J Endocrinol Metab

Journal of Endocrinology and Metabolism, ISSN 1923-2861 print, 1923-287X online, Open Access

Article copyright, the authors; Journal compilation copyright, J Endocrinol Metab and Elmer Press Inc

Journal website http://www.jofem.org

Original Article

Volume 4, Number 3, June 2014, pages 64-77

Safety and Metabolism of AOD9604, a Novel Nutraceutical Ingredient for Improved Metabolic Health

Figure 1. Degradation of AOD9604 in rat plasmain vitro at room temperature. After 56 min, the peptide is completely eliminated. The half-life is approximately 4 min. The appearance of AOD9604 amino-terminal truncated fragments (-xaa) is demonstrated.

Figure 2. After IV (A) or oral (B) application of AOD9604 to two different pigs, the extracted plasma from jugular vein blood was analyzed by MS to quantitate AOD9604 and its degradants. The concentration of AOD9604 and its degradants (lacking 1 - 6 amino acids) in the plasma at different sampling times is indicated.

Figure 3. ¹⁴C-AOD9604 (5 mg/kg), labeled within the cysteines, was administered IV to rats. The animals were subsequently sectioned and subjected to quantitative whole-body radiography. (A-E): serial sections, 5 min after IV administration (slight digital brightness modification of image). Principal sites of localization are the pancreas, pineal body, thyroid, kidney cortex and liver.

Figure 4. ¹⁴C-AOD9604 (5 mg/kg), labeled within the cysteines, was administered by oral gavage to rats. The animals were subsequently sectioned and subjected to quantitative whole-body radiography. (A-E): serial sections, 30 min after oral gavage administration. Principal sites of localization are pancreas, pineal body, thyroid, kidney cortex and liver and the upper gastrointestinal tract (stomach). It should be noted that later time points (for example, 24 h) are likely to represent degradants, so the data is not shown.

Figure 5. On consecutive days, the same 6 pigs received AOD9604 intravenously (A) or with oral gavage (B). Blood serum was analyzed for AOD9604 levels by immunoassay and concentrations are plotted against the sampling time. Error bars for the standard deviation are depicted in grey. *data point of one pig omitted due to an extremely high value indicating a technical error. **top error bar slightly cut off. (A) IV injection (400 µg/kg AOD9604): T_1/2 = 3 min, T_max = 2 min, C_max = 1,944µg/mL, AUC = 12,743.42 (SD ± 625.3) ng/mL/min; (B) Oral gavage (2,000 µg/kg AOD9604): C_max = 1,127 µg/mL, T_max = 60 min, AUC = 108,630 (SD ± 32,654.3) ng/mL/min.

**Table 1.** Overview of Studies Contained in This Report
Study type	Year	Species	Mode of Administration	(Run-in Phase) Study duration	Brief outcome summary
Genotoxicity - Ames test	1999	S. typhimurium E. coli	in vitro	ca. 2 weeks	No genotoxicological concerns
Genotoxicity - chromosomal aberration assay	1999	CHO cells	in vitro	ca. 3 weeks	No clastogenic activity,inconsistent evidence of interfering with cell cycling
Toxicology and Genotoxicity - bone marrow micronucleus	2002	Rat	IV	(15 days) 28 days repeated dose	No genotoxicological concerns, reduced body mass gain
¹⁴C-AOD9604: Quantitative whole-body radiography	2004	Rat	in vitro, IV or oral	1 day; 1 day	Label of various tissues and organs after IV or oral administration
Pharmacokinetics	2000	Pig	IV and oral with food	(2 weeks) 3 days of dosing	IV: rapid plasma clearance oral: slower kinetics
Toxicology	2006	Rat	Oral gavage	(11 days) 6 months repeated dose	No toxicological concerns
Toxicology	2004	Cynomolgus monkey	Oral gavage	(5 - 11 weeks) 9 months repeated dose	No toxicological concerns

Table 1. Overview of Studies Contained in This Report

Study type

Year

Species

Mode of Administration

(Run-in Phase) Study duration

Brief outcome summary

Genotoxicity - Ames test

1999

S. typhimurium E. coli

in vitro

ca. 2 weeks

No genotoxicological concerns

Genotoxicity - chromosomal aberration assay

1999

CHO cells

in vitro

ca. 3 weeks

No clastogenic activity,inconsistent evidence of interfering with cell cycling

Toxicology and Genotoxicity - bone marrow micronucleus

2002

Rat

(15 days)
28 days repeated dose

No genotoxicological concerns, reduced body mass gain

¹⁴C-AOD9604: Quantitative whole-body radiography

2004

Rat

in vitro, IV or oral

1 day; 1 day

Label of various tissues and organs after IV or oral administration

Pharmacokinetics

2000

Pig

IV and oral with food

(2 weeks) 3 days of dosing

IV: rapid plasma clearance
oral: slower kinetics

Toxicology

2006

Rat

Oral gavage

(11 days) 6 months repeated dose

No toxicological concerns

Toxicology

2004

Cynomolgus monkey

Oral gavage

(5 - 11 weeks)
9 months
repeated dose

No toxicological concerns

**Table 2.** Cytotoxicity to CHO Cells by Colony Forming Capacity, AOD9604 in Experiment 1
Without S-9 (3 h)	With S-9 (3 h)
¹Precipitation observed.
0	100	0	100
20	91.56	20	91.81
50	92.29	50	96.58
100	85.80	100	97.88
200¹	108.05	200¹	108.44

Table 2. Cytotoxicity to CHO Cells by Colony Forming Capacity, AOD9604 in Experiment 1

Without S-9 (3 h)

With S-9 (3 h)

Dose µg/mL

Relative growth %

Dose µg/mL

Relative growth %

¹Precipitation observed.

100

91.56

91.81

92.29

96.58

100

85.80

100

97.88

200¹

108.05

200¹

108.44

**Table 3.** Cytotoxicity to CHO Cells by Colony Forming Capacity, AOD9604 in Experiment 2
Without S-9 (1.5 cell cycle)	With S-9 (3 h)
¹Precipitation observed.
0	100	0	100
20	162.72	20¹	62.88
100	84.90	100¹	48.79
200	70.29	200¹	46.54
500¹	3.55	-	-
1,000¹	0.00	-	-

Table 3. Cytotoxicity to CHO Cells by Colony Forming Capacity, AOD9604 in Experiment 2

Without S-9 (1.5 cell cycle)

With S-9 (3 h)

Dose µg/mL

Relative growth %

Dose µg/mL

Relative growth %

¹Precipitation observed.

100

162.72

20¹

62.88

100

84.90

100¹

48.79

200

70.29

200¹

46.54

500¹

3.55

1,000¹

0.00

**Table 4.** Abbreviations
Adrenal cortex	adc
Adrenal medulla	adm
Aorta	ao
Bile ducts	bd
Bladder	bdr
Blood	bl
Bone marrow	bm
Brain	br
Brown fat	bf
Bulbo-urethral gland	bug
Caecum	ca
Choroid plexus	cp
Epididymis	ed
Epithelial lining (tongue)	elt
Exorbital lachrymal gland	elg
Harderian gland	Hd
Hepatic portal vein	hpv
Intra-orbital lachrymal gland	ilg
Kidney cortex	kdc
Kidney medulla	kdm
Kidney pyramid	kdp
Large intestine	li
Lens	le
Liver	lv
Lung	lu
Mandibular lymph nodes	lma
Mucus gland	mg
Muscle	mu
Myocardium	my
Nasal mucosa	nm
Non-pigmented skin	skn
Oesophagus	oe
Pancreas	pa
Parotid salivary gland	sgp
Peridontal membrane	pdm
Periosteum	pm
Peri-renal fat	far
Pineal body	pb
Pituitary	pt
Preputial gland	pg
Prostate	pr
Rectum	re
Salivary glands	sg
Seminal vesicle contents	svc
Seminal vesicle wall	svw
Small intestine	si
Spinal cord	sc
Spleen	sp
Stomach	st
Testis	ts
Thymus	th
Thyroid	ty
Tongue	to
Tooth pulp	tp
Uveal tract	uvt
Vena cava	vc
White fat	fa

Table 4. Abbreviations

Adrenal cortex

adc

Adrenal medulla

adm

Aorta

Bile ducts

Bladder

bdr

Blood

Bone marrow

Brain

Brown fat

Bulbo-urethral gland

bug

Caecum

Choroid plexus

Epididymis

Epithelial lining (tongue)

elt

Exorbital lachrymal gland

elg

Harderian gland

Hepatic portal vein

hpv

Intra-orbital lachrymal gland

ilg

Kidney cortex

kdc

Kidney medulla

kdm

Kidney pyramid

kdp

Large intestine

Lens

Liver

Lung

Mandibular lymph nodes

lma

Mucus gland

Muscle

Myocardium

Nasal mucosa

Non-pigmented skin

skn

Oesophagus

Pancreas

Parotid salivary gland

sgp

Peridontal membrane

pdm

Periosteum

Peri-renal fat

far

Pineal body

Pituitary

Preputial gland

Prostate

Rectum

Salivary glands

Seminal vesicle contents

svc

Seminal vesicle wall

svw

Small intestine

Spinal cord

Spleen

Stomach

Testis

Thymus

Thyroid

Tongue

Tooth pulp

Uveal tract

uvt

Vena cava

White fat