Humanized Animal Models for Next Generation Translational ...€¦ · Case Study 2 – Diclofenac...

1 Confidential. All Rights Reserved. 2012

John Bial – Chief Executive Officer 2012

Humanized Animal Models for Next Generation Translational Research

Humanized Animal Models

2

Humanized Models Quasi-Humanized Models

Transgenic, knockout, or otherwise suppressed systems that allow for primary human cell xenografts

Transgenic, knockout, or otherwise suppressed systems that mimic human processes, but do not contain primary human cells

CXR/Taconic transADMETTM Model YecurisTM FRGTM KO Model

A Brief History of Humanized Mice

3

1990

1995

2000

2005

2010

Humanized Quasi-Humanized

•  Alb/uPA Transgenic - Heckel •  Controllable FAH-/- KO - Grompe •  Repop uPA - Rhim •  Repop FAH-/- - Grompe

•  Hu uPA/SCID / HCV - Mercer

•  Hu uPA/SCID / Metabolism - Tateno

•  Hu FRGTM / Metabolism - Azuma •  Hu uPA/SCID / Validation - Katoh

•  Hu FRGTM / HCV - Bissig

•  Hu FRGNTM / L/B Malaria - Vaughan 2012

•  Hu uPA/SCID / 3A4 DDI - Hasegawa

•  2D6(+) + HNF4α(-) - Corchero •  Intestinal 3A4 - Granvil

•  Liver 3A4 - van Herwaarden •  3a(-), 3A4(+) - van Herwaarden •  3a(-), 3A4(+) - van Waterschoot •  CAR/PXR - Sheer •  3a(-)/3a13(+)/3A4/3A7 and CAR/PXR/

3A4/3A7 DDI - Hasegawa •  2d6(-), 2D6 Variants - Sheer

Why Chimeric Systems?

4

“If you do what you’ve always done, you’ll get what you’ve always gotten.”

- Tony Robbins

Efficacy, 66%

Safety, 21%

Commercial, 7%

Other, 6%

Failure by Type, Phase III

•  Drugs are failing in new and increasingly creative ways

•  Traditional tools are useful, but gaps are forming

0

10

20

30

40

50

60

1 3 5 7 9 11 13 15 17 19 21 23 25 27 29

Comparison of Spend vs. Output

PhRMA R&D Spend ($B) Approved NME's

•  Drugs are failing late in the process

•  New tools must be more physiologically relevant, be more predictive, and on net reduce costs

Chimeric Tools in Preclinical Development

5

Early Tox Screening

Clearance Induction / Inhibition

Metabolite ID

Efflux

Toxicology -  Genotox -  Immunotox -  Reprotox

Drug-Drug Interactions C

linic

Uni-Dimensional Systems Biology

•  Primary Cell Bioreactors •  Rate Determination for Low Clearance Drugs •  Identification of Human Specific Metabolism •  Species Specific Toxicology •  ADME/Tox in Disease Model Context

•  Data Continuity

•  Increasingly Translational

6

Yecuris Corporation Founded in 2007 by Markus Grompe

-  Primary Human Hepatocytes -  FRG on C57Bl/6 -  FRG on NOD -  FAH Rat and Pig

Location: Portland, Oregon

Products and Property

Company Aim: To create a method of culturing high quality human hepatocytes and other primary cells for use in cell therapy applications utilizing animal bioreactor technologies.

In this presentation:

Part I: Introduction and Background •  Tyrosine catabolic pathway •  The Yecuris FRG/c57 Bl6 mouse •  The Yecuris FRG/NOD mouse

Part II: Liver repopulation with human hepatocytes •  Principals of Humanization •  Characterization of FRG KO Humanized Livers •  Liver Model Comparison

Part III: Application Areas •  In Vitro Cells •  Infectious Disease •  Safety/Toxicology •  Cancer •  Stem Cells

7

8

Tyrosine Catabolic Pathway

Yecuris FRG KO/c57 Bl6 Model

Yecuris FRG KO/NOD Model

Part I: Introduction and Background

Tyrosine Catabolic Pathway

9

Tyrosine

4-Hydroxyphenylpyruvic

Acid

Homogentisic Acid

Maleylacetoacetate

Fumarylacetoacetate

Fumarate

Acetoacetate

Succinate

Succinylacetoacetate Succinylacetone

TAT

PPD

HGD

MAI

Acetoacetate

Liver Disease & Renal Injury

FAR

+ +

HT1 FAH FAH

Fumarylacetoacetate

NTB

C


10

Core Technology – The Yecuris FRG Mouse


Yecuris FRG KO/c57 Bl6 mouse contains three mutations:

•  FAH knockout induces liver disease •  Rag2 (recombinant activating gene 2) knockout induces T and B cell deficiency •  Il2rg (Interleukin 2 subunit γ-chain) knockout induces NK cell deficiency

11

Core Technology – Next Generation Yecuris Models


Yecuris FRG KO/NOD (Beta Testing in Process)

•  FAH-/- / Rag2-/- / Il2rg-/-

•  Ability to integrate humanization of liver and immune systems •  Improved model health and robustness

12

Core Technology – Next Generation Yecuris Models


New Species of Humanized Models

•  FRG KO Rat: •  Large single animal cell isolation •  Industry standard species for toxicology studies •  Larger liver and body weight for DMPK studies

•  FAH KO in the Pig: •  Hepatocytes for cell therapy and liver assist •  Human liver transplant

13

Principles of Humanization

Part II: Liver Repopulation with Human Hepatocytes

Characterization of FRG KO Humanized Livers

Liver Model Comparison

14

Cell Engraftment .5-1M Cells

4 Weeks 1-5M Cells

8 Weeks 5-10M Cells

12+ Weeks 50-150M Cells

Human Hepatocyte Repopulation in FRG Mice

20-50 µg/mL HSA >> 1% Liver Repopulation

200-500 µg/mL HSA 1-5% Liver Repopulation

2000-5000+ µg/mL HSA 20-95% Liver Repopulation


14

15

log2 Scale of Human Albumin vs. Percent Repopulation


16

Histology of Various Liver Repopulation Levels


< 2% repopulation 20-30% repopulation 70-80% repopulation 70-80% repopulation

10-20% repopulation 20-30% repopulation 30-50% repopulation 70-80% repopulation

17

Integration of Human Hepatocytes into Mouse Liver


Human hepatocytes

Mouse hepatocytes

•  Human cell expansion occurs in an ordered fashion, and does not exhibit invasive growth characteristics

Tertiary structure

18

Integration of Human Hepatocytes into Mouse Liver


•  Electron Microscopy imaging of both bile duct and tight junctions between human cells in a human repopulated FRG KO mouse.

Bile Cannaliculi

Tight Junctions

19

Yecuris Models Span a Wide Range of Donors


•  Yecuris has accumulated a large repertoire of donors that range in age from 5M to 83 years of age.

0

10

20

30

40

50

60

70

80

90

Don

or A

ge (y

ears

) Donors by Age and Gender

Females Males

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Recapitulation of Genetic Disorders for Studies


Gender Age BMI Banked Cells Notes

Male 12 UK Yes Maple Syrup Urine Disease (MSUD) Female 25 UK Yes Ornithine Transcarbamylase Deficiency

(OTC) Female 5M UK Yes Carbamoyl Phosphate Synthetase I

Deficiency

•  Cryopreserved cells of rare genetic disorders have been archived and are being used to create new models for rare diseases. •  Custom models are frequently created to meet specific research requirements.

21

Comparison of Humanized Mouse Models

Attribute FRG uPA/SCID

•  Robust expansion of hepatocytes of all ages

•  Stable transgenic knockout yielding no reversion of mouse cells

•  Ability to easily serially transplant and expand hepatocytes

•  Ability to ship and use models at client sites or third party CRO’s

•  Large healthy animals offer improved survivability, increased serial sampling, and lower cost per data point


22

In Vitro

Part III: Application Areas

In Vivo

Infectious Disease

Safety & Toxicology

Stem Cells and Lines

23

Yecuris Model Allows for Serial Expansion

In Vitro Stem Cells and Lines Safety / Toxicity Infectious

Disease In Vivo

Human Donor


20 Mice

1 Vial 5M Cells

200 Vials 2B Cells

4K Mice

40000 Vials 400B Cells

800K Mice

8M Vials 80T Cells

4 Months 4 Months 4 Months

0%

20%

40%

60%

80%

100%

120%

140%

1A2 2D6 3A4

120 Donor Data

1° Hepatocyte Activity

0%

20%

40%

60%

80%

100%

120%

140%

1A2 2D6 3A4



0%

20%

40%

60%

80%

100%

120%

140%

1A2 2D6 3A4



24

Humanized Mouse Model Yields High Quality Cells


•  Cryopreserved human hepatocytes from FRGTM KO mice, plated on collagen, no Percol, no Matrigel •  100% of isolations yield plateable cryopreserved cells

.25 Day 7 Days 12 Days


Disease In Vivo

25

Cryopreserved Cells Exhibit Normal Phase II Activity

•  Cryopreserved human hepatocytes from FRGTM KO mice demonstrate normal transporter and robust Phase II activity


(UGT1A1, UGT1A6, UGT1A9, UGT2B15)

(SULT1A1, SULT1A3/4, SULT1E1)

0

5

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25

30

Paracetamol glucuronidation activity (1mM)

Paracetamol sulfation activity

(1mM)

nmol

e/h/

mill

ion

cells

Phase II Dependent Activity Donor A Donor B1 Donor B2 Pool

0

200

400

600

800

1000

1200

Estrodiol, 25µM MPP+, 250µM Taurocholate, 25µM

pmol

e/min

/milli

on ce

lls

Transporter Activity Donor A Donor B1 Pool


Disease In Vivo

26

Hu-FRG KO Mice Express Human-Like Lipid Profiles – In Vivo

•  Serum analysis conducted on control mice versus human cell repopulated FRG mice

Animal Chloresterol mmol/L VLDL (%) LDL (%) HDL (%) LDL/HDL

Ratio

Human 4.7 7.7 57.3 35.1 1.6

Hu-FRG (90%) 1.0 6.2 56.5 37.4 1.5

Hu-FRG (88%) 5.8 1.3 49.9 48.8 1.0

Hu-FRG (45%) 1.9 8.6 43.5 47.9 0.9

FRG KO 5.7 6.6 38.1 55.2 0.7

FRG KO 5.2 1.3 24.5 74.2 0.3

FRG KO 3.9 0.4 22.4 77.1 0.3


Disease In Vivo


27

Human Apo-E is detected in the serum of Hu-FRG KO Mice

Apo

E -/

- Mou

se

c57B

l6 m

ouse

Hum

an s

erum

Apo

E 3

/4

Hum

an s

erum

Apo

E 2

/3

FRG

KO

FRG

KO

45%

Hum

aniz

ed F

RG

KO

88%

Hum

aniz

ed F

RG

KO

90%

Hum

aniz

ed F

RG

KO

human ApoE

mouse ApoE


Disease In Vivo


Mouse % Repopulation Donor % T-CA %G-CA %CA

T1 94% 1 80.8 8.44 11.07

T2 90% 2 81.5 6.96 11.53

T3 88% 3 87.4 1.5 11.12

T4 78% 4 99.4 0.47 0.08

T5 45% 3 95.7 0.14 4.11

C1 0 NA 99.8 0.17 0.02

C2 0 NA 98.6 0.15 1.30

C3 0 NA 99.4 0.11 0.52

28

Hu-FRG KO Mice Express Human-Like Bile Acid Profiles – In Vivo

•  LC-MS/MS analysis of conjugated cholic acid in the gallbladder bile of human repopulated and control FRG KO mice. •  Humanized FRG KO mice show glycine conjugation of cholic acid which is human specific.

T-CA = Taurine conjugated cholic acid; G-CA = Glycine conjugated cholic acid; CA = total cholic acid


Disease In Vivo


29

Case Study 2 – Diclofenac Clearance


Disease In Vivo

Diclofenac

•  Diclofenac is more rapidly cleared in humans than in rodents. Chimeric FRG mice recapitulate this clearance with high reproducibility

0

0.5

1

1.5

2

2.5

0 100 200 300 400 500 600

Con

cent

ratio

n (µ

M)

Time (Min)

Mouse 7254 Mouse 7272 Mouse 8502 Mouse 8527 Control_1 Control_2 Human Theoretical


30

Case Study 1 – Lamotrigine Metabolism


Disease In Vivo

WT

C57

Bl/6

R

ag2-

/- / I

l2rg

-/-

Fah-

/- / R

ag2-

/-

/ Il2

rg-/-

Lamotrigine

M8 M3 M2/M7 P M6 M1/M5

Major Human Metabolite M3 (N2 Glucuronide)

Routes of Formation: UGT, GST, 2A6 (epoxide)


31

Case Study 1 – Lamotrigine Metabolism


Disease In Vivo

Fah-

/- / R

ag2-

/-

/ Il2

rg-/-

Lamotrigine

M3 P

Major Human Metabolite M3 (N2 Glucuronide)

hFR

G (5

0%)

hFR

G (9

0%)

•  At low levels of chimerism, the major human metabolite is observed in modest quantities •  At levels of chimerism in excess of 80%, high levels of human metabolite are observed


32

Case Study 3 – Propafenone Metabolism


Disease In Vivo

Propafenone

•  Propafenone metabolism is 2D6 mediated, and is highly conserved between species. It is cleared at least an order of magnitude more rapidly by mouse hepatocytes than human.

4OH Derivatives 5OH Derivatives


33



Disease In Vivo

Propafenone

•  Despite efforts by Astellas, Merck, and Phoenix Bio, the 5-hydroxy human metabolite has never been observed in chimeric models.


Drug Metab. Pharmacokinet. 25 (3): 223-235 (2010)

16th North American ISSX Meeting, 2009

0 0.2 0.4 0.6 0.8

1 1.2 1.4 1.6

0 50 100 150 200 250 300

Con

cent

ratio

n (%

Ref

)

Time (Min)

Mouse 7254 - Donor 1 - hAlb 5.2 mg/mL

4-Hydroxy 5-Hydroxy

34



Disease In Vivo

•  5-hydroxy metabolite is observed in highly chimerized animals, with no observed metabolite generation in control animals.

0 0.2 0.4 0.6 0.8

1 1.2 1.4 1.6

0 50 100 150 200 250 300

Con

cent

ratio

n (%

Ref

)

Time (Min)

Mouse 7272 - Donor 1 - hAlb 4.7 mg/L

4-Hydroxy 5-Hydroxy

0 0.2 0.4 0.6 0.8

1 1.2 1.4 1.6

0 50 100 150 200 250 300

Con

cent

ratio

n (%

Ref

)

Time (Min)

FRG Control Mouse 1

4-Hydroxy 5-Hydroxy

0 0.2 0.4 0.6 0.8

1 1.2 1.4 1.6

0 50 100 150 200 250 300

Con

cent

ratio

n (%

Ref

)

Time (Min)


4-Hydroxy 5-Hydroxy

0 0.2 0.4 0.6 0.8

1 1.2 1.4 1.6

0 50 100 150 200 250 300

Con

cent

ratio

n (%

Ref

)

Time (Min)


4-Hydroxy 5-Hydroxy

0 0.2 0.4 0.6 0.8

1 1.2 1.4 1.6

0 50 100 150 200 250 300

Con

cent

ratio

n (%

Ref

)

Time (Min)

FRG Control Mouse 2

4-Hydroxy 5-Hydroxy


35

FRG as a Model for Hepatitis B and C – In Vivo


• Chimeric FRG KO mice were susceptible to both HBV and HCV infections. •  HBV infections could be detected in mice that had only 10% human liver repopulation.

• HCV infections persisted for more than 34 weeks: - Treatment with known HCV drugs reduced viral RNA. -  Serial infection from mouse to mouse demonstrated.

HCV

HBV

FAH: Green; Hepatitis: Red


Disease In Vivo

36

FRG as a Model for Malaria – In Vivo


• P. falciparum sporozoites were injected by tail vein into a Chimeric FRG KO mouse and imaged at days three and seven. • Current efforts involve recapitulation of full cycle liver to blood stage infections in FRG-NOD KO mice.


Disease In Vivo

37

Summary


²  Large-scale single donor primary hepatocyte culture utilizing chimeric animals as bioreactors produces consistent high quality cells in large quantities

²  Use of same donor pools for cells and in vivo models provides a new level of data parity and integration of preclinical workflows

²  Chimeric systems can be used for clearance, metabolism, and toxicology, but:

- Quasi-humanized systems have shown compensatory mechanisms that may confound analysis - Not all parameters and correction factors are known and much more study is necessary before - Humanized systems are not (yet) fully human, so care must be taken to develop experimental parameters that isolate effects

²  Next generation systems are bringing disease state, metabolism, and toxicology into juxtaposition, which will allow for the development of novel platforms for biologics and vaccine development

38

Acknowledgements and Accolades

Collaborators Milton Finegold, Department of Pathology, Texas Children’s Hospital Stephen Strom, University of Pittsburgh Cedo Bagi and Jiri Aubrecht at Pfizer Ashley Vaughan and Stefan Kappe at Seattle Biomed Xenotech, BioPredic, and Xenoblis for Analytical Studies Industrial Images Certain images and content courtesy of Pfizer and Seattle Biomed

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Humanized Animal Models for Next Generation Translational ...€¦ · Case Study 2 – Diclofenac...

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