us food and drug administration: 2005-4165s1 06
TRANSCRIPT
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Division of Hematology
Basil Golding M.D.,
Division Director
Office Site Visit 2005
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Division of Hematology
PIs: 3 2 2 4 Total =
11
LBVB
Biochemistry &
Vascular Biology
LCH
Cellular
Hematology
LH
Hemostasis
LPD
Plasma
Derivatives
CRB
Clinical Review
Branch
Research/Review Units
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Division of Hematology
Scope of Regulation
Products (Biologics, Drugs, Devices)
• Cellular components of blood e.g. platelets
• Plasma-derived (Cohn-Oncley Fractionation)
• Analogous recombinant e.g Factor VIII
Clinical indications• Bleeding disorders
• Shock/hypovolemia
• Infectious diseases/immunological deficits
• Replacement therapy in congenital or acquired deficiencies
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Research Priorities:
Critical Path
• Safety
– Product toxicity (HBOCs)
– Contaminants (Microbial)
– Viral transmission (HCV)• Efficacy
– Standards, assays (HBOCs)
– Animal models (platelets)• Counter-Terrorism (anthrax, smallpox)
– In vitro assays of potency
– Animal models
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Hemoglobin-Based O2 Carriers
Public Health Impact:
Oxygen delivery in situations when bloodis not immediately available or acceptable
– Trauma (battlefield, rural areas)
–Religious reasons
– Blood shortages
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Hemoglobin-Based O2 Carriers
(LBVB)
Regulatory and Scientific Challenges:
•Characterization of HBOC structure-function
• Effects of chemical modifications
• Development of pre-clinical models to
evaluate HBOC safety
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O -R-PolyHbA0
Hallmarks of Functional Abnormality
• Non-sigmoidal oxygen
equilibrium curve
• Non-saturating
• Non-cooperative (Hill
coefficient = 1.0 vs. 2.5)
• pH insensitivity
log PO2
-0.5 0.0 0.5 1.0 1.5 2.0
0.00
0.25
0.50
0.75
1.00
BloodO
-R-PolyHbA0
HbA0
Biochemistry (2002)
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Biochemistry (2002), Biochemical J. (2004)
O -R-PolyHbA0
Identification of the Origin of Altered Function
(1) Heme
Destabilization
(2) Protein
Destabilization
(locked T state)
O2 O2
O2
O2 O2
O2 O2
Tense (T) Deoxy
Tense (T) Oxy
Locked (T) State
O2 O2
O2 O2
Relaxed (R) Oxy
Normal Conformational
Change
Tetragonal Heme Fe
HbA0
Rhombic Heme Fe
O-R-
PolyHbA0(EPR)
MALDI-MS
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O -R-PolyHbA0:
Actual Chemical Modification
Boykins,Buehler, Alayash. Proteins (2005)
•Non-specific cross-link
•Non-uniform O-raffinos•Modified cysteines
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HBOC-Induced Endothelial Cytotoxicity
MediumRedox Active
ααHb
D’Agnillo, Am J Physiol. (2004), Blood (2001)
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• Establishing methodologies that
distinguish between functional andnon-functional HBOCs
• Development of an endothelium-basedassay that correlates with HBOC
toxicity in vivo
HBOCs: Outcomes
(LBVB)
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Alpha-1-Proteinase Inhibitor (α1-PI)
(LBVB)
Public Health Impact:
• Most patients with hereditary α1-PI deficiencydevelop fatal emphysema and about 15%develop severe liver disease.
• Such deficient patients (approx. 4,000) canbenefit from augmentation therapy.
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α1-PI: SafetySafety
Regulatory and Scientific Challenges:
Aggregated α1-PI can cause adverse
events and decreases potency of α1-PI.
• How do α1-PI polymers form?
• How can polymer formation be avoidedduring manufacture?
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reactive
center
loop
freesulfhydryl
A β-sheet
Crystal Structure of α1-PI
flexiblereactive loop
Original Loop-Sheet Model, based on indirect evidence
α 1-PI polymerization isinitiated by partial
unfolding of monomer.
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New model explains spontaneous polymerization of dimer
to form aggregate.
Marszal, Danino, and Shrake: J. Biol. Chem. (2003)
Dimer Formed By Partial Unfolding of Monomer Continues to
Polymerize After Refolding to Form Aggregate.
High Aggregate
SE-HPLC
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α1-PI: Outcomes
• New understanding of α1-PI polymer formation.
• This knowledge can be used to minimizeunfolding of monomers and dimer formationduring manufacturing, e.g. adding appropriatestabilizers during heat treatment .
Future plansInvestigate the conformational change in dimers
that results in spontaneous polymerization.
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Immune Globulin Intravenous
(LPD)
Public Health Impact:
Product Contamination• Infusion of IGIV causes adverse events – 25%
– Sterile filtration of final product does notremove microbial components, e.g. LPS,
DNA.
• Microbial contaminants proinflammatorycytokines side effects
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Immune Globulin Intravenous
Product Contamination
Regulatory and Scientific Challenges:
• Need for rapid high throughput assays to
detect microbial components
• Adapt cell lines expressing Toll-like
receptors as a detector system
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Nucleus
Toll-Like Receptors (TLRs) and Their Ligands
Takeda and Akira Inf. & Imm. 2005
Pathogen
Recognition
NFκ -B NF- κ B-
luciferase
Nucleus
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TLR2 TLR4/MD2 TLR90
5
None
PGNEC-LPS
CpG ODN
12.515.0
17.5
20.0
22.5
R L U
HEK-293 Cells Transfected with TLRs and a
Reporter Gene Respond to Microbial Ligands
Huang et al, J. Immunol. 171(3):1441-6. 2003
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IGIV: Outcomes
• Cell lines expressing multiple TLRs
and a reporter gene can detectmicrobial components.
• These cell lines will be used to
develop a rapid high throughputsystem for testing IGIVs and other CBER products for microbialcontaminants.
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Cellular Components: Platelets
(LCH)
Public Health Impact:
• 10 million PLT units are transfused annually
in the US.• Transfusion of suboptimal products leads todecreased circulation time more transfusions increased risk of infection and
alloimmunization.
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Regulatory and Scientific Challenges:
• Testing for Efficacy
• In vitro tests - informative but not predictive
• In vivo survival of radio-labelled platelets in
humans is burdensome
• Adverse Effects
• Possibly due to microparticles
Cellular Components: Platelets
(LCH)
A i l M d l f E l ti D t
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Animal Model for Evaluating Damage to
Human Platelets During Collection,
Processing and Storage
Survival of human platelets in
Control and SCID mice
0 5 10 15 20 25 30 p e r c e n t h u m a n p l a t e l e t s i n c i r c u l a t i o
n ( % )
-10
0
10
20
30
40
50
60
70
8090
100
Control (FVB) MiceSCID Mice
* p < .03
p < 0.34
p < 0.12
Control SCID
In vivo performance of
1 and 7 day old human platelets
in SCID mice
Time (hours)
0 1 2 3 4 5
p e r c e n t h u m a n p l a t e l e t s i n S C I D m o u s e c i r c u l a t i o n ( % )
0
20
40
60
80
100
* p < 0.001 * p < 0.001Day 1, Filled SymbolsDay 7, Open Symbols
1 day 7 day
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Platelets: Outcomes
(LCH)
• New in vivo assay in SCID mice was
developed for measuring platelet survival.
• Flow cytometric assays were developed to
detect microparticles in platelet products:
this will enable us to determine whether
MPs in products are associated withadverse events (thrombosis,
inflammation).
Vi l D i d A i i l
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Viral Detection and Antiviral
Antibodies: Immune globulins
(LPD) Public Health Impact:
An estimated 2 million Americans suffer fromhepatitis C infection.
• ~ 70% chronic hepatitis
• Sequelaeliver fibrosis cirrhosis HCC
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Viral Detection and Antiviral
Antibodies: Immune globulins
(LPD)
Regulatory and Scientific Challenges:
• Screening plasma for HCV• Measuring HCV neutralizing Abs in HCIGIV
– no in vitro system or small animal model is
available for HCV infectivity – chimpanzee is the only model
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HCV Pseudoparticle System
Step 3: Quantification of infection by FACS
Step 1: Transfection and particle production
CMV
gag-polψ GFP
HCV E1/E2
CMV
Step 2: Infection of target cells
HCVpp entryGFP
expression
293 T cell(s)
Huh-7 cells
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Pseudoparticle
<1 : 20
≥1 : 320
≥1 : 320
Sample
Anti-HCV
neg
pos
pos
Control IGIV
HCIGIV
HCIGIV
(HCV RNA+)
Chimpanzee
Protected
Not infectious
Not protected
V.I.
S/D
S/D
None
Pseudoparticle Assay Correlates
With In Vivo Chimp Data
Yu et al, PNAS 2004
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PseudoparticleHepatitis
Cases
≥1:320≥1:320
≥1:320
≥1:320
<1: 20
<1: 20<1: 20
<1: 20
18
0
42
60
Anti-HCV Screening Removed Nt Abs to
HCV and Compromised Safety of IGIV
Implicated lots made fromanti-HCV screened plasma
in 1993
Non implicated lots made
from anti-HCV unscreenedplasma 1988-1990
IGIV (Not V.I.)
Yu et al, PNAS 2004
0
00
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HCIGIV: Outcomes
(LPD)
• HCV neutralization in pseudoparticle assaycorrelates with protection or lack of infectionin chimps.
• Pseudoparticle neutralization assay willfacilitate development of new HCIGIVproducts.
Future Plans• Identification and characterization of
neutralizing epitopes
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Inhibitory Antibodies to Factors VIII/IX
(LH)
Public Health Impact:
Neutralizing antibodies to coagulation
factors complicate the use of Factor VIII in
~20% of patients with severe hemophilia.
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Inhibitory Antibodies to Factors VIII/IX
(LH)
Research and Regulatory Challenges:
• Understanding the genetic factors thatcontrol whether patients make
antibodies to Factor VIII/IX
• Developing pre-clinical models to predictproduct efficacy
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Mouse Pre-Clinical Models
Using genetically well-characterizedinbred mouse strains we showedthat:
– MHC genes, T-cell receptors, andzinc-α-2-glycoprotein 1 genesinfluence the antibody responseto human factor VIII.
– MHC genes and to a lesser extent,
cytokine genes (IL10, Interferon-γ)control the antibody response tohuman factor IX.Lozier, et al., Blood
2005;105:1029-1035.
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Hemophilia A Dog
Pre-Clinical ModelThe Chapel Hill hemophilia A dogs have
a genetic defect identical to that in~40% of humans with severe
hemophilia A.
The bleeding phenotype is identical tohuman hemophilia A.
The dogs make inhibitors when treatedwith dog factor VIII.
Lozier et al, PNAS
2002;99:12991-12996
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Factors VIII/IX: Outcomes
(LH)
• Mouse genetic studies provide clues for probing the genetic predisposition to
induction of inhibitory antibodies inhumans.
• The Chapel Hill hemophilia A dogs are anideal model for preclinical evaluation of
product potential to induce inhibitoryantibodies.
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Immune Globulins:
Counter-Terrorism (LPD)
Public Health Impact:
• Anthrax is a major threat to public health
and security.• Smallpox poses a potential bioterrorism
threat. Widespread vaccination isexpected to cause fatalities in susceptible
individuals – Immune-compromised – Eczema
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Immune Globulins:
Counter-Terrorism (LPD)
Regulatory and Scientific Challenges:
In vitroand
in vivomodels are required toassess efficacy of:
• Anthrax Immune Globulins
• Vaccinia Immune Globulins
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Anthrax Immune Globulins
• Inhalational anthrax
– In 2001, 5/11 patients died despite
antibiotic treatment.
• Combined antibiotics and anti-anthrax
antibodies may improve survival
– Antibiotic targets the bacillus. – Antibodies target the toxins.
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Toxins of Bacillus anthracis
Lethal Factor Lethal Factor Edema Factor Edema Factor AndAnd
Or Or
ProtectiveProtective
AntigenAntigen
(PA)(PA)
Protective AntigenProtective Antigen
(PA) heptamers(PA) heptamers
Cell lysisCytokines
L T
E T Edema
Pil t t di i h
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Pilot studies in sheep
Sheep were immunized with various proteins (PA or LF in
adjuvant) or the Sterne strain agricultural vaccine.
(FEMS Immunology and Med Micro, 2003)
Various anthrax immunogens
Purified sheepantibodies
DBA/2
Sh D i d A th IG P t t DBA 2 i F
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Sheep-Derived Anthrax IG Protect DBA-2 mice From
a Lethal Sterne Spore (1x106) Challenge (IP)
10
N u m
b e r o f m i c
e s u r v i v i n g
Antibodies (25 mg/kg) given day of challenge and Cipro (10mg/kg) given day after challenge-bothgiven daily
01
2
3
4
5
6
7
8
9
1 2 3 4 5 6 7 8 9 10 11
Control Ab
Anti PA
CiproAnti PA + Cipr
Days after challenge
P i Eff f A h I Gl b li
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Protective Effects of Anthrax Immune Globulin
(LBVB)
Time (h)
R e s i s t a n c e ( %
c o n t r o l )
Medium LT + AIGLT
MEK1 Cleavage Assay
0
20
40
60
80
100
120
0 20 40 60 80
Medium
LT
LT + AIG
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Anthrax Immune Globulins: Outcomes
(LPD/LBVB)
• “Proof of Concept” that polyclonal antibodies
made in animals can protect against anthrax
toxins
• Established in-house in vitro and in vivo assays
for testing efficacy of Anthrax Immune Globulin
products
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Vaccinia Immune Globulins
Public Health Impact:
• Complications of smallpox vaccination: – Progressive vaccinia –
• VIG reduces fatality: 100% 50%
– Eczema Vaccinatum• VIG reduces fatality: 30% 3%
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VIG Products: Efficacy
(LPD)
Regulatory and Scientific Challenges:
• How can efficacy and potency for VIG
products be assessed?
• Need for an animal model of severe
vaccinia in an immunodeficient host
SCID Mouse Model of Progressive Vaccinia:
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Day 7
Day 21 Day 28
Day 14
SCID Mouse Model of Progressive Vaccinia:
Similarities to Human Disease
• Mimics human route of
exposure
• Non-healing primary
lesion
• Systemic spread of
virus
• Lethality
VIGIV Testing in Scarified SCID Mice:
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SCID Mouse Dryvax Scarification at 106, 105 with 10mg X 4 VIG Treatment
0 10 20 30 40 50 60 70 80 90 100 110 120 1300
50
100
VIG+10e6
Virus Only 10e5
VIG+10e5
Virus Only 10e6
Days
P e r c e n t S u r v i
v a l
VIGIV Testing in Scarified SCID Mice:
Post-exposure Prophylaxis Efficacy
Vaccinia day 0 - VIGIV day 2, 5, 10, 15
4 long-term
disease-freesurvivors
VIG O t
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VIG: Outcomes
(LPD)
• SCID model used to demonstrate that
VIGIV can reduce vaccinia lethality inpre- and post-exposure treatment.
• SCID model adopted by industry in
support of licensure.
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• 1st International Standard for von Willebrand
Factor Concentrate
• Mega 2/EP BRP Batch 3 InternationalWorking Standard for FVIII Concentrates
• 7th International Standard for Factor VIII
Concentrate
• 2nd International Standard/FDA Standard Lot
K for Thrombin
Reference Standards Established Through
International Collaborations (I)
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Reference Standards Established Through
International Collaborations (II)
• 2nd International Standard/EP BRP1/CBER Lot
4 for Potency of Anti-D Immunoglobulins
• 1st International Standard/CBER Standard for
Parvovirus B19 NAT Assays
• 1st International Standard/CBER Standard for
Hepatitis A Virus NAT Assays• 1st International/CBER Reference Reagents to
Limit the Anti-D Levels in IGIV Products
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DH: Future Directions (I)
• Safety• Studying the association of IGIV on pro-
inflammatory cytokine responses.• Developing NAT and infectivity assays to
determine and quantify HCV and B19 viral
variants in plasma-derived products.
• HBOCs: pre-clinical models to evaluate
oxidative stress and vasoactivity.
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DH: Future Directions (II)
• Efficacy
• Establishing WHO α1-PI reference standard
• Identifying/characterizing HCV neutralizingepitopes and enriching HCV neut. Abs
• Assessing neut. Abs to HAV, B19, HBV and
other viral pathogens byin vitro
culturesystems
• VWF: novel assay for evaluation of activity
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DH: Future Directions (III)
• Counter-terrorism• Anthrax: Human antibodies from trans-
chromosomal cows will be tested for protective antibodies by in vitro
neutralization of toxin (macrophages,
endothelial cells) and in mouse models.
• Vaccinia: develop a mouse model for eczema vaccinatum