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Monica Zepeda AAPS 2014
National Biotechnology Conference
San Diego, CA
The Mini-pig As a Model for Subcutaneous
Administration
2
• Skin: Man vs pig
• Pig Model • In-line (tissue) pressure • Induration at the local tissue site • Hypo-perfusion • PK and PET Scan for dispersion of proteins
• Summary and Conclusions • Resources
Overview
3
The Skin
• Skin makes up 3.7% of the human body by weight
• Skin is not uniform – varies by gender, age,
ethnicity, hormonal levels, and location
• Functions: • Physical barrier • Containment (prevent loss of fluid) • Thermoregulation • Sensation (tactile) • Metabolism (biodegradation of xenobiotics) • Communication (pheromones)
J.A. Mahl – 2008 Minipig Research Forum
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Similarities Between Human and Minipig Skin
J.A. Mahl – 2008 Minipig Research Forum
Comparable Physical Parameters • Thickness of the epidermis
• Similar anatomical structures (ex Rete ridges)
• Sparse density of hair follicles
• Pigmentation
• Similarities in vascularization
• Tight attachment of the skin
Comparable Cell Kinetic Parameters
• Mitotic labeling index
• Rate of proliferation
• Turnover rate
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Differences: Minipig to Human Skin
• Hyperkeratotic stratum corneum (pig is deeper)
• Less vascular dermis
• Absence of eccrine cutaneous glands (only in primates)
• Apocrine skin glands are not involved in thermoregulation
• Found only in foot pads and snout
• Skin is more acidic
J.A. Mahl – 2008 Minipig Research Forum
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Histology of Normal Skin
H&E Stain of Tissue Samples from the Abdominal Region
Human Minipig Epidermis Stratum corneum Stratum luteum
Stratum granulosum Stratum spinosum
Stratum germinativum
Dermis Papillary and
reticular layers Collagen, reticulin, elastin fibers, and
glucosaminoglycan-rich ground substance
Subcutis
(hypodermis) Adipose tissue
Loose connective tissue (collagen)
Elastin and GAG
• 3 areas of the pig skin that translate best to human: 1) Snout 2) behind the ear and 3) abdomen (near inguinal fold)
7 7
§ Rapid hydrolysis of hyaluronan
§ Local removal of the barrier to bulk
fluid flow
§ Increase dispersion of drugs/fluids
§ Relative to traditional SC
§ Accelerates absorption
§ Increases bioavailability
§ Increases maximum volume
§ Effect is temporary
§ rHuPH20 locally metabolized in
minutes
§ Hyaluronan barrier reconstitutes in
1-2 days
Recombinant Human Hyaluronidase (rHuPH20) A Well Characterized Enzyme
Human body turns over more than 5 grams/day
(1/3rd of total body pool)
+ Hyaluronidase
O
NH
OOH
O O
OH
OH
CH3
O
OH
OOH
n
n = 2,000 – 25,000
(0.2 – 10 MDa)
N-‐Acetylglucosamine Glucuronic Acid
8 8 8
PH20 Mechanism Of Ac=on
9 9 9
PH20 Mechanism Of Ac=on
10 10 10
PH20 Mechanism Of Ac=on
11 11 11
PH20 Mechanism Of Ac=on
12 12
rHuPH20 Reduces Local Swelling and Induration What is happening inside and around the bleb?
Control rHuPH20 Control rHuPH20
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Pig Subcutaneous Pressure Model What is occurring in the local tissue during SC administration ?
• 1-4 infusion sites per pig • 18 ga “winged” infusion set
• large bore needle à reflects “tissue pressure” • small bore needleà reflects “system pressure”
• Endpoints: • In-line pressure (tissue pressure) • Local skin visco-elasticity (pliability) • Cutaneous blood flow
Pressure Transducer
Stopcock
18 ga “winged” infusion set
14 14
rHuPH20 Reduces Local Interstitial Pressure During SC Infusions
0 300 600 900 1200 1500 18000
50
100
150
200ControlrHuPH20
Time (sec)
Mea
n In
-Lin
e P
ress
ure
(mm
Hg±
SEM
)
n = 8; p < 0.0001
10 20 30 40 50 Volume Delivered (mL)
• 50 mL IgG (15%) infusion • 2mL/minute (25 minutes) • 2,000 U/mL rHuPH20 • Control (no rHuPH20)
15 15
rHuPH20 Increases Local Fluid Dispersion Surface Area and Reduces Infusion Site Volume
Control rHuPH200
20
40
60
80
Mea
n Vo
lum
e (c
m3±
SEM
) n = 8; p < 0.0001
Control rHuPH2040
50
60
70
80
90
100
Mea
n A
rea
(cm
2±
SEM
) n = 8; p < 0.05
16 16
Skin Elasticity by Cutometer Measurement rHuPH20 Maintains Skin Pliability Following SC Infusions
Control rHuPH20-1.0
-0.5
0.0
0.5
1.0
Mea
n D
iffer
ence
in S
kin
Exte
nsio
nB
etw
een
Bas
elin
e an
d P
ost-I
nfus
ion
(mm±
SEM
)
n = 8; p < 0.05
17 17
Local Cutaneous Blood Flow after SC Delivery in the Pig Laser Doppler Imaging (over time and volume)
Control rHuPH20
Pre-Infusion
Post-Infusion
10 mL 20 mL 30 mL
40 mL 50 mL
18 18
rHuPH20 Maintains Better Cutaneous Blood Flow During SC Infusions
Control rHuPH20
Control histograms shift to the left (less blood flow; hypo-perfusion) throughout the course of the infusion while rHuPH20 maintains blood flow
0 5 10 15 20 25 30 35-20
-10
0
10
20 ControlrHuPH20
~ Time (min)% C
hang
e in
Flu
x fr
om B
asel
ine±
SEM
0 5 10 15 20 25 30 350
10
20
30
40
50ControlrHuPH20
~ Time (min)
Num
ber o
f Pi
xels
In L
owes
tPe
rfus
ion
Cat
egor
y±
SEM
(0-6
2.5
Flux
Uni
ts)
19 19
rHuPH20 Reduces Local Swelling and Induration at the Infusion Site
Control rHuPH200
1
2
3
4
Mea
n Sw
ellin
g/In
dura
tion
Inde
x (S
II)
n = 8; p > 0.05
Control rHuPH20
20
Volumetric Dispersion is Linked to Increased Absorption of Proteins
0 6 12 18 24 30 36 42 48 54 60 66 720
100000
200000
300000
400000
ControlrHuPH20
Time (hr)
Mea
n Pl
asm
a Co
ncen
trat
ion
ofHu
man
IgG
(ng/
mL±
SEM
)
rHuPH20 Control
High IntensityLow Intensity
0 25 50 75 100 125 1500
250
500
750
1000
1250
1500
ControlrHuPH20
Time (min)
PE
T C
ompu
ted
Vol
ume
(Num
ber
of V
oxel
s)
• 15 % IgG soluCon + radiolabeled Zevalin®
• 2,000 U/mL rHuPH20 • Volume = 10 mL • 4-‐10mL/minute flow rate
Volumetric Dispersion and Clearance aRer SC AdministraCon
Transaxial PET Imaging: rHuPH20 Dispersion and Infusion Site Clearance 2-‐hr Post-‐Infusion
ConcentraCon vs. Time
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Preclinical Models for Subcutaneous Delivery Rodent vs Primate vs Swine A>ribute Rodent Primate Swine
Volume No Yes Yes
PK Yes
(large body of experience)
Yes YesΨ (Increasing)
Device Feasibility No Maybe Yes
Clinical De-‐risking (Administra=on/Delivery) No Some Yes
Handling/Housing Easy Limited/complex Moderate
(social, single or group housed)
Expense Low High Low
Toxicology Experience High High Low-‐moderate
RRR • Replacement • Refinement • Reduc=on
Lower impact Higher impact Moderate to low impact
ΨClaudia Suenderhauf, N Parro_ A physiologically based PK model of the Minipig: Data CompilaCon and Model ImplementaCon
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Summary and Conclusions • The mini-pig is a translational large animal model which can be used for
subcutaneous drug delivery evaluation • Similar architecture and physiological properties to that of the human • Clinically relevant volumes can be delivered for risk mitigation • PK can be performed in the pig
• Bleb formation and size of bleb translates to the human • In-line (tissue) pressure can characterize in vivo stress parameters for SC
delivered therapeutics • Skin pliability (cutometer) at the local injection site characterize induration
• Laser Doppler in the mini pig can determine blood flow rate changes during SC delivery
• PET Scan technology can assess local clearance and retention after SC delivery in the mini pig
• rHuPH20 increases dispersion, reduces tissue pressure, and maintains
local blood flow at the injections site
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Resources
• Minipig Research Forum (MRF) h_p://minipigresearchforum.org/
• David Kang and Monica Zepeda [email protected] [email protected]
• The minipig in dermatotoxicology: Methods and challenges
J.A. Mahl et al. /Experimental and Toxicologic Pathology 57 (2006) 341–345
• Immunogenicity tesCng of Adalimumab and Infliximab in Göjngen Minipigs. van Mierlo G et al. Society of Toxicology. Washington DC: Society of Toxicology (2011)
• Immunogenicity tesCng of Kineret in Göjngen minipigs Penninks A et al Society of Toxicology. Washington DC: Society of Toxicology (2011)
• Porcine model to evaluate local Cssue tolerability associated with subcutaneous delivery of protein D.W. Kang et al J Pharm and Tox Meth (2013)
24
Acknowledgements
David Kang
Dan Maneval
Beth Clark
Fred Drake
Gerald Fu
Alex Oh
Genaro Ronquillo
Gio Roxas
James Skipper
Tara A. Nekoroski
Marie A. Printz
Carl K. Hoh and David R. Vera
