1

16 20 24 28 32 36 40 4430

35

40

45

50

55Growth curves in mice housed in Standard Temperature

Weeks on Diet

BW

(g)

Comprehensive characterization of an off-the-shelf AMLN mouse model and considerations for experimental design in NASH pre-clinical researchBarbara Bernardo1, Robert Barnes1, Darla Dash1, Cheryl Tyszkiewicz1, Magalie Boucher1, Megan MacBride2, Janell Richardson2, Trent Ross11 Internal Medicine Research Unit, WRD, Pfizer Inc. Groton, CT & Cambridge MA2 Taconic Biosciences, Inc. Rensselaer, NY

INTRODUCTION RESULTS

Using specific acclimation strategies and study designs which focus on disease stage, the OTS Taconic AMLN mouse serves as a reliable metabolically-driven NASH model which permits efficacy assessment for drugs targeting steatosis, inflammation and fibrotic endpoints in a single model and reduces the time and cost burden to researchers, providing greater flexibility in study planning.

To compare the disease progression of an off-the-shelf (OTS)Taconic AMLN model, relative to internally grown mice (IG), toassess the feasibility of using an OTS AMLN mouse for preclinicaldrug development.

Boland et al. World J Gastroenterology (2019) 25:33Giles et al. Nature Medicine (2017) 23:7 Hansen et al. BMC Gastroenterology (2020) 20:210

CONCLUSION

AIM

MATERIAL & METHODS

DISCLOSURESBB, RB, DD, CT, MB and TR are employees at Pfizer Inc.MM and JR are employees at Taconic Biosciences

REFERENCES

There is still an unmet need for a reproducible, efficient metabolically driven preclinical NASH model with clinical translatability. The AMLN NASH mouse model recapitulates the multifactorial disease mechanisms of human NASH, however, this model requires lengthy time on diet and substantial space requirements, limiting its utility. The availability of an off-the-shelf model could provide space, cost and time savings for NASH pre-clinical researchers.

• “internally grown cohort” = C57BL/6N Mice arrived at 4 weeks of age to Pfizer and started on Chow or modified AMLN diet at 6 weeks

• “OTS cohorts” = C57BL/6N Mice arrived from Taconic at 16, 24 and 32 weeks on modified AMLN diet or chow

• All cohorts divided into housing at standard lab temperature (STD; 72°F) or thermoneutral (TN; 80°F)

• Diet used = Research Diets # D09100310 (modified AMLN diet)• 40% kcal fat (primarily palm oil)• 20% kcal fructose• 2% w/w cholesterol

• Disease Progression evaluated by• Body Weight• Shear Wave Elastography (SWE)• Plasma biomarkers (Lipid panel, Liver Function Tests (LFTs))• Histopathology

• Picrosirius Red (PSR)• Ionized calcium-binding adaptor molecule 1 (Iba1)• Alpha Smooth Muscle Actin (αSMA)

STUDY CONSIDERATIONS

• OTS mice develop all the hallmarks of NASH as indicated bySWE and histopathology though not to the severity ofinternally grown mice

• OTS mice demonstrate reduced body weight and LFTs uponarrival, relative to internally grown mice, resulting fromshipping stress

• Longer acclimation times and housing in thermoneutralitymitigates the shipping induced stress insult observed in theOTS mice and results in a similar phenotype to internally grownmice

• Thermoneutral housing accelerates early disease progressionand results in increased hallmarks of NASH, including liverstiffness and fibrosis

• Shear Wave Elastography, which measures liver stiffness as aresult of elevated fibrosis and inflammation, correlates wellwith histopathology

SUMMARY

• Receive mice based on the NASH disease stage of interest(< lipotoxicity < inflammation < fibrosis). Study goals and endpointsof interest should be determined ahead of ordering mice to achieveadequate window of therapeutic significance

• Utilize appropriate acclimation strategies based on desired studyoutcomes. Recommend minimum acclimation of 4 weeks. Housingin TN will broaden the window for key fibrosis endpoints

• Power group size based on degree of changes in specific endpointsdesired. Based on this study, an n=10-15/group would providesufficient power to detect biologically relevant therapeutic effects.

• Utilize non-invasive methods (SWE, biomarkers) to monitor diseaseprogression and to identify target stages of disease for interventionstrategies

OTS mice shipped at 16 weeks in STD housing were significantly smaller than the other cohorts. Housing in TN and extended duration on diet results in similar growth to IG mice

Chow – Internal - STD AMLN – Internal - STD

AMLN – OTS 16 wk - STD

Chow – Internal - TN AMLN – Internal - TN

AMLN – OTS 16 wk - TN

16 20 24 28 32 36 4030

35

40

45

50

55Growth curve in mice housed in Thermoneutrality

Weeks on Diet

BW

(g)

Steatosis was similar in OTS mice relative to internally grown mice. TN housing drives increased steatosis in chow fed mice.

0

50

100

150Liver Triglycerides - Week 42

Live

r TG

(mg/

g)

0

50

100

150

Liver Triglycerides - Week 42

Live

r TG

(mg/

g)

OTS mice demonstrated lower LFTs upon arrival however TN housing and extended duration of diet mitigates this effect. TN accelerates increases in LFTs.

12 16 20 24 28 32 36 400

100

200

300

400

500

600

700

800ALT in mice housed in Standard Temp

Weeks on Diet

ALT

(U/L

SEM

)

12 16 20 24 28 32 36 400

100

200

300

400

500

600

AST - mice housed in Standard Temp

Weeks on Diet

AST

(U/L

SEM

)

12 16 20 24 28 32 36 400

50

100

150

200

250

GLDH in mice in Standard Temp

Weeks on Diet

GLD

H(U

/LSE

M)

12 16 20 24 28 32 36 400

100

200

300

400

500

600

700

800ALT - mice housed in Thermoneutral

Weeks on Diet

ALT

(U/L

SEM

)

12 16 20 24 28 32 36 400

100

200

300

400

500

600AST - in mice housed in Thermoneutral

Weeks on Diet

AST

(U/L

SEM

)

12 16 20 24 28 32 36 400

50

100

150

200

250GLDH in mice housed in Thermoneutral

Weeks on Diet

GLD

H(U

/LSE

M)

Circulating analytes in OTS mice trended similar to Internally grown mice in both standard and TN housing conditions

12 16 20 24 28 32 36 40

100

200

300

400

500Total Cholesterol in mice housed in Thermoneutral

Weeks on Diet

Cho

lest

erol

(mg/

dL±

SEM

)

12 16 20 24 28 32 36 40

100

200

300

400

500Total Cholesterol in mice housed in Standard Temp

Weeks on Diet

Chol

este

rol(

mg/

dL±

SEM

)

12 16 20 24 28 32 36 4050

75

100

125

150

175

200

225Plasma Triglycerides in mice housed in Standard Temp

Weeks on Diet

Trig

lyce

rides

(mg/

dL±

SEM

)

12 16 20 24 28 32 36 4050

75

100

125

150

175

200

225Plasma Triglycerides in mice housed in Thermoneutral

Weeks on Diet

Trig

lyce

rides

(mg/

dL±

SEM

)

12 16 20 24 28 32 36 400.0

0.5

1.0

1.5

2.0

2.5

3.0Free Fatty Acids in mice housed in Standard Temp

Weeks on Diet

NEFA

(mm

ol/L

)

12 16 20 24 28 32 36 400.0

0.5

1.0

1.5

2.0

2.5

3.0Free Fatty Acids in mice housed in Thermoneutral

Weeks on Diet

NEF

A (m

mol

/L)

OTS mice reach the same degree of liver stiffness in STD housing but not all OTS mice in TN housing. TN housing significantly drives more severe liver stiffness in all AMLN mice.

12 16 20 24 28 32 36 403

4

5

6

7

8

9SWE - mice housed in Standard Temp

Weeks on Diet

kPa

12 16 20 24 28 32 36 403

4

5

6

7

8

9SWE - mice housed in Thermoneutral

Weeks on Diet

kPa

AMLN – OTS 24 wk - STD AMLN – OTS 24 wk - TN

At 42 week on diet, OTS mice have significantly less fibrosis (lower PSR staining), inflammation (less Iba1 staining) and stellate cell activation (lower αSMA staining) compared with internally grown mice, statistically determined using fit linear models (P < 0.001 for weeks on diet as a predictor of the endpoints). OTS mice have significant increases in all these markers of NASH compared with chow fed mice. Thermoneutral housing results in greater fibrosis and stellate cell activation compared with mice housed in standard conditions.

Chow – Internal - TN

AMLN – Internal - TN AMLN – OTS 16 wk - TN

Chow – Internal - TN

AMLN – Internal - TN AMLN – OTS 16 wk - TN

Chow – Internal - TN

AMLN – Internal - TN AMLN – OTS 16 wk - TN

0

3

6

9

12

15

18

Picrosirius Red Staining

PSR

Stai

n Ar

ea %

0

6

12

18

24

30 Iba1 Staining

Iba1

IHC

Area

%

0

2

4

6

8αSMA Staining

αSM

A IH

C Ar

ea %