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Drug Development
Lynnda Reid, Ph.D.
Pharmacology/Toxicology Reviewer
Center for Drug Evaluation and Research (CDER)
Rafael Ponce, Ph.D., DABT
Senior Scientist
ZymoGenetics, Inc.
Outline
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Regulatory Overview
 Drug/biologic development process
 Resources
 Questions (and answers?)
Parties involved in Drug Development
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FDA
Sponsor
Contract Labs
Clinical Sites
Manufacturing Sites
Consultants
Other…
Sponsors
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Pharmaceutical/Biotechnology Firms
Practicing Physicians and Dentists
Academic Institutions
NIH
Other
Proteins
Small Molecules
Drug substance
Heterogenous mixture
Broad specs during development
Specs may change
Drug product
Usually IV or SC
Single entity; high chemical
purity
Exception: racemic mixtures
Specs well-defined early
Usually oral
Impurities
Difficult to standardize
Standards well established
Bridging
requirements
Significant for drug substance
Bioequivalence procedures
Biological activity
May mimic naturally occurring
molecules
Primary MOT
Predictive based on MOA
Variable significance
Less predictive
Chronic Toxicity
Lack of models; species specificity
and antigenicity
Models sometimes relevant
Impurities
Toxicity not a major issue, may
impact immunogenicity
May be significant
Purity standards well
established
Nonspecificity
Usually significant
Drug-drug interaction
What Types of Nonclinical
Studies Should Sponsors Conduct?
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ICH (International Conference on Harmonization) Guidelines
 Drug class specific guidance
Generaltoxicity?
Toxicology?
 FDA Consultations
General
Genotoxicity?
Genotoxicity?
Carcinogenicity?
Carcinogenicity?
The ICH S6 Guidance Coverage
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“Preclinical Testing of Biotechnology-Derived Pharmaceuticals”
– Two species
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see Olson (2000) Reg. Toxicol. Pharm., 32:56-67
– Use relevant over non-relevant species
 Mechanism of safety/efficacy distinct form small molecules
 Demonstrate appropriateness
– Animal models (disease/TG-XO) may provide meaningful insight
– Immunogenicity testing and its implications
– Genotoxicity testing
 Generally not appropriate
– Chronic toxicity testing
 see Clarke J et al (in press) Reg. Toxicol. Pharm.
– Carcinogenicity testing
 Generally unnecessary unless growth factor or immunosuppressive
Darren Warren, SNBL
Designing a Tox Study
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Species selection
Dose Selection
Route of administration
Duration of study
Group sizes
Recovery/non-treatment groups needed?
Endpoints/parameters to evaluate
Katie Sprugel, Amgen
Designing a Tox Program
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ICH M3
 ICH S6
 Precedence
– http://www.fda.gov/cder/biologics/biologics_table.htm
Species Selection
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Goal: Identify most appropriate rodent and nonrodent species for safety evaluations
 Appropriate means what?
– Not the cheapest, the smallest, the easiest…
– Best model scientifically for the human biology
– Suitable for safety assessment studies
Katie Sprugel, Amgen
Species Selection-Data
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Sequence homology
Tissue expression
Binding data
In vitro functional assay
– Ideally reconstituting entire signaling pathway
with species-appropriate reagents
In vivo response
Katie Sprugel, Amgen
Types of Toxicology Studies Recommended
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General Toxicology
– acute and repeat dose toxicology studies
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Special Toxicology Studies
– local irritation studies, e.g., site specific, ocular
– hypersensitivity studies for inhalation and dermal drug products
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Reproductive and Developmental Toxicology Studies
– male and female fertility
– embryonic and fetal development
– post-natal reproductive and developmental effects
Purpose of Preclinical Studies
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Eliminate excessively toxic compounds from development
Identify potential target organs
Provide data to guide selection of safe initial starting dose
in humans
Evaluate potential safety and pharmacodynamic
biomarkers
Impact of Nonclinical Studies on Drug
Development
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Setting Initial Doses in Humans
Identification of Possible Adverse Effects
Identification of Reversible vs Irreversible Effects
Identification of Useful Biomarkers for Monitoring Toxicity
during Clinical Trials
Drug Labeling
Drug Development Process
PRELEAD
IND
Investigational New Drug
NDA/BLA
New Drug Application
Biologics License Appl.
Research
“Discovery”
Development
Toxicology Testing Process
PRELEAD
IND
NDA/BLA
Discovery
Development
Nonclinical tox studies in animals
P1
P2
P3
Clinical trials
Nonclinical Information Flow
In vitro/Animal Models
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Hypothesis testing
 Mechanism of
action
 Safety assessment
 Develop surrogate
markers
 ADME/PK
Application
Trial
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Potential for effect
 Toxicity profile
 Dose/regimen
 Route of administration
J. Lipani, 1998
Contract Research Organizations
• Formulation/Manufacture/Fill and Finish
• Metabolism/distribution (ADME/PK)
• In vitro
– Activity/high throughput screening
– Toxicity (non-GLP and GLP)
• In vivo
– Research
– Model development
– Proof of concept/efficacy
– Development
– GLP toxicology testing for regulatory submission
Types of Nonclinical Studies Reviewed by FDA
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Basic pharmacology
– primary and secondary mechanisms of action
– nonclinical efficacy studies
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Safety pharmacology
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Pharmacokinetics
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Toxicology
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Genotoxicology
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Carcinogenicity
What Does FDA Expect from Nonclinical Studies?
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Pharmacology
– proposed mechanism of action
– identification of secondary pharmacologic effects
– Proof of Concept studies for serious indications
Safety Pharmacology
– effects on neurological, cardiovascular, pulmonary, renal, and
gastrointestinal systems
– abuse liability
What Does FDA Expect from Nonclinical Studies?
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Pharmacokinetics
– comparison of ADME in species used for toxicology studies
– identification of bioaccumulation potential
– identification of potential differences in gender
– generation of PK parameters, e.g., Cmax, Tmax, AUC(o-inf.),
half life
What Does FDA Expect
in General Toxicology Studies?
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Acute and repeat toxicology studies in two species
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Duration of repeat dose nonclinical studies should be at least equal or greater
than the duration of the proposed clinical study
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A control and at least 3 drug concentrations
– identification of the NOAEL and high-dose multiple toxicity
– identify shape of the dose-response curve
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Doses/systemic exposure should exceed clinical dose/exposure
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See also ICH M3
What Does FDA Expect
in General Toxicology Studies?
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Formulation should be the same as the clinical formulation
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Route of exposure:
– should be the same as clinical route
– additional routes of exposure may be needed to achieve systemic toxicity
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Histopathology examination of all animals and standard tissues
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Lymphoproliferative tissues should be assessed for unintended effects on the
immune system
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Toxicokinetic information
Timing of Nonclinical Studies - Phase 1
(see ICH M3)
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Prior to “First Time in Humans”
– Pharmacokinetics/toxicokinetics (exposure data)
– Safety in a rodent and a non-rodent
 single dose toxicity studies in 2 mammalian species
 expanded acute or repeat dose toxicity studies
 safety pharmacology
– Local tolerance
– (In vitro evaluation of mutations and chromosomal damage)
– (Hypersensitivity for inhaled and dermal drugs)
Timing of Nonclinical Studies - Phase 1/2
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Phase 1-2 Clinical Trials
– repeat dose toxicity studies of appropriate length
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Phase 2 Clinical Trials
– (complete genotoxicity assessment (in vivo and in vitro))
– repeat dose toxicity studies of appropriate length
Timing of Nonclinical Studies - Phase 3
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Phase 3 Clinical Trials
– Repeat dose toxicity studies of appropriate length
– Male and female fertility
– Post-natal development
– Carcinogenicity
Questions Asked by
Review Pharmacologist/Toxicologist
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Validity of study design:
– Was the appropriate animal model used?
– Were dose(s) and duration sufficient to support
the proposed clinical study or labeling?
– Were adequate systemic exposures achieved?
– Was the route of administration relevant to
clinical used?
More Questions:
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Did the test system exhibit any effects?
 Were the effects treatment-related?
 Are the effects biologically significant?
 Are the effects reversible?
 Are the effects clinically relevant?
 Can the effects be monitored clinically?
Preclinical Development of rFXIII
Rafael Ponce
Jenn Visich
Toxicol Pathol. 2005;33(4):495-506
Toxicol Pathol. 2005;33(6):702-10
ZYMOGENETICS
Clotting and Fibrinolysis are in balance
Clotting
Contact Pathway
(Intrinsic pathway)
Tissue Factor
(Extrinsic pathway)
Prothrombin (II)
Thrombin
Fibrinogen
FXIII
Fibrinolysis
Cross-linked Fibrin Clot
Plasminogen
Plasmin
Fibrin Degradation products
D-dimers
ZYMOGENETICS
Tissue Factor
(Extrinsic pathway)
Contact Pathway
(Intrinsic pathway)
Neg. Charged Surface (collagen)
HMWK
PK
Kallikrein
XII
XIIa
XI XIa
IX IXa
Tissue damage
Tissue Factor
VIIa
VIIIa
VIII
X
Xa
Prothrombin (II)
Va
V
Thrombin-Antithrombin Complex (TAT)
Thrombomodulin
XIII
XIIIa
Plasminogen
Antithrombin III + Heparin (heparan, etc.)
Thrombin + F 1.2
Fibrinogen
tPA-PAI complex
VII
Serine protease
Thrombin-Thrombomodulin Complex
Protein C
Fibrin
tPA
Plasminogen Activator Inhibitor
Plasmin
Plasmin-antiplasmin complex
a 2 -Antiplasmin
Fibrin Degradation products, D-dimers
Activated Protein C
+ Protein S Inhibit
VIIIa and Va
FXIII in Normal Hemostasis
Prothrombin (II)
Thrombin
Fibrinogen
XIII (rA2B2)
(rA2*B2)
rXIII (rA2)
rXIII (2B)
XIIIa (rA2*)
Fibrin
Fibrinsoluble
Plasmin
Fast
Fibrincross-linked
Slow
ZYMOGENETICS
Preclinical Model Species Selection
Cynomolgus Monkeys
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Coagulation system similar to humans (general lit)
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Humans:
2B + rA2
Cynomolgus: 2cnB + rA2
rA2B2
rA2cnB2
- Binding of rFXIII to B-subunit across species
ZYMOGENETICS
Species Selection
Cynomolgus Monkeys
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Coagulation system similar to humans (general lit)
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Humans:
2B + rA2
Cynomolgus: 2cnB + rA2
rA2B2
rA2cnB2
- Binding of rFXIII to B-subunit across species
- Formation of FXIII rA2cnB2 in cynos in vivo
ZYMOGENETICS
Species Selection
Cynomolgus Monkeys
 Humans:
rFXIIIa + fibrin
Cynomolgus: rFXIIIa + cyno fibrin
x-linked fibrin
x-linked fibrin
ZYMOGENETICS
Species Selection
Cynomolgus Monkeys
 Humans:
rFXIIIa + fibrin
Cynomolgus: rFXIIIa + cyno fibrin
x-linked fibrin
x-linked fibrin
- In vitro FXIII cross-linking in human and cyno plasma
ZYMOGENETICS
Species Selection
Cynomolgus Monkeys
 Humans:
rFXIIIa + fibrin
Cynomolgus: rFXIIIa + cyno fibrin
x-linked fibrin
x-linked fibrin
- In vitro FXIII cross-linking in human and cyno plasma
- Formation of cross-linked fibrin(ogen) in cynos in vivo
ZYMOGENETICS
Design of rFXIII Toxicology Studies
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Dosing regimen support for CD and CPB indications
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Identify initial safe dose and dose escalation scheme
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Identify toxicity endpoints and their reversibility
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Evaluate immunogenicity
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Identify safety parameters for clinical monitoring
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Study evaluating the safety of rFXIII after 2 hr ECC
ZYMOGENETICS
Toxicology Studies
Study
Duration No/Sex /Gp
Dose (mg/kg)
SNBL.002.02
14 d
1F
1x 12.8 and 25.5
SBi-1220-175
7d
1F
1x 0, 10, 17.5, 20
SBi-1278-175
7d
1F
1x 0, 20, 21.2, 22.5, 25, 30
SBi-1249-175
14 d
1 M/F
2x 0, 12.5, 17.5, 22.5
SBi-1266-175
29d + 29d
3-5 M/F
3x 0, 5, 8, 12.5
SBi-1394-175
14d + 28d
3-5 M/F
14x 0, 0.3, 3.0, 6.0
CRP 1184
1d
3M
0.7, 2.1, 7.1
ZYMOGENETICS
FXIII-related Toxicity (> 22 mg/kg)
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Clinical signs
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Variable
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Included loss of consciousness, malaise, and poor food and
water consumption
Hematology
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Decreased platelet counts (<50,000/mL)
Serum chemistry
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Increased blood urea nitrogen, creatinine, LDH, AST, ALT,
and C-reactive protein
ZYMOGENETICS
FXIII-related Toxicity (> 22 mg/kg)
Gross pathology
Hemorrhage in a variety of tissues including:
Adrenal glands
Lung
Kidneys
Heart
Live
Gastrointestinal tract
ZYMOGENETICS
FXIII-related Toxicity (> 22 mg/kg)
Histologic observations
Intravascular congestion, thrombosis, and subsequent
necrosis in:
Adrenal glands Eye
Kidneys
Lung
Heart
Gastrointestinal tract
Pancreas
Spleen
Liver
Brain
Pituitary
Bone marrow
ZYMOGENETICS
IND-Enabling Toxicology Study (CD)
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28-day GLP repeated dose toxicology study
Measurement endpoints
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clinical observations
food consumption
blood pressure
heart rate
histopathological evaluation
- body weight
- clinical pathology
- body temperature
- gross necropsy
Bioanalytical evaluations
 PK (A2, A2B2, free B)
 Anti-FXIII antibody
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Necropsy
 48 hours after the last dose (all groups)
 after four-week dose-free period (vehicle and high dose group)
ZYMOGENETICS
FXIII-related Toxicity
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Clinical signs
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Variable
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Included loss of consciousness, malaise, and poor food and
water consumption
Hematology
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Decreased platelet counts (<50,000/mL)
Serum chemistry
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Increased blood urea nitrogen, creatinine, LDH, AST, ALT,
and C-reactive protein
ZYMOGENETICS
FXIII-related Toxicity
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Gross pathology
Hemorrhage in a variety of tissues
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Histologic observations
Intravascular congestion, thrombosis, and subsequent necrosis
ZYMOGENETICS
Margin of Safety for CD Indication
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Normal animals
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Doses for the CD1 study were 2, 6, 20, 50 and 75 units/kg
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Doses for the UKHV1 study were 2, 5, 10, 25 and 50 units/kg
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Potency of rFXIII = 140 U/mg
Dose associated with mild, reversible pathology
8.0 mg/kg x 140 U/mg = 1750 U/kg
560-fold (2 U/kg) to 15-fold (75 U/kg)
ZYMOGENETICS
Clinical Dosing Regimen
Currently covered by toxicology studies
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Single dose or split dose within 24 hours
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Repeated dose once/month for CD for 3 months
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Twice a month dosing
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Daily rFXIII dosing over 14 days in cynomolgus monkeys
 0.3-6 mg/kg rFXIII
 NOAEL = 6.0 mg/kg
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Post-cardiopulmonary bypass study in progress
ZYMOGENETICS
Mechanism of Toxicity
ZYMOGENETICS
Hypothesis
•
Free [A2] dimer (rFXIII) may be activate in vivo (does not
exist naturally)
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rFXIIIa can cross-link plasma fibrinogen and other proteins
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Cross-linked complexes can accumulate
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Large cross-linked complexes can result in coagulopathy and
ischemia
•
Ischemia/tissue damage can release tissue factor, activate
clotting cascade via classical means
•
Clotting system activation initiates fibrinolytic system
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Loss of compensatory control over clotting/fibrinolysis…
ZYMOGENETICS
End of the story?
ZYMOGENETICS
Pharmacokinetics of rFXIII
Jenn Visich
ZYMOGENETICS
Circulating FXIII Species
cnA2cnB2
cnB
Before Dosing
rA2
cnA2cnB2
rA2cnB2
rA2
cnB
After Dosing
ZYMOGENETICS
FXIII Molecular Species Detected by ELISA and Activity Assays
Assay
Total A2
FXIII Molecular Species Detected
rFXIII [A2]
cnA2cnB2 (endogenous cynomolgus FXIII)
rA2cnB2
(rFXIII complexed with cynomolgus
FXIII-B subunit)
FXIII A2B2
tetramer
cnA2cnB2
rA2cnB2
Free B
cnB
FXIII Activity
(Berichrom®)
rA2
cnA2cnB2
rA2cnB2
rA2
(free cynomolgus FXIII-B subunit)
ZYMOGENETICS
Total A2 (rA2B2 and rA2) Plasma Concentration
versus Time Profiles by Dose
Total A2 Plasma Conc (mg/mL)
(One Intravenous Dose)
1000.0
100.0
10.0
1.0
0.1
0
48
96
144
192
240
288
336
Time (hrs)
vehicle
0.5 mg/kg
1.0 mg/kg
5.0 mg/kg
Series5
ZYMOGENETICS
Plasma Concentration versus Time Profiles, All ELISAs
(One Intravenous Dose rFXIII)
Plasma Conc. (mg/mL)
1000
100
FXIII A2B2
10
Total A2
1
Free cnB
0.1
0
48
96
144
192
240
288
336
Time (hours)
5 mg/kg A2
5 mg/kg A2B2
5mg/kg B
ZYMOGENETICS
Mean Free cnB Plasma Concentration versus Time Profiles
(One intravenous dose)
1.6
1.2
0.8
vehicle
0.5 mg/kg
0.4
1.0 mg/kg
5.0 mg/kg
672
504
336
240
168
120
72
24
8
4
2
1
0.25
0.0
0
Free B Plasma Conc (mg/mL)
2.0
Time (hrs)
vehicle
0.5 mg/kg
1.0 mg/kg
5.0 mg/kg
ZYMOGENETICS
Total A2 Noncompartmental PK Estimates Mean
(Standard Deviation)
Parameter
Units
0.5 mg/kg
1.0 mg/kg
5.0 mg/kg
C0
mg/mL
10.84 (1.55)
18.97 (3.99)
103.24 (9.78)
t1/2, lz
h
194.75 (99.93)
142.67 (70.22)
131.08 (33.94)
AUCINF
h*mg/mL
655.71 (201.22)
985.37 (400.31)
2676.60 (781.02)
AUC(0 to t)
h*mg/mL
588.84 (173.78)
915.81 (356.18)
2535.96 (710.75)
CL
mL/h/kg
0.87 (0.41)
1.15 (0.42)
1.98 (0.43)
Vss
mL/kg
172.75 (76.04)
187.94 (88.64)
275.08 (42.63)
Plasma concentration versus time data were corrected for individual pre-dose FXIII Total A2 levels
ZYMOGENETICS
Pharmacokinetics of 125I-rFXIII in
Male and Female Cynos
•
Iodinated rFXIII characterized
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
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Radiopurity
Activity
Structural Integrity
Ability to bind Free B subunit
•
Bioanalytical Assay for analysis of plasma
•
Iodinated rFXIII administered to cynos, plasma collected
over 72 hours
 SE-HPLC with radiodetection
 Standard curve was created by spiking rA2 into cyno plasma
ZYMOGENETICS
5000
0.25 hr
3750
rA22
rA 2 cnB 2
2500
Counts
1250
0
0
2
4
5000
6
8
10
12
14
16
Elution time (minutes)
SE-HPLC coupled with
radiodetection
72 hr
3750
2500
rA 2 cnB 2
1250
0
2
4
6
8
10
12
14
Elution time (minutes)
Mean Unbound rFXIII
Plasma Concentrations vs.Time
Single Dose, 5.0 mg/kg (125I-) rFXIII
16
(mg/mL)
Concentration
FXIII rFXIII
Unbound
(µg/mL)
Concentration
Unbound
0
1000
100
t
= 3.57 h
1/2,λZ
10
1
0.1
t1/2, lz = 3.57 hr
0.01
0
4
8
12
Time (hr)
Time (hours)
ZYMOGENETICS
Toxicokinetics of rFXIII
•
At high doses, Free B subunit is saturated
•
Uncomplexed rA2 may be activated and cause
cross-linking of fibrin(ogen)
•
Kinetics of rA2 vs. rA2B2
 Circulating half-life of rA2
 Threshold concentration or duration of exposure of rA2
ZYMOGENETICS
Kinetics of rA2 vs rA2B2
•
rA2B2 has a half-life of ~ 4-7 days
•
Total A2 – A2B2  rA2
•
Assay independent way to measure rA2 kinetics
ZYMOGENETICS