Acotiamide hydrochloride hydrate (Acofide®) 盐酸阿考替胺 Z-338 in Zeria; YM-443 in Astellas Tablet, oral, EQ 100 mg acotiamide

 Acotiamide is a peripheral inhibitor, indicated for the treatment of functional dyspepsia (FD), which was first-in-class drug to treat FD in the world and approved in 2013 by Japan PMDA.  It was originally discovered by Zeria, and co-developed with Astellas. The drug is co-marketing in Japan with a single brand name.  The human recommended starting dose is 100 mg at a time, and 3 times a day before meals. Worldwide Key Approvals Global Sales ($Million) Key Substance Patent Expiration 2016-May (US5981557A) 2016-May (EP0870765B1) 2013-Mar (JP) Not available 2021-May (JP3181919B2) 2016-May (CN1063442C) Mechanism of Action Acotiamide hydrochloride hydrate is an acetylcholinesterase (AChE) inhibitor and enhanced the (ACh)-induced contraction and motility of the gastric antrum and the gastric body. Target Binding Selectivity In vitro Efficacy In vivo Efficacy

Mixed pattern: Ki1= 0.61 µM Effect dose of contraction in gastric sample: Significantly improved the gastrointestinal motility:

Ki2= 2.7 µM ACh-induced: at 1 µM In normal and gastric hypomotility dogs: at 10 mg/kg.

Inhibition: IC50= 3 µM Electrical-induced: at 0.3 µM In gastric hypomotility rats: at 100 mg/kg. Parameters Rats Dogs Healthy Humans 3 10 3 10 50 mg 100 mg 200 mg 400 mg 800 mg Dose (mg/kg) (i.v.) (p.o.) (i.v.) (p.o.) (p.o.) (p.o.) (p.o.) (p.o.) (p.o.)

Tmax (hr) - 0.08 - 0.5 2.75 2.42 2.08 2.25 2.13

Cmax (ng/mL) - 282.1 - 839.4 16.37 30.82 72.4 185.9 318.4

AUCinf (ng·hr/mL) 586.3 271.5 1179.6 1470.5 52.2 171.3 312.8 752.3 1655 T (hr) 0.8 3.7 1.3 3.0 12.9 13.31 10.92 21.69 17.14 In 1/2 Vivo CL or CL/F (L/hr/kg) 4.3 30.7 2.1 6.0 823.1 L/hr 537 L/hr 580.8 L/hr 464.1 L/hr 440.3 L/hr Vd or Vd/F (L/kg) 4.9 165.6 4.2 26.5 NA 1.694 L NA NA NA F (%) - 13.9 - 37.5 NA NA NA NA NA Major Component in Plasma (%) M-1 (70.09) Parent (74.22) Parent Major Metabolite in Plasma (%) M-1 (70.09) M-1 (12.75) M-1, M2 (inactive) Excretion by Urine/Feces (%) 7/91 7/93 5/87 -6 Permeability Papp (A→B) = 0.8 ~1.3 ×10 cm/s at concentrations of 1 – 500 µM in Caco-2 cells. In PPB (%Bound) 75.16-77.52 53.39-61.02 84.21-85.95 Vitro Metabolic stability in LM (%Remaining) 90.3 100 42.3 Drug-Drug Interaction CYP Enzymes Other Enzymes Transporters Substrate CYP2C8 UGT1A8, UGT1A9 P-

Inhibitor Not NA P-gp (low) Inducer Not NA NA Non-clinical Toxicology Single Dose Rats: 2000 mg/kg Safety • Nothing special observed in a bunch of safety pharmacology studies Dogs: 2000 mg/kg Pharmacology Toxicity (MTD) Genotoxicity • No genetic toxicity was found.

• Fertility: NOAEL: 1000 mg/kg/day in rats. Rats: 300 mg/kg/day • Embryo-fetal developemt: maternal NOAEL: 300 & 100 mg/kg/day (13 29 & 12×MRHD Repeated Dose Reproductive & 2.2×MRHD); developmental NOAEL: 1000 & 300 mg/kg/day (19 & Dogs: 30&100mg/kg/day Toxicity Toxicity 24×MRHD). (rats & rabbits) 6 & 10×MRHD (NOAEL) • Postnatal development: NOAEL: 1000 mg/kg/day for F0 and F1 in rats. (Male & Female) • Transferred through placenta and excreted through milk. • Endometrial adenocarcinoma incidence increased was found in 2-year Carcinogenicity rat tests, but not found in rasH2 transgenic accelerating tests. Acotiamide hydrochloride hydrate 1

Acotiamide hydrochloride hydrate (Acofide®) 盐酸阿考替胺 Research code: Z-338 in Zeria; YM-443 in Astellas

§1 General Information

 Acotiamide is a peripheral acetylcholinesterase (AChE) in- hibitor, is the first-in-class drug to treat FD in the world, which was approved in 2013 by PMDA of Japan.  It was originally discovered by Zeria, and co-developed with Astellas. The drug is co-marketing in Japan with a sin- gle brand name.  Acotiamide prevents the hydrolysis of acetylcholine by in- hibiting peripheral acetylcholinesterase, and produces the . . improvement of impaired gastric motility and delayed gas- Molecular formula: C21H30N4O5S HCl 3H2O tric emptying, and consequently the symptoms of FD. Molecular weight: 541.06  Acotiamide is indicated for the treatment of postprandial CAS No.: 185106-16-5 (acotiamide) fullness, upper abdominal bloating, and early satiation due 773092-05-0 (acotiamide hydrochloride trihy- to functional dyspepsia. drate)  Available as orally tablets, containing 100 mg of acoti- amide hydrochloride hydrate and the recommended start- Parameters of Lipinski’s “Rule of 5”

ing dose is 100 mg at a time, and 3 times a day before meals. a b b b MW HD HA FRB PSA CLogP

--Key Approvals around the World-- 450.55 3 9 10 141Å2 4.873±0.434 a: molecular weight of acotiamide; b: Calculated by ACD/Labs software V11.02 Country Japan (PMDA) (or Area) --Drug Product-- First Ap-

2013/3/25 proval Date Dosage route: Oral.

NDA NO. 22500AMX00868000 Strengths: 100 mg (EQ to Acotiamide) Dosage forms: Tablet, film coated Brand Name Acofide Inactive ingredients: Indication Functional dyspepsia Lactose hydrate, Crystalline cellulose, Low-substituted Authorization hydroxypropyl cellulose, Hydroxypropyl cellulose, Zeria/Astalles Holder Light anhydrous silicic acid, Magnesium stearate, *: Till May. 2015, it has not been approved by FDA (US), EMA (EU) and CFDA Hypromellose, Carnauba wax and Titanium oxide. (China). Recommended dose: --Worldwide Sales--  In general, for adults, take 1 tablet at a time, 3 times a day before meals. * Sourced from the Japan PMDA drug label information *: The sale data of Acofide® was not available.

--Active Ingredient-- The Pharmaceutical Index – Worldwide 2013 NCEs 2

--Key Patents--

Patent 1

Title

Aminothiazole derivatives, drug containing the same and intermediate in the production of the compounds

International International Patent Type Applicants WO Patent NO. Priority Date Application NO. Filling Date

Product(Substance) Zeria Pharm Co Ltd WO9636619A1 WOJP96001297 1995/5/18 1996/5/16

Drug Approval National Patent NO. Application Date Granted Date Patent Extension Patent Expiration Date

US5981557A 1996/5/16 1999/11/9 NA / 2016/5/16

EP0870765B1 1996/5/16 2003/11/19 NA / 2016/5/16

JP3181919B2 1996/5/16 2001/7/3 2013/3/25 5 years 2021/5/16

CN1063442C 1996/5/16 2001/3/21 NA / 2016/5/16

Patent 2

Title

Method for treatment of climacteric disorders in woman during or after the menopause

International International Patent Type Applicants WO Patent NO. Priority Date Application NO. Filling Date

Uses Hormos Medical Co. WO0207718A1 PCT/FI01/00414 2000/7/21 2001/5/2

Drug Approval National Patent NO. Application Date Granted Date Patent Extension Patent Expiration Date

US6245819B1 2000/7/21 2001/6/12 2013/2/26 0 day 2020/7/21

US6984665B2 2001/5/2 2001/6/12 2013/2/26 354 days 2022/4/21

EP1305014B1 2001/5/2 2006/1/10 NA / 2021/5/2

JP5575351B2 2001/5/2 2005/7/27 2013/3/25 NA 2021/5/2

JP5629746B2 2001/5/2 2014/8/20 2013/3/25 NA 2021/5/2

CN1287775C 2001/5/2 2014/11/26 NA / 2021/5/2

Acotiamide hydrochloride hydrate 3

§2 Chemistry

Route 1: Original Discovery Route[1]

Journal Citation/ Reference Title Comments Patent NO. Aminothiazole derivatives, drug containing the same and intermedi- WO9636619A1 1 Key Ref. ate in the production of the compounds US5981557A

Route 2[2, 3]:

Journal Citation/ Reference Title Comments Patent NO. Preparation of 2-bromo-4,5-dialkoxybenzoic WO2012077673A1 2 Key Ref. acid in high efficiency at low cost US20130253222A1 Method for producing aminothiazole derivative and production inter- WO2006022252A1 3 mediate US8772528B2

The Pharmaceutical Index – Worldwide 2013 NCEs 4

Route 3[4, 5]:

Journal Citation/ Reference Title Comments Patent NO. WO9858918A1 4 Process for producing 2-hydroxybenzamide derivatives Key Ref. US6197970B1

5 Synthetic method of acotiamide hydrochloride hydrate CN103709191A

Route 4[6]:

Journal Citation/ Reference Title Comments Patent NO.

6 One-pot method for preparing acotiamide hydrochloride CN104045606A Key Ref.

Acotiamide hydrochloride hydrate 5

Route 5[7]:

Journal Citation/ Reference Title Comments Patent NO.

7 Method for preparing acotiamide hydrochloride CN103387552A Key Ref.

The Pharmaceutical Index – Worldwide 2013 NCEs 6

§3 Pharmacology

 Mechanism of Action  Acotiamide hydrochloride hydrate is an acetylcholinesterase (AChE) inhib- itor and enhanced the acetylcholine (ACh)-induced contraction and motility of the gastric antrum and the gastric body.  Acotiamide inhibited AChE with mixed pattern (Ki1=0.61±0.03 µM , Ki2=2.7±0.2 µM , and IC50=3 µM) and suppressed the degradation of acetyl- (ACh) released from nerve terminals[8].  The AChE inhibitory effect of Acotiamide almost disappeared by dialysis, it was shown to be reversible[8].  Acotiamide did not show a high affinity to the muscarinic M1, M2, M3 re- ceptors, dopamine D2S receptors and serotonin 5-HT4 receptors involved in the regulation of gastrointestinal motility[8].  In Vitro Efficacy  Acotiamide improved ACh-induced contraction in gastric sample in vitro at 1 µM[8].  Acotiamide improved electrical-induced contraction in gastric sample in vitro at 0.3 µM[8].  In Vivo Efficacy [8]  Acotiamide significantly improved the gastrointestinal motility :  In normal and clonidine-induced gastric antrum hypomotility dogs: at 10 mg/kg dose.  In normal rats: at 30 mg/kg dose.  In clonidine-induced gastric antrum hypomotility rats: at 100 mg/kg. [8]  Significantly improved clonidine-induced delayed gastric emptying in rats: at 100 mg/kg, s.c. . [8]  Significantly increased gastric juice secretion (acid output) in 2 hrs post-administration in rats: at 100 mg/kg, s.c. .

--Mechanism of Action--

Molecular Target Binding Affinity, Selectivity and Efficacy

Table 1: Effects of Acotiamide on AChE[8] Inhibition Assay Acotiamide

Inhibition Pattern Mixed Non-competitive Mixed

a c Binding and Inhibition of Ki1 (μM) 0.61±0.03 NA 0.094±0.004

Human Recombinant AChE b c Ki2 (μM) 2.7±0.2 NA 0.26±0.01

c c Ki (μM) NA 1.1±0.0 NA Human Recombinant 3.0 1.2 0.21

IC50 against Different De- Rat Gastric-derived 2.3 1.6 0.092 rived AChE (μM) Guinea pig Gastric-derived 3.6 1.4 0.19 Dog Gastric-Derived 1.2 1.2 0.36 Human Recombinant 3.0 1.2 0.21 (IC50, μM) Selectivity against AChE via Human Globulins Cohn frac- BuChEd tion IV-4 derived BuChE >1000 430 2.4 (IC50, μM) Ratioe >330 360 11 Dose (μM) 100 60 0.03

Dialysis sample (%) 75.1±1.4 72.0±1.4 77.0±0.8 % Inhibition of AChE Non-dialysis sample (%) 1.0±1.0 1.4±1.4 3.5±2.1

Inhibition type Reversible Reversible Reversible AChE: acetylcholinesterase. Itopride, a prokinetic benzamide, inhibited dopamine and had a gastrokinetic effect. Neostigmine was a parasympathomimetic that acted as a reversible

acetylcholinesterase inhibitor. a: dissociation constants as competitive inhibitors. b: dissociation constants as competitive inhibitors. c: NA, not available. d: BuChE. e: ratio=IC50 of

BuChE/IC50 of AChE.

[8]. Japan, PMDA. Acotiamide hydrochloride hydrate 7

Table 2: Binding Selectivity and Inhibition of Acotiamide on Other Receptors[8]

a Ki (μM) Inhibition of Acotiamide against Receptors Receptor Origin Acotiamide % Acotiamide Itopride Receptor Origin (μM) Inhibition

human re- Adrenaline Β2 Muscarinic M1b 27 67 14 Guinea pig 10 51 combinant Receptor human re- Adrenaline Β2 Muscarinic M2c 31 33 (96%)d Guinea pig 100 92 combinant Receptor human re- Muscarinic M4 human re- Muscarinic M3e 270 (69%)d (75%)d 100 79 combinant Receptor combinant human re- Adenosine Dopamine D f >100 3.7 14 Rat 100 83 2S combinant A2A Receptor Guinea pig Dopamine D2 5-HT g >100 (57%)d 0.067 Rat 100 86 4 striatum Receptor human re- Dopamine D3 5-HT g >100 >100 0.18 Rat 100 59 4c combinant Receptor human re- GABAA Re- 5-HT g >100 >100 0.13 Rat 100 66 4d combinant ceptor Muscarinic M1 Rat 100 97 Receptor g human re- 5-HT4e >100 >100 0.14 combinant Muscarinic M2 Rat 100 93 Receptor a: The inhibition of acotiamide was tested in 41 different receptors and the receptors with significant (>50%) inhibition were showed in the table. b: labeled ligand, [3H] Pirenze- pine. c: ligand, [3H] AF-DX 384. d: inhibition at 10 μM. e: ligand, [3H] 4-DAMP. f: ligand [3H] Spiperone. g: ligand, [3H] GR113808.

--In Vitro Efficacy--

Table 3: In Vitro Contraction by Acotiamide in Isolated Guinea Pig Gastric Strips[8]

Effect on Contraction of Muscle Strips Acoti- Effective Dose (μM) amide Target Origin Stimulator (μM) Acotiamide Itopride Mosapridea

Gastric antrum ACh-induced Contractionb 0.3-3 1 3c No improvement

Muscle Strips Gastric body ACh-induced Contractionb 0.3-3 0.3 1d Not Determined

Gastric antrum/body CCh-induced Contractione 3 No improvement No improvement No improvement

Muscle Strips Gastric body Transmural electrical stimulationf 0.1-1 0.3 1d No improvement

a: tested at 10 μM mosapride. b: 1×10-8~1×10-4 M ACh were added 5 min before drugs dosing. c: tested at 3 μM acotiamide. d: tested at 0.3-3 μM itopride. e: CCh, carbachol 3×10- 9~3×10-7 M was added 10 min before drugs addition. f: electrical stimulation, frequency 1 Hz, pulse width 1 ms, voltage 10 V, time 2 mins, for 4 times, and the test compounds were added 10 min before the 4th time stimulation.

[8]. Japan, PMDA. 8 The Pharmaceutical Index – Worldwide 2013 NCEs

--In Vivo Efficacy--

Table 4: In Vivo Efficacy of Acotiamide[8]

Acotiamide-Stimulated Gastrointestinal Motility Administration Effective Dose Dose Study Target Animal Stimulator Test Route (mg/kg) (mg/kg) Fasting gastric antrum Dog AChb SGTc 3, 10 i.d./single 3

Gastric body Rat Electricald SGTe 3, 10, 30 s.c./single 10 Postprandial gastric an- Dog Foodf SGTc 30 i.d./single 30 trum Postprandial gastric antral Dog Foodf SGTc 3, 10, 30 p.o./single 10g Acotiamide Postprandial gastrointesti- Stimulated Dog Foodf SGTh 3, 10, 30 p.o./single 10i nal Gastrointestinal a Motility Postprandial gastric antral Dog Foodf SGTc 30 i.d./QD×7 No Effect

clonidine-induced Gastric antrum Dog SGTc 3, 10, 30 i.d./single 10 hypomotility

Gastric antral Rat No SGTc 10, 30, 100 s.c./single 30

clonidine-induced Gastric antrum Rat SGTc 10, 30, 100 s.c./single 100 hypomotility PR in Gastric emptying Rat No 3, 10, 100 s.c./single No Effect stomachj Gastric Emptying clonidine-induced PR in Gastric emptying Rat 3, 10, 100 s.c./single 100 delayed stomachj Indwelling Gastric Juice Se- Acid output Rat No gastrostomy 1, 10, 100 s.c./single 100k cretion tube Ach: acetylcholine. SGT: strain gauge force transducer. i.d.: intraduodenal. s.c.: subcutaneously. i.v.: intravenous. p.o.: oral. a: The gastrointestinal motility was calculated with the index of contractile movement, and the motion coefficients by integrating of the contraction wave (V·s). b: 0.05 mg/kg/min ACh. i.v. injection for 15 min. c: SGT, strain gauge force transducer (SGT), sutured onto the gastric antrum. d: stomach vagus nerve electrical stimulation branches at frequency 5 Hz, pulse width 1 ms, voltage 20 V for time 10 seconds. e: Strain gauge force transducer sutured onto the duodenum. f: 0~2 hrs and 2~4 hrs after a meal. g: 30 mg/kg acotiamide significantly improved the motility at 0~2 hrs and 10 mg/kg at 2~4 hrs. h: sutured onto the duodenum, ileum, and colon. i: Significantly improved duodenal and colonic motility at 10 mg/kg acotiamide in 0-2 hrs post-administration. j: amount of PR (phenol red) remaining in the stomach. k: significant increase in 1 or 2 hrs post-administration.

Figure A. Effect of Acotiamide in Clonidine-Induced Gastric Antrum Hypomotility the Dogs

Study: Acotiamide stimulated gastric antrum motility

Animal: Dogs with clonidine-induced gastric antrum (n=8)

Model: 15 μg/kg, clonidine s.c. 30 min before.

Administration: Intraduodenal single dose: 3, 10, 30 mg/kg acotiamide, 0.5% w/v methylcellulose solution.

Test: Strain gauge force transducer sutured onto the gastric antrum, and movement co- efficient rate, from the average kinetic coefficient of per 30 min up to 60 min.

Result: Acotiamide significantly increased the gastric antrum motility at 10 mg/kg. (###: P<0.001, ***: P<0.001)

[8]. Japan, PMDA. Acotiamide hydrochloride hydrate 9

§4 ADME & Drug-Drug Interaction

 Absorption • Oral dosing produced linear increased in AUC and Cmax exposures in humans in the dose range of 50 to 800 mg acoti- amide. • Acotiamide had a low oral bioavailability in rats (13.9%) and a moderate oral bioavailability in dogs (37.5%). • Acotiamide was absorbed rapidly with the Tmax occurring 0.08 ~ 3 hrs in non-clinical species and humans. • The half-life of acotiamide in humans (13 hrs) was longer than them in rats (3.7 hrs) and dogs (3 hrs). • The clearance of acotiamide in rats (4.3 L/hr/kg) and humans (537 L/hr) were higher comparable to liver blood flow, but low in dogs (2.1 L/hr/kg). • The apparent volume of distribution was low in humans (1.7 L), but moderate in rats (4.9 L/kg) and dogs (4.2 L/kg), so acotiamide had an extensive distribution in tissues in non-clinical species. -6 • Acotiamide had a low permeability with Papp (A→B) between 0.8~1.3 ×10 cm/s at 5~100 μM in Caco-2 model.  Distribution • Acotiamide exhibited moderate plasma protein binding in humans (84-85%), rats (75-77%) and dogs (53-61%), and the Cb:Cp were close to 1 in all species, suggesting penetration into red blood cells. • Albino and Pigmented Male Rats following a single oral administration:  Acotiamide was rapidly and well distributed from blood into most of the tissues except for the central nervous sys- tem (CNS) since the blood-brain barrier was crossed by a very small extent.  The high concentration tissues were small intestine, stomach, urinary bladder, kidneys, liver, and artery at 0.5 hr postdose.  The radioactivity concentration in the eyeballs was 12% of that in the plasma at 0.5 hr postdose, and the concentra- tion remained, albeit at a low level, suggesting the possibility that acotiamide was distributed in melanin-containing tissues.  No significant difference was observed in the changes over time in the radioactivity concentration in the white der- mal tissue and that in the pigmented dermal tissue.  Radioactivity concentrations decreased below the lower limit of quantitation in all tissues at 24 hrs postdose.  High radioactivity was detected in the gastrointestinal content. The concentration was higher than that in plasma, and was detected at 6 hrs postdose.  • The metabolism of acotiamide was low in rat and mouse liver microsomes, moderate in human and rabbit liver micro- somes, but no metabolite was detected in dog liver microsomes. • The major component was parent and the major metabolites was the glucuronide conjugate of acotiamide (M1) in liver microsomes of all species except dogs. • Unchanged acotiamide and metabolites M-2 and M-1 were detected in human plasma. • The major component was parent and the major metabolites was the glucuronide conjugate of acotiamide (M1) in plasma of dogs. The major component was M1 in plasma in rats and mice. • Acotiamide was metabolized mainly by UGT1A8 and UGT1A9, and only slightly by UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A10, UGT2B7, and UGT2B15. • CYP2C8 mainly contributed to the formation of M-4. • CYP1A1, CYP3A4, CYP2C9, CYP2C18, CYP2C19, CYP2D6, CYP3A5, or CYP4A11 made a little contribution to the metabolism of acotiamide.  Excretion • The predominant elimination route of acotiamide was via feces and the major component in feces was parent in all spe- cies. The reabsorption rate of biliary excreted radioactivity was calculated to be approximately 7% in rats.  Drug-Drug Interaction • Acotiamide did not inhibit and induce most of the CYP molecular species, indicating a pharmacokinetic drug interac- tion was low. • Acotiamide was the substrate of P-gp and had low inhibition of P-gp.

10 The Pharmaceutical Index – Worldwide 2013 NCEs

--Absorption--

Table 5: Pharmacokinetic Parameters of Unchanged Acotiamide in Different Species after Intravenous or Oral Administration[8]

Dose T C AUC T CL or CL/F V or V /F F Species/ Gender Route max max inf 1/2 d d (mg/kg) (hr) (ng/mL) (ng·hr/mL) (hr) (L/hr/kg) (L/kg) (%)

i.v. 3 - - 586.3 0.8 4.3 4.9 - Rats/Male p.o. 10 0.08 282.1 271.5 3.7 30.7 165.6 13.9

i.v. 3 - - 1179.6± 53.4 1.3± 0.2 2.1± 0.1 4.2± 0.7 - Dogs/Male p.o. 10 0.5± 0.1 839.4± 139.5 1470.5± 147.8 3.0± 0.4 6.0± 0.8 26.5± 5.5 37.5± 3.6

50 mg 2.75±2.59 16.37±13.49 52.20 ±9.744 12.90±4.96 823.1±167.9 NA NA

100 mg 2.42±0.97 30.82±13.33 171.3 ±59.43 13.31±6.91 537.0±181.5 1.694±549.5 L NA Healthy Humans/ p.o. 200 mg 2.08±1.24 72.40±34.44 312.8±101.5 10.92±4.44 580.8±184.6 NA NA Male or Female 400 mg 2.25±1.21 185.9±116.9 752.3 ±164.5 21.69±12.18 464.1±119.2 NA NA

800 mg 2.13±0.89 318.4±122.9 1655 ±560.8 17.14±13.16 440.3±136.8 NA NA Mean±SE. The unit of CL/F for humans was L/hr. Vehicle: saline (1.2% DMSO) for i.v. methyl cellulose suspension for p.o. Each parameter was calculated from the mean plasma concentration in 5 animals at each measuring time point for rats.

Figure B: Mean Concentration-Time Profile in Plasma Acotiamide after Oral Dose of Acotiamide Administration to Humans[8]

Figure C: Mean Concentration-Time Profile in Plasma Acotiamide after Introvenous and Oral Dose of Acotiamide Ad- ministration to Rats[8]

[8]. Japan, PMDA. Acotiamide hydrochloride hydrate 11

Figure D: Mean Concentration-Time Profile in Plasma Acotiamide after Introvenous and Oral Dose of Acotiamide Ad- ministration to Dogs[8]

Table 6: In Vitro Assessment of Permeability of Acotiamide using Caco-2 Cells[8]

-6 Donors Conc. (μM) Papp ( A→B ) (1×10 cm/s ) 1 0.834±0.383 5 1.23±0.05 10 0.999±0.158 Acotiamide HCl 50 1.16±0.07 100 1.04±0.03 500 1.29±0.25 [3H] Digoxin 1 0.577±0.116 [14C] Mannitol 1 2.00±0.52

--Distribution--

Table 7: In Vitro Plasma Protein Binding and Blood Partitioning of [14C] Acotiamide in Several Species[8] Plasma Protein Binding Blood Partitioning Species Conc. (μg/mL) %Bound Conc. (μg/mL) Cb:Cp Rats 0.03-1 75.16-77.52 0.14-13 0.84-0.95 Dogs 0.03-1 53.39-61.02 0.14-13 1.21-1.29 Humans 0.03-1 84.21-85.95 0.14-13 0.85-0.95

Table 8: In Vivo Distribution study of [14C] Acotiamide in Rats after a Single Oral Dose of 10 mg/kg [14C] Acotiamide Administration[8] Species/Gender Rats/Male Partially Pigmented Rats/Male

Radioactivity (μg eq./mL or g) Tissue/Plasma Radioactivity (μg eq./mL or g) Tissue/Plasma Organs Organs 0.5 hr 6 hrs 24 hrs AUC0-t Ratios 0.5 hr 24 hrs 360 hrs AUC0-t Ratios Plasma 828.1±192.4 69.7±10.8 ND 1.0 Plasma 251 4a 0a 1.0 Brain ND ND ND 0.0 Blood 148 1a 0a 0.6 Kidney 4426.8±655.0 653.0±172.9 ND 7.2 Skin 121 3a 0a 2.3 Liver 2347.7±314.8 303.6±96.2 ND 3.6 Skinb 136 2a 0a 1.4 bladder 4868.5±2309.3 406.9±165.4 ND 5.1 Kidney 884 13 0a 7.7 Lung 602.6±134.1 135.3±26.5 ND 1.4 Liver 296 7 0a 2.1 Stomach 15519.8±883.7 263.1±54.5 ND 10.2 Lung 120 2a 0a 0.8 Small Intestine 81770.3±47998.5 465.6±348.1 ND 40.5 Eyeballs 31 42 30 8.0 a: mean±SE (n=3). b: pigmented skin. a: calculated by the measured value above the back ground and below the 30 dmp.

[8]. Japan, PMDA. 12 The Pharmaceutical Index – Worldwide 2013 NCEs

Figure E: The Gastric Tissue and Plasma Distribution of Rats after Oral Dose Administration of Acotiamide

--Metabolism-- Table 9: Metabolites in Humans . Non-clinical Test Species Liver Microsomes in Vitro and Plasma, Urine, Bile and Feces after Oral Administration[8] % of Total Radioactivity Matrix Species Administration Dose Time （hr) Parent M-1 M-2 M-4

Liver Micro- Mice in vitro 10 μM 1 61.1 21.4 - 2.8 somes SD Rats in vitro 10 μM 1 90.3 9.7 - - Fischer Rats in vitro 10 μM 1 95.3 4.7 - - Dogs in vitro 10 μM 1 100 - - - Rabbits in vitro 10 μM 1 53.9 33.9 - 3 Humans in vitro 10 μM 1 42.3 48.5 - 4 Plasma Mice p.o. 10 mg/kg 0.5 13.4 55.9 3.9 - Rats p.o. 10 mg/kg 0.5 17.7 70.09 2.84 0.76 Dogs p.o. 10 mg/kg 0.5 74.22 12.75 1.54 0.56 Humans p.o. NA 0.5 NA NA NA - Urineb Mice p.o. 10 mg/kg 0-24 0.9 2.1 0.1 Rats p.o. 10 mg/kg 0-24 20.63 57.68 4.09 - Dogs p.o. 10 mg/kg 0-24 62.8 20.18 5.4 2.67 Humanse p.o. NA 0-72 NA 3.63 0.66 - Bilec Mice p.o. 10 mg/kg 0-24 - 11.4 0.5 - Rats p.o. 10 mg/kg 0-8 6.32 76.79 12.06 0.5 Dogs p.o. 10 mg/kg 0-24 13.68 65.05 8.77 3.18 Fecesd Mice p.o. 10 mg/kg 0-24 78.3 - - - Rats p.o. 10 mg/kg 0-24 82.41 0.33 0.23 11.28 Dogs p.o. 10 mg/kg 0-24 90.08 0.21 0.29 5.67 Humanse p.o. NA 0-120 66.85 4.49 3.73 0.59 a: % of total plasma radioactivity. b: % of total urinary radioactivity. c: % of total biliary radioactivity. d: % of total fecal radioactivity. e: of dose administration for human urine and feces.

[8]. Japan, PMDA. Acotiamide hydrochloride hydrate 13

Figure F: Proposed Pathways for the in Vivo Biotransformation of Acotiamide in Mice, Rats, Dogs and Humans

Table 10: Identification of CYP Metabolic Enzyme Involved in the Metabolism of Acotiamide[8]

CYP Isoform Concentration of Deisopropyl Formed (M) UGT Isoform Km (μM)

Acotiamide Conc.(μM) 10 100 Acotiamide Conc.(μM) 1

CYP1A1 263.84±3.31 1215.05±35.84 UGT1A1 -(slightly)

CYP2C8 1390.76±75.84 8830.10±369.73 UGT1A3 -(slightly)

CYP2C9 - 163.28±4.63 UGT1A4 -(slightly)

CYP2C18 - 38.36±2.94 UGT1A6 -(slightly)

CYP2C19 - 100.13±5.33 UGT1A8 1426.51(mainly)

CYP2D6 - 139.89±6.20 UGT1A9 43.13(mainly)

CYP3A4 203.24±9.39 1085.92±34.72 UGT1A10 -(slightly)

CYP3A5 - 139.67±3.21 UGT2B15 -(slightly)

CYP4A11 - 39.88±7.54

CYP1A2,2A6,2B6,2C18, 2E1 - -

[8]. Japan, PMDA. 14 The Pharmaceutical Index – Worldwide 2013 NCEs

--Excretion--

Table 11: Excretion of [14C] Acotiamide after Single Oral Dose in Various Species[8] Dose Time Bile Urine Feces Recovery Species State Route (mg/kg) (hr) (% of dose) (% of dose) (% of dose) (% of dose)

Intact p.o. 10 0-120 - 9.0±7.3 89.2±10.4 98.2 Micea BDC p.o. 10 0-24 12.0±0.8 5.2±4.9 87.6 b 104.8

Intact p.o. 1 0-120 - 7.42±1.29 91.30±1.49 98.7

Rats BDC p.o. 10 0-24 37.16±2.45 24.41±1.14 35.47±4.91 97.0

BDC i.d. 10 0-48 3.82±0.13 3.47%±0.29 90.84%±4.29 97.3

Intact p.o. 2 0-168 - 7.0±0.6 93.0±1.5 100 Dogs BDC p.o. 2 0-48 20.2±2.7 10.3±1.9 61.6±1.5 92.1

Humans Healthy Male p.o. NA 0-216 - 5.3 87.4 92.7 mean±SE, n=3. a: mean±SD. b: value observed in 1 animal.

--Drug-Drug Interaction--

Table 12: Evaluation of Acotiamide as an Inhibitor and an Inductor of Enzymes[8] Acotiamide as an Inhibitor Acotiamide as an Inductora

CYP Items Vehicle Acotiamide Phenobarbitone Ki (μM) Enzyme - 10 mg/kg 100 mg/kg 1000 mg/kg 80 mg/kg

1A1/2 158.9 Liver weight (g) 8.2±0.3 7.7±0.1 7.5±0.3 7.8±0.5 10.0±0.3

2A6 60.7 Protein (mg/mL) 11.18±0.44 11.95±0.42 12.16±0.18 11.74±0.29 13.55±0.68

2B6 No inhibition CYP450(nmol/mg protein) 0.92±0.10 1.15±0.04 0.91±0.08 0.97±0.12 1.33±0.05

2C8 120.5 CYP b5(nmol/mg protein) 0.48±0.03 0.60±0.02 0.56±0.02 0.49±0.04 0.58±0.03

NADPH-Cytochrome C 2C9 964.1 0.16±0.01 0.17±0.03 0.14±0.03 0.14±0.01 0.28±0.03 (U/mg protein) Anline water acidification 2C19 No inhibition 0.50±0.04 0.62±0.03 0.54±0.04 0.51±0.04 1.05±0.08 (nmol/min/mg protein) Aminopyrine N-Demethyl 2D6 115.7 3.59±0.19 4.43±0.26 3.83±0.22 4.20±0.17 9.65±0.63 (nmol/min/mg protein) 7-Ethoxycoumarin O-Deethylation 2E1 No inhibition 0.58±0.03 0.70±0.02 0.62±0.02 0.60±0.01 1.93±0.10 (nmol/min/mg protein) p- nitrophenol Glucuronidation 3A4 267.6 4.75±0.38 4.06±0.32 3.72±0.06 3.85±0.19 6.74±0.35 (nmol/min/mg protein)

a: administration: oral, once daily for 7 days to rats.

Table 13: In Vitro Evaluation of Acotiamide as a Substrate and an Inhibitor of P-gpTransporters[8] Acotiamide as a Substrate Acotiamide as an Inhibitora

-6 Conc. (μM) Papp (B→A)/Papp (A→B) Ratio Donors Conc. (μM) Papp (1×10 cm/s) Inhibition Rate (%)

1 14.4 Control - 9.93 0

5 11.1 0.5 7.93 20.1

10 13.2 Acotiamide 5 8.83 11.1

50 10.9 50 7.84 21.1

100 11.2 Cyclosporin A 10 3.38 66.0

500 8.1 Verapamil 50 3.53 64.4 a: [3H] digoxin as the substrate.

[8]. Japan, PMDA. Acotiamide hydrochloride hydrate 15

§5 Non-Clinical Toxicology  Single Dose Toxicity • Single dose oral administration of acotiamide in different species:  Rats MTD: 2000 mg/kg.  Dogs MTD: 2000 mg/kg.  Repeated Dose Toxicity • Repeated dose oral administration of acotiamide in different species from 4 to 39 weeks:  For rats: The NOAEL was 300 mg/kg/day (29 and 12 ×MRHD for male and female respectively), determined by 26-week repeated dose toxicity study, no toxicologically significant effect was observed.  For dogs: The NOAEL was 30 mg/kg (6×MRHD) and 100 mg/kg (10×MRHD) for male and female respectively, determined by 39-week repeated dose toxicity study, pharmacological action of acotiamide to inhibit AChE were observed.  Safety Pharmacology • Nothing special was observed in a bunch of safety pharmacology studies.  Genotoxicity • No genetic risk was found in a standard battery of gene toxicity studies. • Very weak positive was found in Chromosomal aberration study in CHL with D20=10.1 mg/mL, TR=2.25.  Reproductive and Developmental Toxicity • Fertility toxicity: The NOAEL was 1000 mg/kg/day for both male and female rats. • Embryonic fetal developmental toxicity: The NOAEL for maternal was 300 mg/kg/day (13×MRHD) and 100 mg/kg/day (2.2×MRHD) in rats and rabbits, respectively. The NOAEL for fetus was 1000 mg/kg/day (19×MRHD) and 300 mg/kg/day (24×MRHD) in rats and rabbits, respectively. • Postnatal developmental toxicity: The NOAEL was determined as 1000 mg/kg/day for F0 and F1 in rats. • Acotiamide can be transferred through placenta and excreted through milk.  Carcinogenicity • Endometrial adenocarcinoma incidence increased found in 2-year rat tests, but not found in rasH2 transgenic accelerat- ing tests.

--Single Dose Toxicity-- Table 14: Single Dose Toxicity Studies of Acotiamide by Oral Administration[8] Species Dose (mg/kg) MTD (mg/kg) Findings  Miosis, vomiting/vomiting-like behavior, loose stools, decreased SD Rats 0, 500, 1000, 2000 2000 locomotor activity. Pharmacological action of acotiamide Beagle Dogs 60, 200, 600, 2000 2000  Twitching. Pharmacological action of acotiamide Vehicle: 0.5 w/v% MC (methylcellulose) solution in rats and gelatin capsules in dogs.

--Repeated Dose Toxicity--

Table 15: Repeated Dose Toxicity Studies of Acotiamide by Oral Administration[8] Duration Dose NOAEL AUC a Safety Margina Species 0-24 hr Findings (weeks) (mg/kg/day) (mg/kg/day) (ng·hr/mL) (×MRHD) 0, 100, 300, 4 100 1555/1970 2.5/3  Miosis was observed in the 300 mg/kg/day group. 1000 SD Rats 0, 10, 30, 18198.2/ 26 300 29/12  No toxicologically significant effect was noted. 100, 300 7420.2  Exposure of nictating membrane, palpebral edema, decreased locomotor activity, abnormal gait, tremor, 0, 100, 300, 38000.6/ 4 100 61/42 and vomiting/vomiting-like behavior, miosis, hypere- 1000 26097.6 mia of bulbar conjunctiva, episclera, increased total Beagle cholesterol and phospholipids salivation. Dogs  Lacrimation and salivation, exposure of nictating 0, 30, 100, 49899.0/ 13 100 80/40 membrane and hyperemia of bulbar conjunctiva and 300 24771.5 episclera were observed. 0, 30, 100, 3612.0/ 39 30/100 6/10  Salivation, lacrimation were observed. 300 6348.1

Vehicle: 0.5 w/v% MC (methylcellulose) solution in rats and gelatin capsules in dogs. Safety margin was calculated with exposure data obtained by tripling the AUClast (207.3 ng·hr/mL) after the last dose (single dose administration on the last day) in the phase I multiple dose study (duration of multiple administrations: 7 days). a: male/female.

[8]. Japan, PMDA

16 The Pharmaceutical Index – Worldwide 2013 NCEs

--Safety Pharmacology--

Table 16: Safety Pharmacology Studies of Acotiamide[8] Study System Dosea Findings Neurobehavioral Ef- SD rats p.o.  1/8 and 4/8 miosis in 100 and 1000 mg/kg re- fects in vivo (FOB) 10, 100, 1000 mg/kg spectively. Unanesthetized Beagle p.o. Dogs  Most observation found in 100 mg/kg. 10, 100, 1000 mg/kg in vivo Isolated guinea pig papil- 0.1, 1, 10, 100 µM  100 µM significant prolong APD50. lary muscle  hERG potassium current in 100 µM was signif- HEK-293-hERG Cells 0.1, 1, 10, 100 µM icantly inhibited. Cardiovascular Function Rabbit Ventricular myo- 3, 10, 30, 100 μM  Inhibition of IKr with IC50=54 μM. cytes Anesthesia rabbits Continuous i.v.  Did not induce VPC, TdP or VT. (Torsade de Pointes) 30 mg/kg Anesthesia guinea pig (ep- i.v.  10 mg/kg significantly prolonged QT, QTcB icardial Mactan-phase ac- 0.1, 0.3, 1, 3, 10 mg/kg and QTcF interval. tion potential duration) Unanesthetized SD rats p.o. 10, 100, 1000 mg/kg  Negative Respiratory Func- in vivo tion Isolated guinea pig trachea 0.1, 1, 10, 100 µM  Negative Kidney Function Unanesthetized SD rats 10, 100, 1000 mg/kg  Na+, K+ excretion at ≥100 mg/kg. Gastrocnemius mus- Anesthetized rats. 10, 100, 1000 mg/kg  Negative cle contraction Effects on Gastric s.c.  Negative for interaction with gastric acid Acid Secretion In- SD Rats 1, 10, 100 mg/kg secretion inhibitors. hibitors 5 mg/body/day (p.o. × 15 or s.c.×3),  Negative for active systemic anaphylaxis Guinea pigs, Antigenicity Study 2 mg/kg/day (p.o.×15 or i.p.×3) in 3 (ASA) reaction, passive cutaneous anaphy- BALB/c mice weeks laxis (PCA) sensitization, or PCA reaction.  Negative and acotiamide was unlikely to have a direct growth-promoting effect on the Study on Uterine 17 to 18 day-old female 200, 600, 1000 mg/kg/day QD×3 uterus or to induce endocrine abnormality Enlargement rats via the hypothalamic-pituitary-gonadal axis (HPG).

Vehicle: 0.5 w/v% MC (methylcellulose) solution in rats and gelatin capsules in dogs. Safety margin was calculated with exposure data obtained by tripling the AUClast (207.3 ng·hr/mL) after the last dose (single dose administration on the last day) in the phase I multiple dose study (duration of multiple administrations: 7 days). a: single dose unless otherwise specified.

--Genotoxicity--

Table 17: Genotoxicity Studies of Acotiamide[8] Assay Species/ System Metabolism Administration Dose Findings Bacterial Reverse Mutation Assay TA98, TA100, TA1535, ±S9 in vitro 313-5000 μg/plate Negative (Ames) TA1537, WP2uvrA Chinese hamster-derived Positive (very weak), lung fibroblast cell line ±S9 in vitro 675-5400μg/mL D =10.1 mg/mL, TR=2.25 Chromosomal Aberration Study in (CHL) 20 Cultured Mammalian Cells Human peripheral ±S9 in vitro 2700-3300 μg/mL Negative lymphocytes

Single p.o. Bone Marrow Micronucleus Test SD Rats + in vivo 0, 500, 1000, Negative 2000 mg/kg/day Single p.o. Measurement of Unscheduled SD Rats + in vivo 0, 1000, 2000 Negative DNA Synthesis in Rat Liver mg/kg Vehicle: 0.5 w/v% MC (methylcellulose ) solution in vivo tests, while DMSO for in vitro tests

[8]. Japan, PMDA

Acotiamide hydrochloride hydrate 17

--Reproductive and Developmental Toxicity--

Table 18: Embryonic and Developmental Toxicology Studies of Acotiamide by Oral Administration[8] Dose AUC Safety Margin Studies Species Gender NOAEL (mg/kg/day) 0-24 hr (mg/kg/day) (ng·hr/mL) (×MRHD)

Male (general) 100 1555~1972 >2.5

Female (general) 100 2242 >3.6 0, 100, 300, Fertility SD Rats Male (fertility) 1000 5575~11460 >8.9 1000 Female (fertility) 1000 15780 >25

Embryo (development) 1000 NA NA

0, 100, 300, Maternal 300 8389~10040 >13 SD Rats 1000 Embryo-fetal Devel- Development 1000 12290~15780 >19 opment 0, 100, 300, Maternal 100 1404~1419 >2.2 NZW Rabbits 1000 Development 300 14960~23240 >24

F0 1000 Pre- and Postnatal 100, 300, SD Rats F1 1000 12290~15780 >17 Development 1000 F2 1000

Vehicle: 0.5 w/v% MC (methylcellulose) solution. Safety margin was calculated with exposure data obtained by tripling the AUClast (207.3 ng·hr/mL) after the last dose (single dose administration on the last day) in the phase I multiple dose study (duration of multiple administrations: 7 days).

Table 19: Placenta Transfer of [14C]Acotiamide by Single Dose Oral Administration 10mg/kg to GD19 Pregnant Rats[8] Radio Activity Converted Concentration (ng eq./mL or g )

Tissue/Organ Mater Fetus

0.5 hr 6 hrs 24 hrs 0.5 hrs 6 hrs 24 hrs

Plasma 420.2±20.9 198.8±34.2 15.9±1.7 NA NA NA Blood 279.6±33.2 127.3±25.1 ND 19.8±4.6 11.2±5.8 12.1±8.3 Kidney 2238.0±306.2 1180.0±199.6 9.9±1.0 17.1±2.7 14.6±4.8 12.4±5.1 Liver 1832.1±131.5 1005.4±323.9 3.1±1.6 13.6±3.0 11.4±5.0 5.8±3.8 Lung 408.3±58.5 277.5±9.6 5.4±0.3 14.7±1.4 7.6±3.9 8.4±5.4 Fetus NA NA NA 15.3±1.0 12.9±4.6 8.1±4.1 NA: not applicable. ND: not detected. Data were presented with mean±SE. vehicle: 0.5% methylcellulose aqueous solution / suspension

Table 20: Milk Excretion of [14C]Acotiamide by Single Dose Oral Administration 10 mg/kg to Lactating Rats 13 Days after Delivery[8] Radio Activity Converted Pharmacokinetics Property Time (hr) Concentration (ng eq./mL) Parameters Plasma Milk Milk/Plasma Plasma Milk Milk/Plasma

0.5 113.2±258.5 530.7±205.6 0.44±0.10 Cmax (ng eq./mL) 113.2±258.5 856.4±214.9 0.77

2 196.8±48.1 856.4±214.9 4.33±0.09 Tmax (hr) 0.5±0.0 2.0±0.0 NA

4 101.2±14.5 589.7±118.5 6.05±1.50 T1/2 (hr) 5.3±1.3 5.0±0.4 NA

8 74.2±9.8 397.0±108.6 5.28±1.14 AUC0-8 hr (ng eq.·hr/mL) 1909.8±359.2 4592.6±1147.5 2.3 NA: not applicable. Vehicle: 0.5% methylcellulose aqueous solution / suspension

[8]. Japan, PMDA

18 The Pharmaceutical Index – Worldwide 2013 NCEs

--Carcinogenicity--

Table 21: Carcinogenicity Toxicity Studies of Acotiamide by Oral Administration[8] Duration Dose AUC at highest Fold of Species 0-24 hr Findings (weeks) (mg/kg/day) dose (ng·hr/mL) MRHD 0, 1000,  A dose-dependent increase in relative ovary weight 4 14698.8~20445.6 >23 1500, 2000 in female mice. B6C3F 0, 1000,  An increase in the relative weight of the heart in 13 10671.0~19516.7 >17 1 Mice 1500, 2000 female mice. 0, 200, 600,  No acotiamide-related neoplastic lesion was ob- 104 10671.0~19516.7a >17 2000 served.  Miosis, loose stools and a decrease in the weight of the spleen in both sexes. increased total cho- 0, 1000, 4 23051.0~23505.0 >37 lesterol level in males while decreases in eryth- 1500, 2000 rocytic parameters and an increase in the absolute weight of the ovary in females  Miosis and salivation in both sexes. a decrease Fischer 0, 1000, 13 16096.1~31338.5 >25 weight of liver in males while an increase weight 344 1500, 2000 in heart in females Rats  The increase incidence in endometrial adenocarci- noma at 600 mg/kg/day group was statistically sig- 0, 200, 600, nificant (16%). As proliferative lesions in the 104 16096.1~31338.5a >25 2000 uterus, cystic endometrial hyperplasia and endome- trial epithelial/glandular hyperplasia were ob- served 15 days 0, 600, 2000 NA NA  No observation.  19 of 50 animals (38%) and 24 of 50 animal (48%) died or moribund-sacrificed in control and treated rasH2 group respectively. Mice 26 0, 2000 NA NA  The incidence of endometrial adenocarcinomas was the same (10%) in the two groups, and none of the findings showed statistically significant differ- ences between the groups.

Vehicle: 0.5 w/v% MC (methylcellulose) solution in rats and gelatin capsules in dogs. Safety margin was calculated with exposure data obtained by tripling the AUClast (207.3 ng·hr/mL) after the last dose (single dose administration on the last day) in the phase I multiple dose study (duration of multiple administrations: 7 days). Transgenic mice trans- duced with proto-oncogene of human origin (c-Ha-ras) (CByB6F1-Tg (HRAS) 2Jic). a: based on 13-week TK in carcinogenicity study.

Reference [1]. Nagasawa, M.; Murata, M.; Nishioka, H.; Kurimoto, T.; Ueki, S.; Kitagawa, O. WO9636619A1, 1996.

[2]. Nakao, R. WO2012077673A1, 2012.

[3]. Nagasawa, M.; Asami, K.; Nakao, R.; Tanaka, N.; Aida, Y. WO2006022252A1, 2006.

[4]. Nagasawa, M.; Nishioka, H.; Suzuki, T.; Nagano, E.; Ishii, K.; Nakao, R. WO9858918A1, 1998.

[5]. Huang, J.; Lu, X.; Song, B.; Yang, Y. CN103709191A, 2014.

[6]. Du, H.; Ding, J.; He, Y.; Wang, W.; Liu, Y. CN104045606A, 2014.

[7]. Xie, Z.; Wang, Z.; Yi, B.; Liu, Q.; Zhong, J.; Shi, H. CN103387552A, 2013. [8]. Japan, PMDA.