Drug Development Lynnda Reid, Ph.D. Pharmacology/Toxicology Reviewer Center for Drug Evaluation and Research (CDER) Rafael Ponce, Ph.D., DABT Senior Scientist

Drug DevelopmentDrug Development

Lynnda Reid, Ph.D.

Pharmacology/Toxicology Reviewer

Center for Drug Evaluation and Research (CDER)

Rafael Ponce, Ph.D., DABT

Senior Scientist

ZymoGenetics, Inc.

OutlineOutline

Regulatory OverviewRegulatory Overview Drug/biologic development processDrug/biologic development process ResourcesResources Questions (and answers?)Questions (and answers?)

Parties involved in Drug DevelopmentParties involved in Drug Development FDA Sponsor Contract Labs Clinical Sites Manufacturing Sites Consultants Other…

SponsorsSponsors

Pharmaceutical/Biotechnology Firms Practicing Physicians and Dentists Academic Institutions NIH Other

ProteinsProteins Small Molecules Small MoleculesProteinsProteins Small Molecules Small MoleculesDrug substance Heterogenous mixture

Broad specs during developmentSpecs may change

Single entity; high chemicalpurityException: racemic mixturesSpecs well-defined early

Drug product Usually IV or SC Usually oral

Impurities Difficult to standardize Standards well established

Bridgingrequirements

Significant for drug substance Bioequivalence procedures

Biological activity May mimic naturally occurringmoleculesPrimary MOTPredictive based on MOA

Less predictive

Nonspecificity Variable significance Usually significantDrug-drug interaction

Chronic Toxicity Lack of models; species specificityand antigenicity

Models sometimes relevant

Impurities Toxicity not a major issue, mayimpact immunogenicity

May be significantPurity standards wellestablished

Drug substance Heterogenous mixtureBroad specs during developmentSpecs may change

Single entity; high chemicalpurityException: racemic mixturesSpecs well-defined early

Drug product Usually IV or SC Usually oral

Impurities Difficult to standardize Standards well established

Bridgingrequirements

Significant for drug substance Bioequivalence procedures

Biological activity May mimic naturally occurringmoleculesPrimary MOTPredictive based on MOA

Less predictive

Nonspecificity Variable significance Usually significantDrug-drug interaction

Chronic Toxicity Lack of models; species specificityand antigenicity

Models sometimes relevant

Impurities Toxicity not a major issue, mayimpact immunogenicity

May be significantPurity standards wellestablished

What Types of NonclinicalWhat Types of Nonclinical Studies Should Sponsors Conduct? Studies Should Sponsors Conduct?

ICH (International Conference on Harmonization) Guidelines Drug class specific guidance FDA Consultations General Toxicology?

Genotoxicity?Carcinogenicity?

General toxicity? Genotoxicity? Carcinogenicity?

The ICH S6 Guidance CoverageThe ICH S6 Guidance Coverage “Preclinical Testing of Biotechnology-Derived Pharmaceuticals”

– Two species 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 StudyDesigning a Tox Study

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 ProgramDesigning a Tox Program

ICH M3 ICH S6 Precedence

– http://www.fda.gov/cder/biologics/biologics_table.htm

Species SelectionSpecies Selection

Goal: Identify most appropriate rodent and non-rodent 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


Species Selection-DataSpecies Selection-Data

Sequence homology Tissue expression Binding data In vitro functional assay

– Ideally reconstituting entire signaling pathway with species-appropriate reagents

In vivo response


Types of Toxicology Studies RecommendedTypes of Toxicology Studies Recommended

General Toxicology

– acute and repeat dose toxicology studies

Special Toxicology Studies

– local irritation studies, e.g., site specific, ocular

– hypersensitivity studies for inhalation and dermal drug products

Reproductive and Developmental Toxicology Studies

– male and female fertility

– embryonic and fetal development

– post-natal reproductive and developmental effects

Purpose of Purpose of PreclinicalPreclinical Studies Studies

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 Impact of Nonclinical Studies on Drug DevelopmentDevelopment

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 ProcessDrug Development Process

PRELEADPRELEAD IND IND NDA/BLA NDA/BLA

““Discovery”Discovery”

DevelopmentDevelopment

Investigational New Drug Investigational New Drug New Drug ApplicationNew Drug Application

Biologics License Appl.Biologics License Appl.

ResearchResearch

Toxicology Testing ProcessToxicology Testing Process

PRELEADPRELEAD IND IND NDA/BLA NDA/BLA

DiscoveryDiscovery

DevelopmentDevelopment

Clinical trialsClinical trials

P1P1 P2P2 P3P3

Nonclinical tox studies in animalsNonclinical tox studies in animals

Nonclinical Information FlowNonclinical Information Flow

In vitro/Animal ModelsIn vitro/Animal Models ApplicationApplication TrialTrial

J. Lipani, 1998J. Lipani, 1998

Hypothesis testingHypothesis testing Mechanism of Mechanism of

actionaction Safety assessmentSafety assessment Develop surrogate Develop surrogate

markersmarkers ADME/PKADME/PK

Potential for effectPotential for effect Toxicity profileToxicity profile Dose/regimenDose/regimen Route of administrationRoute of administration

Contract Research OrganizationsContract Research Organizations

• Formulation/Manufacture/Fill and FinishFormulation/Manufacture/Fill and Finish• Metabolism/distribution (ADME/PK)Metabolism/distribution (ADME/PK)• In vitroIn vitro

– Activity/high throughput screeningActivity/high throughput screening– Toxicity (non-GLP and GLP)Toxicity (non-GLP and GLP)

• In vivoIn vivo– ResearchResearch

– Model developmentModel development– Proof of concept/efficacyProof of concept/efficacy

– DevelopmentDevelopment– GLP toxicology testing for regulatory submissionGLP toxicology testing for regulatory submission

Types of Nonclinical Studies Reviewed by FDATypes of Nonclinical Studies Reviewed by FDA

Basic pharmacology

– primary and secondary mechanisms of action

– nonclinical efficacy studies

Safety pharmacology

Pharmacokinetics

Toxicology

Genotoxicology

Carcinogenicity

What Does FDA Expect from Nonclinical Studies?What Does FDA Expect from Nonclinical Studies?

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?What Does FDA Expect from Nonclinical Studies?

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 What Does FDA Expect in General Toxicology Studies?in General Toxicology Studies?

Acute and repeat toxicology studies in two species

Duration of repeat dose nonclinical studies should be at least equal or greater than the duration of the proposed clinical study

A control and at least 3 drug concentrations

– identification of the NOAEL and high-dose multiple toxicity

– identify shape of the dose-response curve

Doses/systemic exposure should exceed clinical dose/exposure

See also ICH M3

What Does FDA Expect What Does FDA Expect in General Toxicology Studies?in General Toxicology Studies?

Formulation should be the same as the clinical formulation

Route of exposure:

– should be the same as clinical route

– additional routes of exposure may be needed to achieve systemic toxicity

Histopathology examination of all animals and standard tissues

Lymphoproliferative tissues should be assessed for unintended effects on the immune system

Toxicokinetic information

Timing of Nonclinical Studies - Phase 1Timing of Nonclinical Studies - Phase 1(see ICH M3)(see ICH M3)

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 1/2Timing of Nonclinical Studies - Phase 1/2

Phase 1-2 Clinical Trials– repeat dose toxicity studies of appropriate length

Phase 2 Clinical Trials– (complete genotoxicity assessment (in vivo and in vitro))– repeat dose toxicity studies of appropriate length

Timing of Nonclinical Studies - Phase 3Timing of Nonclinical Studies - Phase 3

Phase 3 Clinical Trials– Repeat dose toxicity studies of appropriate length– Male and female fertility– Post-natal development– Carcinogenicity

Questions Asked byQuestions Asked by Review Pharmacologist/Toxicologist Review Pharmacologist/Toxicologist

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:More Questions:

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 rFXIIIPreclinical Development of rFXIII

Rafael PonceJenn Visich

Rafael PonceJenn Visich

ZYMOGENETICS

Toxicol Pathol. 2005;33(4):495-506

Toxicol Pathol. 2005;33(6):702-10

ZYMOGENETICS

Contact Pathway(Intrinsic pathway) Contact Pathway

(Intrinsic pathway) Tissue Factor

(Extrinsic pathway) Tissue Factor

(Extrinsic pathway)

ThrombinThrombin

FibrinogenFibrinogen

Cross-linked Fibrin ClotCross-linked Fibrin Clot

Prothrombin (II)Prothrombin (II)

Clotting and Fibrinolysis are in balanceClotting and Fibrinolysis are in balance

PlasminogenPlasminogen

PlasminPlasmin

Fibrin Degradation productsD-dimers

Fibrin Degradation productsD-dimers

Clo

ttin

gC

lott

ing

Fib

rin

oly

sis

Fib

rin

oly

sis

FXIIIFXIII

Contact Pathway(Intrinsic pathway)

Tissue Factor(Extrinsic pathway)

Tissue Factor

Neg. Charged Surface (collagen)HMWK

PKKallikrein

XII XIIa

XI XIaIX IXa

VIIVIIa

VIIIVIIIa

Tissue damage

X Xa

Thrombin +

Fibrinogen

Fibrin

XIIIa

XIII

tPA

Plasminogen

Plasmin

Fibrin Degradation products, D-dimers

Prothrombin (II)

Va V Antithrombin III + Heparin (heparan, etc.)

1.2F Thrombin-Antithrombin Complex (TAT)

Serine protease

Plasminogen Activator Inhibitor

tPA-PAI complex

-Antiplasmin

Plasmin-antiplasmin complex

2

Thrombomodulin

Thrombin-Thrombomodulin Complex

Activated Protein CProtein C+ Protein S Inhibit

VIIIa and Va

ZYMOGENETICS

ThrombinThrombin

FibrinogenFibrinogen

FibrinFibrin

XIIIa (rA2*)XIIIa (rA2*)

PlasminPlasmin

Prothrombin (II)Prothrombin (II)

FXIII in Normal HemostasisFXIII in Normal Hemostasis

XIII (rA2B2)XIII (rA2B2)

rXIII (rA2)rXIII (rA2)rXIII (2B)rXIII (2B)

(rA2*B2)(rA2*B2)

FibrinsolubleFibrinsoluble Fibrincross-linkedFibrincross-linked

FastFast SlowSlow

ZYMOGENETICS

Preclinical Model Species SelectionPreclinical Model Species Selection

Cynomolgus Monkeys Coagulation system similar to humans (general lit)

Humans: 2B + rA2 rA2B2

Cynomolgus: 2cnB + rA2 rA2cnB2

- Binding of rFXIII to B-subunit across species





ZYMOGENETICS






- Formation of FXIII rA2cnB2 in cynos in vivo





- Formation of FXIII rA2cnB2 in cynos in vivo

ZYMOGENETICS

Cynomolgus Monkeys

Humans: rFXIIIa + fibrin x-linked fibrin

Cynomolgus: rFXIIIa + cyno fibrin x-linked fibrin

Cynomolgus Monkeys




ZYMOGENETICS

Cynomolgus Monkeys



- In vitro FXIII cross-linking in human and cyno plasma

Cynomolgus Monkeys





ZYMOGENETICS

Cynomolgus Monkeys




- Formation of cross-linked fibrin(ogen) in cynos in

vivo

Cynomolgus Monkeys




- Formation of cross-linked fibrin(ogen) in cynos in

vivo


ZYMOGENETICS

Design of rFXIII Toxicology StudiesDesign of rFXIII Toxicology Studies

• Dosing regimen support for CD and CPB indicationsDosing regimen support for CD and CPB indications

• Identify initial safe dose and dose escalation schemeIdentify initial safe dose and dose escalation scheme

• Identify toxicity endpoints and their reversibility Identify toxicity endpoints and their reversibility

• Evaluate immunogenicityEvaluate immunogenicity

• Identify safety parameters for clinical monitoringIdentify safety parameters for clinical monitoring

• Study evaluating the safety of rFXIII after 2 hr ECCStudy evaluating the safety of rFXIII after 2 hr ECC

• Dosing regimen support for CD and CPB indicationsDosing regimen support for CD and CPB indications

• Identify initial safe dose and dose escalation schemeIdentify initial safe dose and dose escalation scheme

• Identify toxicity endpoints and their reversibility Identify toxicity endpoints and their reversibility

• Evaluate immunogenicityEvaluate immunogenicity

• Identify safety parameters for clinical monitoringIdentify safety parameters for clinical monitoring

• Study evaluating the safety of rFXIII after 2 hr ECCStudy evaluating the safety of rFXIII after 2 hr ECC

ZYMOGENETICS

Toxicology StudiesToxicology Studies

Study Duration No/Sex /Gp Dose (mg/kg)

SNBL.002.02 14 d 1F 1x 12.8 and 25.5

SBi-1220-175 7 d 1F 1x 0, 10, 17.5, 20

SBi-1278-175 7 d 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 1 d 3M 0.7, 2.1, 7.1

Study Duration No/Sex /Gp Dose (mg/kg)

SNBL.002.02 14 d 1F 1x 12.8 and 25.5

SBi-1220-175 7 d 1F 1x 0, 10, 17.5, 20

SBi-1278-175 7 d 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 1 d 3M 0.7, 2.1, 7.1

ZYMOGENETICS

FXIII-related Toxicity (> 22 mg/kg)FXIII-related Toxicity (> 22 mg/kg)

• Clinical signs

• Variable

• Included loss of consciousness, malaise, and poor food and water consumption

• Hematology

• Decreased platelet counts (<50,000/mL)

• Serum chemistry

• Increased blood urea nitrogen, creatinine, LDH, AST, ALT, and C-reactive protein

• Clinical signs

• Variable


• Hematology


• Serum chemistry


ZYMOGENETICS

Gross pathology

Hemorrhage in a variety of tissues including:

Adrenal glands

Lung

Kidneys

Heart

Live

Gastrointestinal tract

Gross pathology

Hemorrhage in a variety of tissues including:

Adrenal glands

Lung

Kidneys

Heart

Live

Gastrointestinal tract


ZYMOGENETICS

Histologic observations

Intravascular congestion, thrombosis, and subsequent necrosis in:

Adrenal glands Eye Kidneys

Lung Heart Gastrointestinal tract

Pancreas Spleen Liver

Brain Pituitary Bone marrow

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)IND-Enabling Toxicology Study (CD)

• 28-day GLP repeated dose toxicology study• Measurement endpoints

- clinical observations - body weight- food consumption - clinical pathology- blood pressure - body temperature- heart rate - gross necropsy- histopathological evaluation

• Bioanalytical evaluations PK (A2, A2B2, free B) Anti-FXIII antibody

• Necropsy 48 hours after the last dose (all groups) after four-week dose-free period (vehicle and high dose group)

• 28-day GLP repeated dose toxicology study• Measurement endpoints

- clinical observations - body weight- food consumption - clinical pathology- blood pressure - body temperature- heart rate - gross necropsy- histopathological evaluation

• Bioanalytical evaluations PK (A2, A2B2, free B) Anti-FXIII antibody

• Necropsy 48 hours after the last dose (all groups) after four-week dose-free period (vehicle and high dose group)

ZYMOGENETICS

FXIII-related ToxicityFXIII-related Toxicity

• Clinical signs

• Variable


• Hematology


• Serum chemistry


• Clinical signs

• Variable


• Hematology


• Serum chemistry


ZYMOGENETICS

• Gross pathology

Hemorrhage in a variety of tissues

• Histologic observations

Intravascular congestion, thrombosis, and subsequent necrosis

• Gross pathology

Hemorrhage in a variety of tissues

• Histologic observations

Intravascular congestion, thrombosis, and subsequent necrosis

FXIII-related ToxicityFXIII-related Toxicity

ZYMOGENETICS

Margin of Safety for CD IndicationMargin of Safety for CD Indication• Normal animalsNormal animals

• Doses for the CD1 study were 2, 6, 20, 50 and 75 units/kgDoses for the CD1 study were 2, 6, 20, 50 and 75 units/kg

• Doses for the UKHV1 study were 2, 5, 10, 25 and 50 units/kgDoses for the UKHV1 study were 2, 5, 10, 25 and 50 units/kg

• Potency of rFXIII = 140 U/mgPotency of rFXIII = 140 U/mg

Dose associated with mild, reversible pathologyDose associated with mild, reversible pathology

8.0 mg/kg x 140 U/mg = 1750 U/kg8.0 mg/kg x 140 U/mg = 1750 U/kg

560-fold (2 U/kg) to 15-fold (75 U/kg)560-fold (2 U/kg) to 15-fold (75 U/kg)

• Normal animalsNormal animals

• Doses for the CD1 study were 2, 6, 20, 50 and 75 units/kgDoses for the CD1 study were 2, 6, 20, 50 and 75 units/kg

• Doses for the UKHV1 study were 2, 5, 10, 25 and 50 units/kgDoses for the UKHV1 study were 2, 5, 10, 25 and 50 units/kg

• Potency of rFXIII = 140 U/mgPotency of rFXIII = 140 U/mg

Dose associated with mild, reversible pathologyDose associated with mild, reversible pathology

8.0 mg/kg x 140 U/mg = 1750 U/kg8.0 mg/kg x 140 U/mg = 1750 U/kg

560-fold (2 U/kg) to 15-fold (75 U/kg)560-fold (2 U/kg) to 15-fold (75 U/kg)

ZYMOGENETICS

Clinical Dosing RegimenClinical Dosing Regimen

Currently covered by toxicology studiesCurrently covered by toxicology studies

• Single dose or split dose within 24 hoursSingle dose or split dose within 24 hours

• Repeated dose once/month for CD for 3 monthsRepeated dose once/month for CD for 3 months

• Twice a month dosing Twice a month dosing

• Daily rFXIII dosing over 14 days in cynomolgus monkeysDaily rFXIII dosing over 14 days in cynomolgus monkeys

0.3-6 mg/kg rFXIII

NOAEL = 6.0 mg/kg

• Post-cardiopulmonary bypass study in progress Post-cardiopulmonary bypass study in progress

Currently covered by toxicology studiesCurrently covered by toxicology studies

• Single dose or split dose within 24 hoursSingle dose or split dose within 24 hours

• Repeated dose once/month for CD for 3 monthsRepeated dose once/month for CD for 3 months

• Twice a month dosing Twice a month dosing

• Daily rFXIII dosing over 14 days in cynomolgus monkeysDaily rFXIII dosing over 14 days in cynomolgus monkeys

0.3-6 mg/kg rFXIII

NOAEL = 6.0 mg/kg

• Post-cardiopulmonary bypass study in progress Post-cardiopulmonary bypass study in progress

ZYMOGENETICS

Mechanism of ToxicityMechanism of Toxicity

ZYMOGENETICS

HypothesisHypothesis

• Free [A2] dimer (rFXIII) may be activate in vivo (does not exist naturally)

• rFXIIIa can cross-link plasma fibrinogen and other proteins

• Cross-linked complexes can accumulate

• 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

• Loss of compensatory control over clotting/fibrinolysis…

• Free [A2] dimer (rFXIII) may be activate in vivo (does not exist naturally)

• rFXIIIa can cross-link plasma fibrinogen and other proteins

• Cross-linked complexes can accumulate

• 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

• Loss of compensatory control over clotting/fibrinolysis…

ZYMOGENETICS

End of the story?End of the story?

ZYMOGENETICS

Pharmacokinetics of rFXIIIPharmacokinetics of rFXIII

Jenn VisichJenn Visich

ZYMOGENETICS

ZYMOGENETICS

cnA2cnB2

cnB

cnA2cnB2

cnBcnA2cnB2

rA2cnB2

rA2

cnB

cnA2cnB2

rA2cnB2

rA2

cnB

rA2rA2

Circulating FXIII SpeciesCirculating FXIII Species

After DosingAfter Dosing

Before Dosing

ZYMOGENETICS

FXIII Molecular Species Detected by ELISA and Activity AssaysFXIII Molecular Species Detected by ELISA and Activity Assays

AssayAssay FXIII Molecular Species DetectedFXIII Molecular Species Detected

Total A2Total A2 rFXIII [A2]

cnA2cnB2 (endogenous cynomolgus FXIII)

rA2cnB2 (rFXIII complexed with cynomolgus

FXIII-B subunit)

rFXIII [A2]

cnA2cnB2 (endogenous cynomolgus FXIII)

rA2cnB2 (rFXIII complexed with cynomolgus

FXIII-B subunit)

FXIII A2B2

tetramer

FXIII A2B2

tetramercnA2cnB2

rA2cnB2

cnA2cnB2

rA2cnB2

Free BFree B cnB (free cynomolgus FXIII-B subunit)cnB (free cynomolgus FXIII-B subunit)

FXIII Activity(Berichrom®)FXIII Activity(Berichrom®)

rA2

cnA2cnB2

rA2cnB2

rA2

cnA2cnB2

rA2cnB2

rA2

ZYMOGENETICS

0.1

1.0

10.0

100.0

1000.0

0 48 96 144 192 240 288 336

Time (hrs)

Tot

al A

2 P

lasm

a C

onc

(mg/

mL

)

vehicle 0.5 mg/kg 1.0 mg/kg 5.0 mg/kg Series5

Total A2 (rA2B2 and rA2) Plasma Concentration versus Time Profiles by Dose

(One Intravenous Dose)

Total A2 (rA2B2 and rA2) Plasma Concentration versus Time Profiles by Dose

(One Intravenous Dose)

ZYMOGENETICS

Plasma Concentration versus Time Profiles, All ELISAs (One Intravenous Dose rFXIII)

Plasma Concentration versus Time Profiles, All ELISAs (One Intravenous Dose rFXIII)

0.1

1

10

100

1000

0 48 96 144 192 240 288 336

Time (hours)

Pla

sma

Con

c. (m

g/m

L)

5 mg/kg A2 5 mg/kg A2B2 5mg/kg B

FXIII A2B2

Total A2

Free cnB

ZYMOGENETICS

0.0

0.4

0.8

1.2

1.6

2.0

0

0.25 1 2 4 8 24 72 120

168

240

336

504

672

Time (hrs)

Fre

e B

Pla

sma

Con

c (m g

/mL

)

vehicle 0.5 mg/kg 1.0 mg/kg 5.0 mg/kg

Mean Free cnB Plasma Concentration versus Time Profiles (One intravenous dose)

Mean Free cnB Plasma Concentration versus Time Profiles (One intravenous dose)

0.5 mg/kg

5.0 mg/kg

vehicle

1.0 mg/kg

ZYMOGENETICS

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,z 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)

Total A2 Noncompartmental PK Estimates Mean (Standard Deviation)

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

Pharmacokinetics of 125I-rFXIII in

Male and Female Cynos

• Iodinated rFXIII characterizedIodinated rFXIII characterized Radiopurity Activity Structural Integrity Ability to bind Free B subunit

• Bioanalytical Assay for analysis of plasmaBioanalytical Assay for analysis of plasma SE-HPLC with radiodetection Standard curve was created by spiking rA2 into cyno plasma

• Iodinated rFXIII administered to cynos, plasma collected Iodinated rFXIII administered to cynos, plasma collected over 72 hours over 72 hours

• Iodinated rFXIII characterizedIodinated rFXIII characterized Radiopurity Activity Structural Integrity Ability to bind Free B subunit

• Bioanalytical Assay for analysis of plasmaBioanalytical Assay for analysis of plasma SE-HPLC with radiodetection Standard curve was created by spiking rA2 into cyno plasma

• Iodinated rFXIII administered to cynos, plasma collected Iodinated rFXIII administered to cynos, plasma collected over 72 hours over 72 hours

ZYMOGENETICS

Mean Unbound rFXIII Plasma Concentrations vs.Time

Single Dose, 5.0 mg/kg (Single Dose, 5.0 mg/kg (125125I-) rFXIIII-) rFXIII

SE-HPLC coupled with SE-HPLC coupled with radiodetectionradiodetection

rA2

1250

2500

3750

5000

1250

2500

3750

5000

2 2 rA cnB

rA2

1250

2500

3750

1250

2500

3750

0 2 4 6 8 10 12 14

16 Elution time (minutes)

0 2 4 6 8 10 12 14

16 Elution time (minutes)

2 2 rA cnB

5000

0

0

0.01

0.1

1

10

100

1000

0 4 8 12

Time (hours)

Unb

ound

rFX

III

Con

cent

ratio

n (µ

g/m

L)

t1/2,λZ = 3.57 h

tt1/2, 1/2, z = 3.57 hrz = 3.57 hr

Time (hr)

Unb

ound

FX

III

Con

cent

rati

on (mg

/mL

)

Cou

nts

0.25 hr

72 hr

ZYMOGENETICS

Toxicokinetics of rFXIII Toxicokinetics of rFXIII

• At high doses, Free B subunit is saturatedAt high doses, Free B subunit is saturated

• Uncomplexed rAUncomplexed rA22 may be activated and cause cross- may be activated and cause cross-linking of fibrin(ogen)linking of fibrin(ogen)

• Kinetics of rAKinetics of rA22 vs. rA vs. rA22BB22

Circulating half-life of rA2

Threshold concentration or duration of exposure of rA2

• At high doses, Free B subunit is saturatedAt high doses, Free B subunit is saturated

• Uncomplexed rAUncomplexed rA22 may be activated and cause cross- may be activated and cause cross-linking of fibrin(ogen)linking of fibrin(ogen)

• Kinetics of rAKinetics of rA22 vs. rA vs. rA22BB22

Circulating half-life of rA2

Threshold concentration or duration of exposure of rA2

ZYMOGENETICS

Kinetics of rA2 vs rA2B2Kinetics of rA2 vs rA2B2

• rArA22BB22 has a half-life of ~ 4-7 days has a half-life of ~ 4-7 days

• Total ATotal A22 – A – A22BB22 rA rA22

• Assay independent way to measure rAAssay independent way to measure rA22 kinetics kinetics

• rArA22BB22 has a half-life of ~ 4-7 days has a half-life of ~ 4-7 days

• Total ATotal A22 – A – A22BB22 rA rA22

• Assay independent way to measure rAAssay independent way to measure rA22 kinetics kinetics

Documents

Drug Development Lynnda Reid, Ph.D. Pharmacology/Toxicology Reviewer Center for Drug Evaluation and Research (CDER) Rafael Ponce, Ph.D., DABT Senior Scientist