First-In-Human Safety,Tolerability,and Pharmacokinetics of Single and Multiple Doses of AZP2006,A Synthetic Compound for the Treatment of Alzheimer’s Disease and Related Diseases

Abstract

Background:

Alzheimer’s disease (AD) and progressive supranuclear palsy (PSP) are major neurodegenerative conditions with tau pathology in common but distinct symptoms—AD involves cognitive decline while PSP affects balance and eye movement. Progranulin (PGRN) is a growth factor implicated in neurodegenerative diseases, including AD and PSP. AZP2006, a synthetic compound, targets tauopathies by stabilizing PGRN levels and reducing tau aggregation and neuroinflammation.

Objective:

Evaluate the safety, tolerability, and pharmacokinetics of AZP2006.

Methods:

A first-in-Human phase 1 study comprised a single ascending dose (SAD) and a multiple ascending dose study (MAD). The SAD study included 64 healthy male volunteers and tested singles oral doses of 3 to 500 mg of AZP2006 free base equivalent or placebo. In the MAD study, 24 healthy male volunteers were administered oral doses of 30, 60, and 120 mg per day of AZP2006 or placebo for 10 days.

Results:

No serious adverse events were observed. Clinical, biological, and electrocardiogram findings were non-relevant. Nineteen minor adverse events resolved before study completion. The safety profile indicated no specific risks. The multiple ascending dose study was halted, and the optional dose level of 180 mg was not performed due to high levels of M2 metabolite in plasma that necessitated additional preclinical evaluation of M2. Both AZP2006 and its M2 metabolite were quickly absorbed and widely distributed in tissues. Exposure increased more than proportionally with dose.

Conclusions:

AZP2006 had a favorable safety profile and was rapidly absorbed. Elevated M2 metabolite levels necessitated further studies to clarify excretion and metabolism mechanisms.

Keywords

Alzheimer’s disease AZP2006 ezeprogind multiple ascending dose neurologic degenerative diseases progranulin progressive supranuclear palsy single ascending dose small-molecule drug tauopathies

INTRODUCTION

Alzheimer’s disease (AD) and progressive supranuclear palsy (PSP) are progressive neurodegenerative disorders that significantly impact cognitive and motor functions, respectively. AD is characterized by memory loss, cognitive decline, and daily function deterioration, and remains the most common cause of dementia among older adults, affecting an estimated 47 million people worldwide. PSP is an orphan, debilitating neurodegenerative disease, marked by difficulties in balance, eye movement, and speech [1 –4]. It is characterized by neuronal loss, gliosis, and accumulation of tau-immunoreactive neurofibrillary tangles in specific brain areas. A strong genetic association exists with the tau protein gene (MAPT), making PSP a primary tauopathy [1]. Under pathological conditions, hyperphosphorylated tau detaches from microtubules and forms toxic filaments, accumulating in neurons and glia. Despite the prevalence and considerable burden these diseases place on patients and caregivers, the underlying mechanisms of AD and PSP are not fully understood and effective treatments for both conditions remain limited. This study aims to advance the potential treatment of AD, PSP, and related neurodegenerative diseases by developing the small molecule AZP2006, which mode of action enables a pleiotropic effect through the modulation of progranulin (PGRN) [5].

PGRN is a growth factor highly expressed in central nervous system microglia and implicated in neurodegenerative disorders like frontotemporal dementia and certain forms of AD [6 –8]. PGRN modulates inflammation, oxidative stress, enhances neuronal survival, and plays diverse roles in the clearance amyloid-β protein(Aβ) and modulation of tau phosphorylation [9,10, 9,10]. PGRN interacts with various receptors including EphA2 and Notch [8,11, 8,11] and is vital for lysosomal function [12]. Its deficiency, often due to gene mutations, exacerbates neurodegeneration by affecting microglial activation. Several studies indicate its crucial role in neurodegenerative pathogenesis, making it a potential therapeutic target. Elevated levels of PGRN, and its interacting partner prosaposin (PSAP) [13], offer a promising treatment avenue, although further research is required to clarify mechanisms and therapeutic strategies.

AZP2006 (N-(3-(4-(3-(diisobutylamino) propyl) piperazin-1-yl) propyl)-1H-benzo[d]imidazol-2-amine disulphate salt; INN: ezeprogind) is a small synthetic cationic amphiphilic compound.

The main human metabolite of the AZP2006 parent compound is M2, also known as AZP2045. According to preclinical studies, M2 is produced by the cytochrome P450 3A4 enzyme, which is responsible for the N-dealkylation of the parent compound, specifically the loss of an isobutyl group. The M2 metabolite is pharmacologically inactive as it did not show any significant biological activity in numerous studies.

The compound was originally developed for the treatment of AD since it can reduce the secretion of the Aβ_1 - 42, a key protein involved in the pathogenesis of AD [14]. However additional preclinical studies have shown that AZP2006 has the potential to treat tauopathies according to the following mechanisms of action: (i) decreasing the hyperphosphorylated tau protein,(ii) stabilizing PGRN levels, and (iii) decreasing microglial-induced neuroinflammation [5].

Taking into account the previous enounced AZP2006 in vitro properties and the results obtained from preclinical studies performed in PSP animal models that demonstrated a significant impact on both tau hyperphosphorylation and neuroinflammation [1,15, 1,15], AZP2006 is currently developed for the treatment of PSP.

Through Phase 1 clinical studies, we aimed to assess safety and tolerability and to obtain a first pharmacokinetics (PK) profile of AZP2006 and its metabolite M2 in healthy humans. The encouraging results of the first-in-human Phase 1 study are presented here:

A safety, tolerability, and PK single ascending dose (SAD) study. Primary objectives were to determine the tolerability and safety, and the secondary objectives were to determine the PK of single ascending doses of AZP2006 in healthy volunteers and obtain preliminary data on blood to plasma repartition of AZP2006.

A multiple ascending dose (MAD) study to assess safety, tolerability, and PK of AZP2006 in healthy volunteers. Primary objectives were to evaluate the safety and tolerability of up to four dose levels of AZP2006 and the secondary objectives were to determine the PK parameters of AZP2006 after multiple dose administration and to investigate the pharmacodynamics effects of AZP2006 after multiple dose administration.

METHODS

The study was a first-in-human Phase 1, single-center, double-blind, randomized study of ascending single and multiple doses in healthy male volunteers.

Study 1 – Single Ascending Dose Study (SAD)

AZP2006C01/OP086913.ALZ; EudraCT N° 2014-000247-33.

Study design

The SAD study testedsingles doses of 3, 5, 10, 30, 90, 180, 360, and 500 mg of AZP2006 free base equivalent in healthy male subjects (Fig. 1).

Fig. 1

Phase 1 Study design. Single ascending dose (SAD) study included 64 healthy subjects. Multiple ascending dose study (MAD) included only 32 healthy subjects corresponding to the Part A. The 180 mg dose of the Part A and the whole Part B were not performed (*).

Sixty-four healthy adults were included and 8 were assigned (6 verum and 2 placebos) to each dose level. Participants were all males, age comprised between 18 and 45 years and their healthy status ascertained by a comprehensive clinical assessment with a body mass index between 18.5 and 30 kg/m², smoking less than five cigarettes per day, with normal blood pressure, heart rate and normal electrocardiogram (ECG), as well as laboratory parameters within the normal range (hematological, blood chemistry tests, and urinalysis).

The subjects were screened 21 days prior administration of the first dose, were hospitalized for approximately 96 hours (Day 1 morning to Day 4 morning) and received only one oral dose of verum or placebo. The end-of-study visit for the individual subject was on Day 7. AZP2006 (or placebo) was administered at 8 a.m. in sitting position, fasting condition, and as aqueous solutions prepared at 2 and 10 mg/ml. The doses were 3, 5, 10, 30, 90, 180, 360, and 500 mg free base equivalent. The administration of the investigational product in each dose group of the study was done sequentially within each cohort, on three consecutive days as follows: 2-3-3 subjects each day, respectively, to minimize the risk. For the first 2 subjects of a dose level, the administrations took place with a lag time of 1 h during which the Investigator carefully monitored for adverse events (AEs) or reactions, if any. At the first day of dosing, the first 2 subjects were randomized in a ratio of verum:placebo equal to 1.

Data analysis

The parameters registered to evaluate the primary endpoint (tolerability and safety) were:

Physical examination and vital signs

12-lead ECG

Psychometrics tests of awareness: Computerized Memory Test and Multiple-Choice Reaction Time

Clinical laboratory tests (hematology, clinical chemistry, and urinalysis)

Aes

PK samplings:

Plasma: at pre-dose, 0.5, 1, 1.5, 2, 2.5, 3, 4, 5, 6, 7, 8, 10, 12, 24, 48, and 72 h.

Whole blood: after the administration of 3, 5, 10, and 30 mg of AZP2006, concentration was assessed at 0.5, 1.5, and 5 h only. After administration of 90, 180, 360, and 500 mg AZP2006, concentration was analyzed at all time points (0.5, 1, 1.5, 2, 2.5, 3, 4, 5, 6, 7, 8, 10, 12, 24, 48, and 72 h).

Bioanalytical methods

One set of plasma or blood samples was analyzed using a previously validated method according to the Guidance for Industry – Bioanalytical Validation – FDA May 2001, draft from September 2013 and EMA EMEA/CHMP/EWP/192217/2009 – July 2011 and the requirements of Good Laboratory Practices (OECD ENV/MC/CHEM (98) 17) referenced PKH/MOA/550 for human plasma and PKH/MOA/554 for human blood as detailed in validation study plan 14-184 (human plasma) and 14-229 (human blood).

AZP2006 and M2 concentrations were calculated using Watson^® 7.2.0.03 directly from chromatograms after automatic integration by Analyst 1.5.1. and expressed as ng/mL. Mean concentrations of AZP2006 and M2 were calculated by using individual AZP2006 and M2 concentrations (without considering placebo subjects) and were expressed with the corresponding standard deviation and coefficient of variation values (when AZP2006 and M2 concentrations were not BLQ). The LLOQ was 0.5 ng/mL in blood and 1 ng/mL in plasma. Concentrations below the LLOQ were indicated by BLQ. Individual BLQ values were taken equal to zero. When all concentrations were BLQ, the mean concentration was reported BLQ.

The pharmacokinetic analysis was performed on each kinetic profile (for doses 30, 90, 180, 360, and 500 mg in plasma and for doses 90, 180, 360, and 500 mg in blood) using scheduled sampling time. BLQ values were substituted by zeros, except for the BLQ values between quantifiable concentrations, which were treated as missing values. BLQ values obtained at the last time points were excluded for the pharmacokinetic analysis.

Statistical methods

Descriptive statistics were used for safety parameters, vital signs, ECG, and biological parameters. Quantitative statistics: sample size (N), mean, standard deviation (SD), standard error of the mean (SEM), minimum (Min), median, and maximum (Max).Qualitative statistics: absolute frequency (n) and relative frequency as percentage (%). AEs were described by system organ and preferred term according to MedDRA codification.

Pharmacokinetics

PK parameters of AZP2006 and M2 were determined using plasma and blood concentrations from individual subjects by non-compartmental analysis with Kinetica (4.3) software.

The PK analysis was performed for doses 30, 90, 180, 360, and 500 mg in plasma and for doses 90, 180, 360, and 500 mg in blood, corresponding to complete sampling time profiles.

The parameters registered to evaluate the PK were: C_max, T_max, AUC_0–12, AUC_0–24, AUC_0 - t, AUC_0 - inf, Kel, t_1/2, % AUC_extra, Vd/F, Cl/F. Results were presented as mean, SD, coefficient of variation, median, minimum, and maximum values, and geometric mean.

Study 2 – Multiple Ascending Dose (MAD)

AZP2006C02/OP091914.ALZ EudraCT N° 2014-005175-89

Study design

The MAD study consisted in two parts, A and B (Fig. 1).

Part A was designed to evaluate the safety and tolerability of up to four dose levels of AZP2006 (30, 60, 120, and an optional high dose at 180 mg) and to determine the PK parameters of AZP2006 after multiple dose administration once daily for 10 days in healthy male subjects. The choice of multiple ascending doses was based on the review of the safety/bioavailability and PK data (AUC and C_max) of the previous SAD study. Based on anticipated accumulation from the single-dose study (PK simulation showing x3 accumulation), the selected starting dose was 30 mg, followed by a dose escalation ratio 2-fold for 2 steps, and was reduced for the latest level.

The administration design was planned to secure that approximative expected exposure in terms of AUC and C_max, did not exceed 2,200 ng/ml*h and 90 ng/ml respectively (exposure obtained at level 360 mg).The highest dose (180 mg) was planned to be tested after an addition testing of a dose level of 500 mg in the SAD study and depending on the tolerability and PK of this 500 mg in single dose.

Eight healthy male subjects (6 verum and 2 placebos) were included in each of the four dose levels, resulting in 32 healthy subjects. Inclusion and exclusion criteria were the same as for the SAD study described above. The subjects were screened 21 days prior administration of the first dose and were hospitalized for 11 days (Day 1 morning to Day 11). The end of study visit for the individual subject was on Day 17.Subjects received a daily oral dose of AZP2006 or placebo (in a relation 6 : 2) from Day 1 to Day 10. The AZP2006 administered doses were: 30, 60, 120 mg and up to an optional high dose 180 mg depending on the additional single dose potentially performed at 500 mg. The optional higher dose level of 180 mg was to be evaluated depending on the following conditions:

No adverse effect was observed during the 30, 60, and 120 mg repeated dose study.

A supplementary single dose administration of AZP2006 within the SAD study (Protocol N° AZP2006C01/OP086913.ALZ; EudraCT N° 2014-000247-33) at 500 mg had been performed and no adverse effects had been observed.

The Part B, which was not performed, was to evaluate the safety and tolerability of AZP2006, after multiple dose administration once daily for 10 days in male and female healthy adults aged 50 years or more.

Data analysis

The parameters registered to evaluate the primary endpoints were the same as in the SAD study.

PK samplings:

Plasma: at pre-dose, 0.25, 0.5, 1, 1.5, 2, 2.5, 3, 4, 5, 6, 7, 8, 10, 12, 24 h on Day 10.

Whole blood: at pre-dose, 0.25, 0.5, 1, 1.5, 2, 2.5, 3, 4, 5, 6, 7, 8, 10, 12, 24 h for Day 1 and Day 10 and at pre-dose from Day 3 to Day 9.

Bioanalytical methods

Bioanalytical methods used and mean concentrations of AZP2006 and M2 calculation were the same as for the SAD study described above. The LLOQ was 0.5 ng/mL in plasma and 1 ng/mL in blood. Concentrations below the LLOQ were indicated by BLQ. Individual BLQ values were taken equal to zero. When all concentrations were BLQ, the mean concentration was reported BLQ.

The pharmacokinetic analysis was performed on each kinetic profile (for doses 30, 60, and 120 mg in plasma and blood) using scheduled sampling time. BLQ values were substituted by zeros, except for the BLQ values between quantifiable concentrations, which were treated as missing values. BLQ values obtained at the last time points were excluded for the pharmacokinetic analysis.

Statistical methods

Descriptive statistics were used for safety parameters, vital signs, ECG, and biological parameters as for the SAD study described above.

Pharmacokinetics

PK parameters of AZP2006 and M2 were determined using plasma and blood concentrations from individual subjects by non-compartmental analysis with Kinetica (4.3) software.

PlasmaC_max, T_max, AUC_0–12, AUC_0–24, AUC_t, AUC_inf, Kel, t_1/2, % AUC_extra, Vd/F, and Cl/F results were presented as mean, SD, coefficient of variation, median, minimum, maximum values, and geometric mean.

Safety

AEs were to be reported descriptively and classified as serious AEs or non-serious AEs and as related or non-related AEs by the Investigator and the Sponsor. AEs for which the Investigator or the Sponsor considered that a causal link with the study product could reasonably be envisaged were classified as suspected AEs. Should the evaluations of the Sponsor and the Investigator differ regarding causality, then both were reported in the declaration of suspected AE.

RESULTS

Demographics of the patient populations

Hundred and six (106) male healthy volunteers were screened for the SAD study and 64 subjects were included. Mean age was 31.2±8.1 years ranging from 19 to 45 years, and 79.7% (51/64) were Caucasian, 18.8% (12/64) were Black, and 1.6% (1/64) were Asian. The first subject was included on 29 July 2014 and the last subject, last visit was on 2 July 2015. The duration of the study for each subject was 4 weeks maximum. All subjects completed the study.

For the MAD study, 43 healthy volunteers were screened, and 24 subjects were included. Mean age was 32.0±8.1 years ranging from 22 to 45 years, and 70.8% were Caucasian, 20.8% were Black, 8.3% were Amerindian and Mixed-race. The first subject was included on 16 March 2015 and the last subject, last visit was on 23 June 2015. All subjects completed the study.

Safety results

SAD Study: 5 of 64 (7.8%) subjects reported the occurrence of 7 AEs. Six of these were treatment-emergent adverse events (TEAEs): 1 in the placebo group with back pain, moderate in intensity and unlikely related to treatment administration, 1 in the 3 mg group with presyncope, moderate in intensity and unlikely related to treatment administration, 1 in the 30 mg group with rash maculo-papular, mild in intensity and unlikely related to treatment administration, 1 in the 180 mg group with presyncope, mild in intensity and unrelated to treatment administration, and 2 in the 360 mg group (by the same subject) with 1 episode of nausea and 1 episode of headache, mild and moderate in intensity, respectively, and both unrelated to treatment administration (Table 1).

Table 1

SAD: Treatment EAEs by system organ class and preferred term – By dose group – safety set (N = 64)

		Placebo			3 mg			30 mg			180 mg			360 mg			Overall
		(N = 16)			(N = 6)			(N = 6)			(N = 6)			(N = 6)			(N = 64)
System organ class	Preferred term	NAE	n	%	NAE	n	%	NAE	n	%	NAE	n	%	NAE	n	%	NAE	n	%
All SOCs	All PTs	1	1	6.3	1	1	16.7	1	1	16.7	1	1	16.7	2	1	16.7	6	5	7.8
Gastrointestinal disorders	All PTs	.	.	.	.	.	.	.	.	.	.	.	.	1	1	16.7	1	1	1.6
	Nausea	.	.	.	.	.	.	.	.	.	.	.	.	1	1	16.7	1	1	1.6
Musculoskeletal and connective tissue disorders	All PTs	1	1	6.3	.	.	.	.	.	.	.	.	.	.	.	.	1	1	1.6
	Back pain	1	1	6.3	.	.	.	.	.	.	.	.	.	.	.	.	1	1	1.6
Nervous system disorders	All PTs	.	.	.	1	1	16.7	.	.	.	1	1	16.7	1	1	16.7	3	3	4.7
	Headache	.	.	.	.	.	.	.	.	.	.	.	.	1	1	16.7	1	1	1.6
	Presyncope	.	.	.	1	1	16.7	.	.	.	1	1	16.7	.	.	.	2	2	3.1
Skin and subcutaneous tissue disorders	All PTs	.	.	.	.	.	.	1	1	16.7	.	.	.	.	.	.	1	1	1.6
	Rash maculo-papular	.	.	.	.	.	.	1	1	16.7	.	.	.	.	.	.	1	1	1.6

NAE, Number of adverse events. n, Number of subjects with at least one adverse event. %, (n/N) *100 (N: number of subjects by treatment). No adverse events reported in groups of 5, 10, 90, and 500 mg.

MAD study: 8 of 24 (33.33%) subjects reported the occurrence of 12 AEs. Eleven of these occurred after treatment initiation (emergent): 4 TEAEs experienced in the placebo group, 2 TEAEs in the 60 mg group, 5 TEAEs in the 120 mg group with abdominal pain and possibly related to the study drug. No TEAE was reported in the 30 mg group. Eight TEAEs were judged unlikely related to study drug administration and 2 were judged unrelated to study drug (Table 2). All were of mild to moderate intensity.

Table 2

MAD: Treatment EAEs by system organ class and preferred term – By dose group – safety set (N = 24)

		Placebo			60 mg			120 mg			Overall
		(N = 6)			(N = 6)			(N = 6)			(N = 24)
System organ class	Preferred term	NAE	n	%	NAE	n	%	NAE	n	%	NAE	n	%
All SOCs	All PTs	4	3	50.0	2	1	16.7	5	3	50.0	11	7	29.2
Gastrointestinal disorders	All PTs	.	.	.	.	.	.	1	1	16.7	1	1	4.2
	Abdominal pain upper	.	.	.	.	.	.	1	1	16.7	1	1	4.2
Infections and infestations	All PTs	.	.	.	.	.	.	1	1	16.7	1	1	4.2
	Nasopharyngitis	.	.	.	.	.	.	1	1	16.7	1	1	4.2
Injury, poisoning, and procedural complications	All PTs	1	1	16.7	.	.	.	.	.	.	1	1	4.2
Wound		1	1	16.7	.	.	.	.	.	.	1	1	4.2
Musculoskeletal and connective tissue disorders	All PTs	.	.	.	.	.	.	1	1	16.7	1	1	4.2
Myalgia		.	.	.	.	.	.	1	1	16.7	1	1	4.2
Nervous system disorders	All PTs	2	2	33.3	2	1	16.7	2	1	16.7	6	4	16.7
	Dizziness postural	1	1	16.7	.	.	.	.	.	.	1	1	4.2
	Headache	.	.	.	2	1	16.7	1	1	16.7	3	2	8.3
	Presyncope	1	1	16.7	.	.	.	.	.	.	1	1	4.2
	Tremor	.	.	.	.	.	.	1	1	16.7	1	1	4.2
Vascular disorders	All PTs	1	1	16.7	.	.	.	.	.	.	1	1	4.2
	Orthostatic hypotension	1	1	16.7	.	.	.	.	.	.	1	1	4.2

NAE, Number of adverse events. n, Number of subjects with at least one adverse event. %, (n/N) *100 (N: number of subjects by treatment).

All the TEAEs occurring during both studies were resolved before the end of the studies. No serious AEs were reported during the studies and no clinically relevant findings were observed in the clinical examination, biological parameters, or ECG parameters. The safety and tolerability results of both studies demonstrated that there are no specific risks related to the use of the product.

Though the product was well tolerated, it was decided to stop the MAD study after the third cohort (120 mg) was completed, based on the high levels of the M2 metabolite observed in plasma. The pharmacological and potential toxicological effects of the M2 metabolite were studied in the following year (and will be published elsewhere). The steps taken included pharmacology studies using AZP2006 as a reference, optimizing the M2 synthesis method, good manufacturing practice (GMP) production, and good laboratory practice (GLP) toxicology studies in dogs. The necessary studies to investigate the effects of the M2 metabolite took up to 18 months to complete. Due to time constraints and the need to address concerns regarding the M2 metabolite, part B of the study was not conducted. Instead, considering the proven safety of the M2 metabolite, it was decided to directly test the compound in PSPsubjects.

SAD Study – Pharmacokinetic

AZP2006 and M2 concentrations were below the lower limit of quantification in plasma and blood samples from subjects receiving AZP2006 at doses lower than 10 mg. At 10 mg, concentrations above the lower limit of quantification could be measured in some of the blood and plasma samples, but PK parameters could not be calculated.

Figure 2 shows the AZP2006 and M2 plasma and blood concentrations after single oral ascending doses. Tables 3 and 4 show the mean PK parameters of AZP2006 and M2 in plasma and blood. AZP2006 and M2 concentrations increased more than proportionally with the dose. Plasma peak concentration was reached after 0.5 to 3 h following single administration and terminal half-life was long: about 30 h for AZP2006 and longer for M2 (median 41 h for a 500 mg dose). However, mainly for M2, sampling times up to 72 h after administration did not allow to calculate accurately the T_1/2.

Fig. 2

Mean plasma and blood concentrations (ng/mL) of AZP2006 (A and C) and M2 (B and D) measured in male healthy volunteers after single oral ascending doses of AZP2006. Mean profiles are presented on a log-linear scale.

Table 3

SAD: Pharmacokinetic parameters of AZP2006 in plasma and blood (mean±SD)

Dose (mg)	C_max (ng/ml)	T_max¹ (h)	T_1/2¹ (h)	AUC_0–12 (ng/ml*h)	AUC_0–24 (ng/ml*h)	AUC_t (ng/ml*h)	C_max/dose	Cl/F (L/h)	AUC_0–24/dose	AUC_t/dose
Plasma
30	1.8±0.5	1.25	4.95	6.3±2.4	6.3±2.4	6.3±2.4	0.06±0.02	2869±508.5	0.2±0.08	0.4±0.06
90	6.9±3.0	0.75	9.53	30.1±14.6	36.0±20.7	45.0±36.5	0.08±0.03	2349±1325	0.4±0.2	0.5±0.4
180	18.5±12.2	0.75	22.00	67.7±28.6	85.6±35.8	111.4±57.7	0.1±0.07	1608±923	0.5±0.2	0.6±0.3
360	55.8±34.2	0.5	39.38	174.2±91.4	220.6±118.8	315.8±189.8	0.2±0.09	1369±1137	0.6±0.3	0.9±0.5
500	97.3±44.8	1.0	41.45	379.4±88.5	487.9±129.7	696.7±196.8	0.2±0.09	621.2±192	1.0±0.3	1.4±0.4
Blood
90	24.7±9.3	1.0	34.48	157.9±78.3	239.6±142.0	369.0±234.4	0.3±0.1	250.4±131.3	2.7±1.6	4.1±2.6
180	61.8±27.1	1.5	25.88	373.8±86.7	538.1±122.4	747.6±133.3	0.3±0.2	197.3±37.53	3.0±0.7	4.2±0.7
360	173.2±89.2	1.0	28.10	1089±489.3	1635±813.3	2455±1292	0.5±0.3	159.5±83.56	4.5±2.3	6.8±3.6
500	263.8±77.4	1.0	24.79	1820±465.7	2641±652.3	3989±1106	0.5±0.2	117.77±38.61	5.3±1.3	8.0±2.2

¹Median.

Table 4

SAD: Pharmacokinetic parameters of M2 in plasma and blood (mean±SD)

Dose (mg)	C_max (ng/ml)	T_max¹ (h)	T_1/2¹ (h)	AUC_0–12 (ng/ml*h)	AUC_0–24 (ng/ml*h)	AUC_t (ng/ml*h)	C_max/dose	AUC_0–24/dose	AUC_t/dose
Plasma
30	2.1±1.6	0.5	1.33	4.6±3.8	4.6±3.8	4.6±3.8	0.07±0.05	0.2±0.1	0.2±0.1
90	10.2±3.6	0.75	3.57	23.2±5.6	25.5±8.9	30.1±19.1	0.04±0.01	0.3±0.1	0.3±0.2
180	34.3±30.8	0.5	14.25	67.1±47.0	74.6±51.5	86.2±66.9	0.2±0.2	0.4±0.3	0.5±0.4
360	140.0±98.1	0.5	54.98	267.3±136.7	318.9±156.0	446.0±203.8	0.4±0.3	0.9±0.4	1.2±0.6
500	252.7±82.1	1.0	41.53	690.0±100.1	839.1±120.3	1163±154.1	0.5±0.2	1.7±0.2	2.3±0.3
Blood
90	8.4±3.4	1.0	2.07	18.1±3.8	18.1±3.8	18.1±3.8	0.09±0.04	0.2±0.04	0.2±0.04
180	26.9±24.0	0.8	7.26	67.7±45.2	86.7±78.5	86.7±78.5	0.2±0.1	0.5±0.4	0.5±0.4
360	105.8±60.6	0.5	68.77	255.3±117.0	318.9±144.3	490.6±225.1	0.3±0.2	0.9±0.4	1.4±0.6
500	186.8±37.3	1.0	50.03	554.7±115.8	705.1±144.2	1099±224.1	0.4±0.07	1.4±0.3	2.2±0.5

¹Median.

AZP2006 clearance decreased with ascending doses. Mean Vd/F was important (more than 2,000 L). AZP2006 accumulated in blood and not in plasma, as blood AUC_t values were 5 to 9 times higher than plasma AUC_t values. This is not the case for the M2 metabolite, whose concentration in plasma represented up to 200% of the AZP2006 exposure. In blood, due to AZP2006 accumulation in blood cells, metabolite M2 exposure represented about 10% to 30% of AZP2006 exposure. This percentage increased with ascending doses. Subsequently, it was demonstrated that the M2 metabolite does not penetrate red blood cells, unlike AZP2006, which was previously observed. This finding may provide an explanation for the different PK profiles of M2 in plasma and blood.

MAD Study – Pharmacokinetic

Figure 3 shows the AZP2006 and M2 plasma and blood concentrations after multiple oral ascending doses, and the PK parameters are summarized in Tables 5–7. The PK for AZP2006 and M2 was not linear in the dose range investigated with a C_max and AUC increasing more than proportionally with the administered dose, mainly between the 60 and 120 mg dose level. Steady state was not reached after 10 days of repeated oral administration in any of the doses explored.T_1/2 for AZP2006 and M2 was long (median 21.62–20.6 h and 22.63–29.28 h, respectively) as described in the SAD study.

Fig. 3

Mean plasma and blood concentrations (ng/mL) of AZP2006 (A and C) and M2 (B and D) measured at day 10 in male healthy volunteers after repeated oral ascending doses of AZP2006. Mean profiles are presented on a log-linear scale.

Table 5

MAD: Pharmacokinetic plasma parameters (mean±SD) of AZP2006 and M2 after 10 days of repeated oral administration

Analyte	Dose (mg)	C_max (ng/ml)	T_max¹ (h)	T_1/2¹ (h)	AUC_t (ng/ml*h)	C_max/dose	AUC_t/dose
AZP2006	30	3.16±1.39	1.5	21.62	37.36±17.9	0.11±0.05	1.24±0.59
	60	7.1±2.1	0.5	22.74	48.70±30.8	0.12±0.03	0.81±0.51
	120	24.81±5.1	0.5	20.06	276.60±66.4	0.21±0.04	2.30±0.55
M2	30	5.34±2.72	1.0	22.63	40.02±17.4	0.18±0.09	0.13±0.58
	60	12.63±7.2	0.5	21.96	63.88±67.0	0.21±0.12	0.11±1.11
	120	41.40±6.6	0.5	29.28	432.60±115.0	0.34±0.05	3.60±0.96

¹Median.

Table 6

MAD: Pharmacokinetic blood parameters (mean±SD) of AZP2006 and M2 at Day 1 after single oral administration of AZP2006

Analyte	Dose (mg)	C_max (ng/ml)	T_max¹ (h)	T_1/2¹ (h)	AUC_t (ng/ml*h)	C_max/dose	AUC_t/dose
AZP2006	30	5.1±0.9	2.0	15.32	54.3±24.6	0.17±0.03	1.8±0.80
	60	14.1±1.8	2.0	10.27	156.3±21.9	0.23±0.03	2.6±0.36
	120	40.3±13.8	1.0	10.85	393.6±126.2	0.34±0.11	3.3±1.10
M2	30	3.1±1.6	1.0	2.40	5.9±4.4	0.10±0.05	0.2±0.15
	60	5.1±0.9	0.50	2.26	10.7±2.0	0.08±0.02	0.18±0.03
	120	16.5±6.6	0.75	2.72	43.2±8.8	0.14±0.05	0.36±0.07

¹Median.

Table 7

MAD: Pharmacokinetic blood parameters (mean±SD) of AZP2006 and M2 at Day 10 after a 10-day repeated oral administration of AZP2006

Analyte	Dose (mg)	C_max (ng/ml)	T_max¹ (h)	T_1/2¹ (h)	AUC_t (ng/ml*h)	C_max/dose	AUC_t/dose
AZP2006	30	10.6±3.5	2.0	27.57	185.6±71.5	0.35±0.12	6.2±2.4
	60	37.2±7.2	1.75	29.34	523.3±80.5	0.62±0.12	8.7±1.3
	120	99.6±12.7	2.0	32.04	1717±341.5	0.83±0.11	14.3±2.8
M2	30	3.9±2	1.25	33.32	38.7±19.8	0.13±0.065	1.3±0.66
	60	10.2±4.5	0.5	29.96	94.0±46.8	0.17±0.075	1.6±0.78
	120	33.8±4.8	0.5	33.86	447.2±130.4	0.28±0.04	3.7±1.1

¹Median.

AZP2006 accumulated in blood and not in plasma (blood AUC_t were 5 to 8 times higher than plasma AUC_t at Day 10) confirming the results of the SAD study. Accumulation ratios calculated between Day 1 and Day 10 for AZP2006 and M2 in blood were important (about 4 and 10, respectively).

In plasma at Day 10, the relative M2 exposure (AUC_t in plasma) represented 143 to 184% of AZP2006. In blood, due to accumulation of AZP2006 in blood cells, M2 amount represented about 10 to 20% of AZP2006 exposure (AUC_t in blood) at Day 1 and 20 to 30% at Day 10 (Fig. 4).

Fig. 4

Mean blood concentrations (ng/mL) of AZP2006 (solid lines) and M2 (dotted lines) measured in male healthy volunteers after repeated oral ascending doses of AZP2006. Mean profiles are presented on a log-linear scale.

Though the product was well tolerated at the three tested doses, it was decided to stop the study after the third cohort (120 mg) of Part A was completed, based on the high levels of the M2 metabolite observed in plasma. Therefore, the fourth cohort (180 mg dose) of Part A and the cohort of Part B of the study were not carried out and nonclinical studies to acquire more information relative to the M2 metabolite tolerance were started.

DISCUSSION

The present study provides important insights into the safety, tolerability, and PK of AZP2006 and its metabolite M2. The SAD and MAD studies included healthy male volunteers with a mean age of 31.2±8.1 years and 32.0±8.1 years, respectively. subjects Most in both studies were Caucasian. The safety results demonstrated that AZP2006 was well tolerated, with only mild to moderate AEs reported in a small percentage of subjects. No serious AEs were reported, and no clinically relevant findings were observed in the clinical examination, biological parameters, or ECG parameters.

The PK results showed that concentrations of AZP2006 and M2 increased more than proportionally with the dose. The terminal half-life was long for both AZP2006 and M2 (30 and 41 h, respectively) and AZP2006 clearance decreased with ascending doses. AZP2006 accumulated in blood and not in plasma, while M2 metabolite was present in both plasma and blood. The accumulation ratio for M2 was higher than that of AZP2006 and the relative M2 exposure in plasma was greater than that of AZP2006.

The safety and PK results of both studies provide valuable information for the further development of AZP2006. Despite the promising results, the MAD study was stopped after the third cohort (120 mg) due to the high levels of M2 metabolite observed in plasma. Nonclinical studies were initiated to investigate the tolerability of the M2 metabolite. After these studies proved the safety of the M2 metabolite, it was decided to directly test the compound in PSP subjects. These findings highlight the importance of continuous monitoring of safety and PK profiles during drug development and the need for further investigation into the tolerability and efficacy of AZP2006 and its metabolite in patients with neurological disorders. Based on the lack of clinical AEs and the results of the nonclinical complementary studies, the high concentration of M2 in plasma is not clinically relevant.

Conclusion

Overall, 66 healthy volunteers have been exposed to AZP2006 in the Phase 1 studies (SAD: 48, MAD: 18).

AZP2006 was rapidly absorbed and found in plasma and whole blood. AZP2006 and M2 distributed extensively into tissues. Both AZP2006 and its metabolite M2, accumulated in blood. The exposure to AZP2006 and M2 in plasma and blood increased more than proportionally with the dose administered.

Safety and tolerability data collected in the SAD and MAD studies showed no specific risks concerning the use of the product, particularly considering that the M2 metabolite is pharmacologically inactive and inert. AZP2006 was very well tolerated in the two studies.

These encouraging results support the further clinical development of AZP2006 as a promising agent for the treatment of PSP and AD. A Phase 2a clinical trial, which included patients with PSP, was successfully completed in December 2022 and is pending publication. Following this, a proof-of-concept study is slated to commence in 2024. Concurrently, AZP2006 is also scheduled to undergo a Phase 2 clinical trial involving patients with AD.

AUTHOR CONTRIBUTIONS

Philippe Verwaerde (Conceptualization; Formal analysis; Project administration; Supervision); Stéphane Burlet (Conceptualization; Formal analysis); Cecilia Estrella (Formal analysis; Writing – original draft; Writing – review & editing); Mathieu Barrier (Formal analysis); Andrée-Anne Marotte (Formal analysis); Gilbert Clincke (Conceptualization; Formal analysis).

Footnotes

ACKNOWLEDGMENTS

We thank Claudia Frumento and Luc Geeraert for their contributions and collaborative efforts in the writing process.

FUNDING

This study has been financed by Finovam, Nord France Amorçage, Participations Besançon and FEDER Fund.

CONFLICT OF INTEREST

The authors are actual or former employees of Alzprotect S.A.S. to the exception of Gilbert Clincke (external consultant).

DATA AVAILABILITY

The data supporting the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy, legal or ethical restrictions.

References

Dickson

, Kouri

, Murray

, Josephs

(2011) Neuropathology of frontotemporal lobar degeneration-tau (FTLD-tau). J Mol Neurosci 45, 384–389.

Tomita

(2017) Aberrant proteolytic processing and therapeutic strategies in Alzheimer disease. Adv Biol Regul 64, 33–38.

, Liu

(2020) Tau in Alzheimer’s disease: Pathological alterations and an attractive therapeutic target. Curr Med Sci 40, 1009–1021.

Williams

, Lees

(2009) Progressive supranuclear palsy: Clinicopathological concepts and diagnostic challenges. Lancet Neurol 8, 270–279.

Callizot

, Estrella

, Burlet

, Henriques

, Brantis

, Barrier

, Campanari

, Verwaerde

(2021) AZP2006, a new promising treatment for Alzheimer’s and related diseases. Sci Rep 11, 16806.

Mendsaikhan

, Tooyama

, Walker

(2019) Microglial progranulin: Involvement in Alzheimer’s disease and neurodegenerative diseases. Cells 8, 230.

Cenik

, Sephton

, Kutluk Cenik

, Herz

, Yu

(2012) Progranulin: A proteolytically processed protein at the crossroads of inflammation and neurodegeneration. J Biol Chem 287, 32298–32306.

Altmann

, Vasic

, Hardt

, Heidler

, Häussler

, Wittig

, Schmidt

MHH

, Tegeder

(2016) Progranulin promotes peripheral nerve regeneration and reinnervation: Role of notch signaling. Mol Neurodegener 11, 69.

Luo

, Lü

, Lu

, Zhang

, Li

, Guo

, Chen

, Wang

, Shao

, Xu

(2014) Effects of hypoxia on progranulin expression in HT22 mouse hippocampal cells. Mol Med Rep 9, 1675–1680.

10.

Beel

, Herdewyn

, Fazal

, De Decker

, Moisse

, Robberecht

, Van Den Bosch

, Van Damme

(2018) Progranulin reduces insoluble TDP-43 levels, slows down axonal degeneration and prolongs survival in mutant TDP-43 mice. Mol Neurodegener 13, 55.

11.

Paushter

, Du

, Feng

, Hu

(2018) The lysosomal function of progranulin, a guardian against neurodegeneration. Acta Neuropathol 136, 1–17.

12.

Lee

, Stankowski

, Chew

, Cook

, Lam

, Almeida

, Carlomagno

, Lau

, Prudencio

, Gao

, Bogyo

, Dickson

, Petrucelli

(2017) The lysosomal protein cathepsin L is a progranulin protease. Mol Neurodegener 12, 55.

13.

, Zhou

, Feng

, Hu

(2022) Regulation of lysosomal trafficking of progranulin by sortilin and prosaposin. Brain Commun 4, fcab310.

14.

Melnyk

, Vingtdeux

, Burlet

, Eddarkaoui

, Grosjean

M-E

, Larchanché

P-E

, Hochart

, Sergheraert

, Estrella

, Barrier

, Poix

, Plancq

, Lannoo

, Hamdane

, Delacourte

, Verwaerde

, Buée

, Sergeant

(2015) Chloroquine and chloroquinoline derivatives as models for the design of modulators of amyloid Peptide precursor metabolism. ACS Chem Neurosci 6, 559–569.

15.

Avila

, Santa-María

, Pérez

, Hernández

, Moreno

(2006) Tau phosphorylation, aggregation, and cell toxicity. J Biomed Biotechnol 2006, 74539.