios of DC20 pharmacokinetic parameters between high-fat diets and fasting after oral DC20 tablets are shown in Table 3. The ratios of DC20 pharmacokinetic parameters between standard diets and fasting after oral DC20 tablets are shown in Table 4.

For the study of elderly subjects (Part C), the pharmacokinetics parameters of DC20 were summarized in Table 5. The plasma concentrations of DC20 after single oral dose (2 and 4 mg) in elderly subjects’ study are shown in Figure 4. DC20 of 2 mg and 4 mg was rapidly absorbed following oral administration with the C_max attained at 3.45 and 6.27 ng/ml, and T_max at 1.5 and 1.25 hour. The t_1/2 of DC20 was calculated as 77.1 hour with 2 mg and 74.2 hour with 4 mg, which was indicative of the slow elimination of DC20 from the body.

Safety and Tolerability

For the study of young subjects, a summary of treatment-emergent adverse events (TEAEs) during treatment was provided in Tables 6-7. The MAD arm and food effect arm results indicated that 6 mg q.d. and 4 mg with a different diet (fast/standard diet/high-fat diet) were well tolerated and did not induce any serious SAEs or the TEAEs of Grade 3 or higher.

For the study of elderly subjects, the results showed that DC20 was well tolerated after single doses of 2 mg and 4 mg. There were no serious AEs and no significant differences in the number or nature of AEs reported across the two cohorts. No subject discontinued participation in the study because of AEs. In addition, there were no clinically significant differences in safety laboratory values across the two cohorts and no differences in changes from baseline. TEAEs based on the safety analysis set is summarized in Table 8. TEAEs related to the study drug, which occurred at least once during the treatment period, is summarized in Table 9. Table 10 summarizes the Common Terminology Criteria for Adverse Events (CTCAE）classification of study drug-related AEs during treatment.

There were no clinically relevant alterations in hematology, clinical chemistry, vital signs, or ECG measurements during all the studies.

Donepezil, which is the first option drug for the clinical treatment of mild or moderate Alzheimer's dementia symptoms, currently accounts for nearly 57% of the market share of AD drugs in China. Fluoropezil (DC20), a new-generation AChE inhibitor, was developed by Shanghai Institute of Materia Medica on the basis of a kinetics-driven drug design strategy [14].

The single ascending-dose (SAD) arm study (double-blind, randomized, placebo-controlled, single ascending-dose, 1 to 12mg, N= 50) of DC20 were described in the previous paper [14]. DC20 was expected to show a dose-proportional pharmacokinetic profile in the dose range in the SAD. According to the previous literature, AUC_0-∞ of donepezil was 498ng*hr in healthy volunteers after 5mg single-dose administration. Preclinical studies have shown that the potency of DC20 in mouse brain and rat cortex and hippocampus was 3 times, 16 times, and 8 times stronger than donepezil in vitro anti-AChE activity, respectively. So, the MAD doses were selected as low-dose 2mg and high-dose 6mg were determined based on the potency ratio between donepezil and DC20. The T_max of DC20 (1.5 hour) was shorter than that of donepezil (4 hour), suggesting that the absorption time of DC20 would be faster. The t_1/2of DC20 was around 30 hours, suggesting that clinical administration could be a once-daily dose for young subjects, faster than that of donepezil which was around 70 hours [15]. The R_AUC of DC20 was approximately consistent across doses in this study. The food effect arm results were carried out to investigate the effect of food on the absorption of DC20 at the same time. An increase of 29% in AUC_0-t was observed under the high fat diet, which was slightly increase from the fasting. Results from the standard diet indicated a slightly increase of 19% in AUC_0-t compared with the fasting condition. However, the T_max of these two feeds was delayed from 1 hour to 1.75 hours.

In the study of young subjects (include SAD arm study in previous paper), reported AEs of DC20 were consistent with an increase in dosage. Based on preclinical pharmacology and toxicology studies of DC20, the expected and most common clinical AEs were diarrhea, muscle cramps, fatigue, nausea, vomiting, and insomnia. The highest incidence of AEs was in the digestive system, with diarrhea occurring the most frequently, which was consistent with the previous studies and the effect of donepezil. In the multiple-dose study, 11.1% and 44.4% of diarrhea occurred in the 2 mg dose cohort and 6 mg dose cohort, respectively, compared with 16.7% in the placebo control. In the food influence study, a total of 41.7% of diarrhea occurred. In contrast to placebo control, the incidence of diarrhea was higher in the 6 mg dosage cohort and fasting cohort; however, the incidence of diarrhea in all 2 mg dosage cohorts was not higher than that in the placebo control. Overall, diarrhea was more likely to occur at higher doses or when administered under fasting conditions. It seemed that there was a drug- or dose-related increase in the incidence of reported AEs. The severity of all instances of diarrhea in this study was grade 1, and all of them resolved spontaneously without medication.

Concerning the study of elderly subjects, Ohnishi et al have conducted pharmacokinetic studies of donepezil in healthy elderly or Alzheimer's patients [16]. Compared with 65-year-old subjects, the clearance rate of 90-year-old subjects decreased by 17%, while the clearance rate of 40-year-old subjects increased by 33% [10,12]. Based on these reports, the T_max and t_1/2of the elderly were longer than that of subjects. Based on the comprehensive clinical dosage of donepezil hydrochloride and preclinical efficacy and toxicity results of DC20 and based on the calculated dosage of multi-administered dose, the 2 mg and 4 mg dose cohorts were selected for the single dose-escalation study in Chinese elderly healthy subjects.

In the study of elderly subjects, 2 mg and 4 mg dose of DC20 as the tablet formulation was found to be rapidly absorbed from the empty gastrointestinal tract, as indicated by the T_max, which was achieved 1.25 to 1.5h after administration. The terminal elimination, t_1/2 of DC20 was slow, ranging from 77 hour to 74 hour. The observed long t_1/2of DC20 in clinical studies was consistent with the kinetic characteristics of DC20 on AChE inhibition in preclinical research, which were reversible, high-affinity, and slow dissociation processes [14]. The pharmacokinetics results of DC20 at a dose of 2 and 4 mg also showed that the C_max, AUC_0-t, and AUC_0-∞ increased proportionally to the dose. V_z/F ranged from 3600 to 3830 liters, which reflected excellent tissue penetration for DC20.

When comparing the young subjects’ SAD arm study (4 mg cohorts) with elderly subjects’ study (4 mg cohorts), a sightly increase in T_max, AUC_0-t,and t_1/2 (T_max 0.75 vs. 1.25 hour, AUC_0-t 59.2 vs. 91.0 ng/ml, t_1/2 30.8 vs. 74.2 h) were observed. A sightly decrease in C_max (8.61 vs. 6.27 ng/ml) was also observed in contrast with young and elderly subjects in 4 mg dose cohorts. The difference in pharmacokinetics parameters indicated that the gastrointestinal absorption or gastric motility of the elderly was much slower than that of the young cohorts.

By combining the data from the preclinical pharmacology and toxicology studies of DC20 and the clinical AEs of donepezil [11], a marketed drug as an AChE inhibitor, the most common AEs that occurred during the clinical trial were: diarrhea, muscle cramps, fatigue, nausea, vomiting, and insomnia. DC20 was well tolerated by the healthy elderly subjects in this study after 2 and 4 mg dose administration. Gastrointestinal diseases, such as nausea, vomiting, diarrhea, and abdominal pain, were the most reported TEAEs across the studies, which was consistent with the MAD arm and food effect arm. Nausea occurred in 1 out of 11 subjects (9.1%) in the 2 mg dose cohort. Dry mouth, abdominal discomfort, and bloating occurred 2 (20%), 1 (10%), and 1 (10%), respectively, in 10 subjects with the 4 mg dose cohort. There were no SAEs, grade 3 or higher TEAEs, or TEAEs that led to withdraw, or deaths that occurred in elderly subjects’ study.

The results of the elderly subjects' study revealed that the blood cell AChE activities (96.22% to 103.65%) after oral 2 mg cohort for 0-48 hours did not significantly change. Similar data were observed in the 4 mg dose cohort (elderly subjects' study). These results suggested that DC20 might have very little influence on peripheral AChE activity, and may therefore resulting in less peripheral AChE inhibition-caused side effects. However, further optimized analytical methods should be used to better determine the inhibitory effects of DC20 on AChE activity in blood cells.

In addition, the incidence of AEs such as elevated alanine aminotransferase and nausea was equivalent to that of donepezil. Compared with donepezil (SAD, 2 mg cohort and 4 mg cohort) [11], DC20 reached the peak faster (T_max of donepezil were 4.5 and 4.7 hour, while that of DC20 1.5 and 0.75 hour), and the volume of distribution was larger (the Vλz/F of donepezil were 8.6 and 12.9 L/kg, while that of DC20 is 32.3 and 40.1 L/kg), which suggested that it might more rapidly and widely distributed in tissues such as the brain. The elimination of DC20 from the body was slower in elderly subjects than in young subjects. The safety risks of DC20 in the clinical treatment of AD could be predicted.

Authors have non-financial interests that are related to the work submitted for publication.

Consent for publication: all authors consent to the publication of the manuscript in Alzheimer's Research & Therapy. No personal or clinical details of participants are presented.

Funding

None

Data availability

All data generated or analyzed during this study are included in this published article.

Acknowledgments

All authors performed data analysis and interpretation, as well as manuscript review and approval. Chengyin Yu is paid employee of the Shanghai Institute of Materia Medica, and simultaneously worked for Central Laboratory, Shanghai Xuhui Central Hospital. Jiao Jing is the sponsor who works for Jiangsu Kanion Pharmaceutical Co., LTD. Xin Xu is worked for Shanghai Kerlin Biomedical Technology Co., LTD. All the other authors were paid employees of Central Laboratory, Shanghai Xuhui Central Hospital, or Shanghai Institute of Materia Medica, at the time of this work.

This study was supported by Jiangsu Kanion Pharmaceutical Co., LTD.

Contributions of Authors statement

Chengyin Yu performed the research and wrote the paper. Hongjie Qian, Huijuan Zhu, Qichen Ding, and Yuting Cai performed the research. Jiao Jing, Xin Xu, and Runcong Guo analyzed the data. Haiyan Zhang, Hong Liu, Xiaoyan Chen, and Yun Liu revised the paper and conceived the study.

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Table 1. Pharmacokinetic parameter of DC20 in MAD arm

Dose	Parameter	Unit	Day 1					Day 9
Dose	Parameter	Unit	N	Mean	SD	CV%	N	Mean	SD	CV%
2 mg	λ_z	1/h	9	0.0334	0.00441	13.2	8	0.0123	0.00263	21.4
	t_1/2z	h	9	21.1	2.73	13.0	8	58.7	12.4	21.2
	*T_max	h	9	1.50(0.750-1.50)			8	1.00(1.00-4.00)
	C_max	ng/ml	9	3.87	1.15	29.8	8	5.67	1.47	25.9
	AUC_0-t	h*ng/ml	9	32.3	4.46	13.8	8	115	22.2	19.2
	AUC_0-24h	h*ng/ml	9	25.1	3.49	13.9
	AUC_0-_∞	h*ng/ml					8	161	37.6	23.3
	T_min	h					8	9.03	12.4	137
	C_min	ng/ml					8	1.27	0.248	19.6
	C_avg	ng/ml					8	2.32	0.416	17.9
	DF						8	189	37.8	20.0
	CL_ss/F	L/h					8	37.1	7.51	20.3
	V_z/F	L					8	3090	612	19.8
	AUC_ss	h*ng/ml					8	55.7	9.98	17.9
	R_AUC						8	2.29	0.348	15.2
	R_Cmax						8	1.60	0.562	35.1
6 mg	λ_z	1/h	9	0.0391	0.00603	15.4	9	0.0130	0.00345	26.5
	t_1/2z	h	9	18.1	2.46	13.6	9	56.8	14.8	26.1
	*T_max	h	90	1.00(0.750-4.00)			9	1.00(0.750-2.00)
	C_max	ng/ml	9	13.1	4.34	33.2	9	18.0	3.24	18.0
	AUC_0-t	h*ng/ml	9	80.1	14.4	18.0	9	267	68.4	25.7
	AUC_0-24h	h*ng/ml	9	64.6	11.2	17.4
	AUC_0-_∞	h*ng/ml					9	357	103	28.8
	T_min	h					9	2.67	8.00	300
	C_min	ng/ml					9	2.77	0.881	31.8
	C_avg	ng/ml					9	5.78	1.29	22.4
	DF						9	269	44.9	16.7
	CL_ss/F	L/h					9	45.2	9.80	21.7
	V_z/F	L					9	3670	1170	31.9
	AUC_ss	h*ng/ml					9	139	31.0	22.4
	R_AUC						9	2.15	0.334	15.5
	R_Cmax						9	1.53	0.664	43.3

Table 2. Pharmacokinetic parameter of DC20 in food effect arm

	Parameter	Unit	N	Mean	SD	CV%
Fasting	λ_z	1/h	12	0.0143	0.00351	24.5
	t_1/2z	h	12	51.9	16.9	32.5
	*T_max	h	12	1.00(0.750-4.00)
	C_max	ng/ml	12	9.32	3.74	40.1
	AUC_0-t	h*ng/ml	12	89.8	33.5	37.3
	AUC_0-∞	h*ng/ml	12	116	66.0	56.8
	V_z/F	L	12	2750	545	19.8
	Cl/F	L/h	12	39.4	10.7	27.1
High-fat meal	λ_z	1/h	11	0.0142	0.00404	28.4
	t_1/2z	h	11	52.2	14.3	27.3
	*T_max	h	11	2.00(1.00-4.00)
	C_max	ng/ml	11	8.79	1.99	22.6
	AUC_0-t	h*ng/ml	11	113	31.9	28.1
	AUC_0-∞	h*ng/ml	11	142	53.0	37.4
	V_z/F	L	11	2240	658	29.4
	Cl/F	L/h	11	30.9	8.57	27.8
Standard meal	λ_z	1/h	12	0.0142	0.00198	13.9
	t_1/2z	h	12	49.8	7.20	14.5
	*T_max	h	12	1.75(0.750-4.00)
	C_max	ng/ml	12	9.11	1.38	15.1
	AUC_0-t	h*ng/ml	12	104	20.7	19.8
	AUC_0-∞	h*ng/ml	12	128	30.9	24.1
	V_z/F	L	12	2310	433	18.7
	Cl/F	L/h	12	32.7	6.76	20.7

Table 3. Ratios of DC20 pharmacokinetic parameters between high-fat meal and fasting after oral DC20 tablets

Analyte	Parameter	High fat meal-GeoLSM	Fasting-GeoLSM	Ratio_% Fed/Fasting	90%CI
DC20	AUC_0-∞	137	107	128.28	116.34-141.44
	AUC_0-t	112	86.0	129.61	120.29-139.64
	C_max	8.52	8.73	97.62	84.11-113.30

Table 4. Ratios of DC20 pharmacokinetic parameters between standard meal and fasting after oral DC20 tablets

Analyte	Parameter	Standard meal-GeoLSM	Fasting-GeoLSM	Ratio_% Std/Fasting	90%CI
DC20	AUC_0-_∞	125	107	116.99	106.46-128.56
	AUC_0-t	103	86.0	119.28	111.00-128.19
	C_max	9.00	8.73	103.09	89.25-119.08

Table 5. Main pharmacokinetic parameter for DC20 in elderly subjects after SAD of DC20

Sample	Dose	PK data	Unit	N	Mean	SD	CV%
DC20	2 mg	^*T_max	h	11	1.50(1.00-3.00)
		C_max	ng/ml	11	3.45	0.861	25.0
		AUC_0-t	h^*ng/ml	11	44.3	14.3	32.2
		AUC_0-120h	h^*ng/ml	11	44.6	12.6	28.2
		AUC_0-∞	h*ng/ml	11	60.7	21.4	35.2
		t_1/2z	h	11	77.1	26.7	34.6
		Cl/F	L/h	11	36.7	12.0	32.7
		V_z/F	L	11	3830	1260	32.9
		lz	1/h	11	0.0100	0.00351	35.0
		MRT_0-∞	h	11	84.4	28.5	33.7

DC20	4 mg	^*T_max	h	10	1.25(1.00-6.00)
		C_max	ng/ml	10	6.27	1.82	29.1
		AUC_0-t	h^*ng/ml	10	91.0	27.0	29.6
		AUC_0-120h	h^*ng/ml	10	88.4	26.3	29.8
		AUC_0-∞	h*ng/ml	10	121	52.4	43.2
		t_1/2z	h	10	74.2	34.1	45.9
		Cl/F	L/h	10	38.8	15.6	40.3
		V_z/F	L	10	3600	961	26.7
		lz	1/h	10	0.0113	0.00523	46.4
		MRT_0-∞	h	10	83.0	38.8	46.7

*: Median (minimum - maximum).

Table 6. Summary of the TEAE for MAD arm in the study of young subjects

	DC20
	2mg N=9 n (%) E*	6mg N=9 n (%) E*	Placebo N=6 n (%) E*
Any TEAE	8 (88.9) 12	7 (77.8) 22	4 (66.7) 8
TEAE related to study drug	7 (77.8) 10	7 (77.8) 21	4 (66.7) 7

Serious adverse event（SAE）	0	0	0
SAE related to study drug	0	0	0

Level 3 and higher TEAE	0	0	0
Level 3 and higher TEAEs related to study drug	0	0	0

Adverse events leading to withdrawal from the trial	1 (11.1) 2	0	0

Adverse events leading to the death of the subjects	0	0	0
Adverse events related to the study drug that led to the death of the subject	0	0	0