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 Regulatory Overview Regulatory Overview Drug/biologic development process Drug/biologic development process Resources Resources Questions (and answers?) Questions (and answers?)

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

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

Proteins Small Molecules

What Types of Nonclinical 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 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 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 Program ICH M3 ICH S6 Precedence –

Species 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 Katie Sprugel, Amgen

Species Selection-Data 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 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 Preclinical 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 Development 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 NDA/BLA “Discovery” Development Investigational New Drug New Drug Application Investigational New Drug New Drug Application Biologics License Appl. Biologics License Appl. Research

Toxicology Testing Process PRELEAD IND NDA/BLA Discovery Development Clinical trials P1P2P3 Nonclinical tox studies in animals

Nonclinical Information Flow In vitro/Animal ModelsApplicationTrial J. Lipani, 1998 Hypothesis testing Hypothesis testing Mechanism of action Mechanism of action Safety assessment Safety assessment Develop surrogate markers Develop surrogate markers ADME/PK ADME/PK Potential for effect Potential for effect Toxicity profile Toxicity profile Dose/regimen Dose/regimen Route of administration Route of administration

Contract 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 screening –Toxicity (non-GLP and GLP) In vivoIn vivo –Research –Model development –Proof of concept/efficacy –Development –GLP toxicology testing for regulatory submission

Types 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? 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? 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? 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 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 1 (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 1/2 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 3 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 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: 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 Rafael Ponce Jenn Visich Z YMO G ENETICS Toxicol Pathol. 2005;33(4): Toxicol Pathol. 2005;33(6):702-10

Z YMO G ENETICS Contact Pathway (Intrinsic pathway) Contact Pathway (Intrinsic pathway) Tissue Factor (Extrinsic pathway) Tissue Factor (Extrinsic pathway) Thrombin Fibrinogen Cross-linked Fibrin Clot Prothrombin (II) Clotting and Fibrinolysis are in balance Plasminogen Plasmin Fibrin Degradation products D-dimers Fibrin Degradation products D-dimers Clotting Fibrinolysis FXIII

Contact Pathway (Intrinsic pathway) Tissue Factor (Extrinsic pathway) Tissue Factor Neg. Charged Surface (collagen) HMWK PK Kallikrein XIIXIIa XIXIa IXIXa VIIVIIa VIIIVIIIa Tissue damage XXa Thrombin + Fibrinogen Fibrin XIIIa XIII tPA Plasminogen Plasmin Fibrin Degradation products, D-dimers Prothrombin (II) VaV Antithrombin III + Heparin (heparan, etc.) 1.2 F Thrombin-Antithrombin Complex (TAT) Serine protease Plasminogen Activator Inhibitor tPA-PAI complex -Antiplasmin Plasmin-antiplasmin complex 2  Thrombomodulin Thrombin-Thrombomodulin Complex Activated Protein C Protein C + Protein S Inhibit VIIIa and Va

Z YMO G ENETICS Thrombin Fibrinogen Fibrin XIIIa (rA 2 *) Plasmin Prothrombin (II) FXIII in Normal Hemostasis XIII (rA 2 B 2 ) rXIII (rA 2 ) rXIII (2B) (rA 2 *B 2 ) Fibrin soluble Fibrin cross-linked Fast Slow

Z YMO G ENETICS Preclinical Model Species Selection Cynomolgus Monkeys  Coagulation system similar to humans (general lit)  Humans: 2B + rA 2 rA 2 B 2 Cynomolgus: 2cnB + rA 2 rA 2 cnB 2 - Binding of rFXIII to B-subunit across species Cynomolgus Monkeys  Coagulation system similar to humans (general lit)  Humans: 2B + rA 2 rA 2 B 2 Cynomolgus: 2cnB + rA 2 rA 2 cnB 2 - Binding of rFXIII to B-subunit across species

Z YMO G ENETICS Species Selection Cynomolgus Monkeys  Coagulation system similar to humans (general lit)  Humans: 2B + rA 2 rA 2 B 2 Cynomolgus: 2cnB + rA 2 rA 2 cnB 2 - Binding of rFXIII to B-subunit across species - Formation of FXIII rA 2 cnB 2 in cynos in vivo Cynomolgus Monkeys  Coagulation system similar to humans (general lit)  Humans: 2B + rA 2 rA 2 B 2 Cynomolgus: 2cnB + rA 2 rA 2 cnB 2 - Binding of rFXIII to B-subunit across species - Formation of FXIII rA 2 cnB 2 in cynos in vivo

Z YMO G ENETICS Cynomolgus Monkeys  Humans: rFXIIIa + fibrin x-linked fibrin Cynomolgus: rFXIIIa + cyno fibrin x-linked fibrin Cynomolgus Monkeys  Humans: rFXIIIa + fibrin x-linked fibrin Cynomolgus: rFXIIIa + cyno fibrin x-linked fibrin Species Selection

Z YMO G ENETICS Cynomolgus Monkeys  Humans: rFXIIIa + fibrin x-linked fibrin Cynomolgus: rFXIIIa + cyno fibrin x-linked fibrin - In vitro FXIII cross-linking in human and cyno plasma Cynomolgus Monkeys  Humans: rFXIIIa + fibrin x-linked fibrin Cynomolgus: rFXIIIa + cyno fibrin x-linked fibrin - In vitro FXIII cross-linking in human and cyno plasma Species Selection

Z YMO G ENETICS Cynomolgus Monkeys  Humans: rFXIIIa + fibrin x-linked fibrin Cynomolgus: rFXIIIa + cyno fibrin x-linked fibrin - In vitro FXIII cross-linking in human and cyno plasma - Formation of cross-linked fibrin(ogen) in cynos in vivo Cynomolgus Monkeys  Humans: rFXIIIa + fibrin x-linked fibrin Cynomolgus: rFXIIIa + cyno fibrin x-linked fibrin - In vitro FXIII cross-linking in human and cyno plasma - Formation of cross-linked fibrin(ogen) in cynos in vivo Species Selection

Z YMO G ENETICS Design of rFXIII Toxicology Studies Dosing regimen support for CD and CPB indications Dosing regimen support for CD and CPB indications Identify initial safe dose and dose escalation scheme Identify initial safe dose and dose escalation scheme Identify toxicity endpoints and their reversibility Identify toxicity endpoints and their reversibility Evaluate immunogenicity Evaluate immunogenicity Identify safety parameters for clinical monitoring Identify safety parameters for clinical monitoring Study evaluating the safety of rFXIII after 2 hr ECC Study evaluating the safety of rFXIII after 2 hr ECC Dosing regimen support for CD and CPB indications Dosing regimen support for CD and CPB indications Identify initial safe dose and dose escalation scheme Identify initial safe dose and dose escalation scheme Identify toxicity endpoints and their reversibility Identify toxicity endpoints and their reversibility Evaluate immunogenicity Evaluate immunogenicity Identify safety parameters for clinical monitoring Identify safety parameters for clinical monitoring Study evaluating the safety of rFXIII after 2 hr ECC Study evaluating the safety of rFXIII after 2 hr ECC

Z YMO G ENETICS Toxicology Studies Study DurationNo/Sex /GpDose (mg/kg) SNBL d1F1x 12.8 and 25.5 SBi d1F1x 0, 10, 17.5, 20 SBi d1F1x 0, 20, 21.2, 22.5, 25, 30 SBi d1 M/F2x 0, 12.5, 17.5, 22.5 SBi d + 29d3-5 M/F3x 0, 5, 8, 12.5 SBi d + 28d3-5 M/F14x 0, 0.3, 3.0, 6.0 CRP d3M0.7, 2.1, 7.1 Study DurationNo/Sex /GpDose (mg/kg) SNBL d1F1x 12.8 and 25.5 SBi d1F1x 0, 10, 17.5, 20 SBi d1F1x 0, 20, 21.2, 22.5, 25, 30 SBi d1 M/F2x 0, 12.5, 17.5, 22.5 SBi d + 29d3-5 M/F3x 0, 5, 8, 12.5 SBi d + 28d3-5 M/F14x 0, 0.3, 3.0, 6.0 CRP d3M0.7, 2.1, 7.1

Z YMO G ENETICS 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 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

Z YMO G ENETICS Gross pathology Hemorrhage in a variety of tissues including: Hemorrhage in a variety of tissues including: Adrenal glands Lung Kidneys Heart Live Gastrointestinal tract Gross pathology Hemorrhage in a variety of tissues including: Hemorrhage in a variety of tissues including: Adrenal glands Lung Kidneys Heart Live Gastrointestinal tract FXIII-related Toxicity (> 22 mg/kg)

Z YMO G ENETICS Histologic observations Intravascular congestion, thrombosis, and subsequent necrosis in: Adrenal glands EyeKidneys LungHeartGastrointestinal tract PancreasSpleenLiver BrainPituitaryBone marrow Histologic observations Intravascular congestion, thrombosis, and subsequent necrosis in: Adrenal glands EyeKidneys LungHeartGastrointestinal tract PancreasSpleenLiver BrainPituitaryBone marrow FXIII-related Toxicity (> 22 mg/kg)

Z YMO G ENETICS IND-Enabling Toxicology Study (CD) 28-day GLP repeated dose toxicology study 28-day GLP repeated dose toxicology study Measurement endpoints Measurement endpoints - clinical observations- body weight - food consumption- clinical pathology - blood pressure- body temperature - heart rate- gross necropsy -histopathological evaluation Bioanalytical evaluations Bioanalytical evaluations  PK (A2, A2B2, free B)  Anti-FXIII antibody Necropsy 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 28-day GLP repeated dose toxicology study Measurement endpoints Measurement endpoints - clinical observations- body weight - food consumption- clinical pathology - blood pressure- body temperature - heart rate- gross necropsy -histopathological evaluation Bioanalytical evaluations Bioanalytical evaluations  PK (A2, A2B2, free B)  Anti-FXIII antibody Necropsy Necropsy  48 hours after the last dose (all groups)  after four-week dose-free period (vehicle and high dose group)

Z YMO G ENETICS FXIII-related Toxicity 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 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

Z YMO G ENETICS Gross pathology Gross pathology Hemorrhage in a variety of tissues Hemorrhage in a variety of tissues Histologic observations Histologic observations Intravascular congestion, thrombosis, and subsequent necrosis Intravascular congestion, thrombosis, and subsequent necrosis Gross pathology Gross pathology Hemorrhage in a variety of tissues Hemorrhage in a variety of tissues Histologic observations Histologic observations Intravascular congestion, thrombosis, and subsequent necrosis Intravascular congestion, thrombosis, and subsequent necrosis FXIII-related Toxicity

Z YMO G ENETICS Margin of Safety for CD Indication Normal animals Normal animals Doses for the CD1 study were 2, 6, 20, 50 and 75 units/kg Doses 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/kg Doses for the UKHV1 study were 2, 5, 10, 25 and 50 units/kg Potency of rFXIII = 140 U/mg 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) Normal animals Normal animals Doses for the CD1 study were 2, 6, 20, 50 and 75 units/kg Doses 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/kg Doses for the UKHV1 study were 2, 5, 10, 25 and 50 units/kg Potency of rFXIII = 140 U/mg 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)

Z YMO G ENETICS Clinical Dosing Regimen Currently covered by toxicology studies Single dose or split dose within 24 hours Single dose or split dose within 24 hours Repeated dose once/month for CD for 3 months Repeated dose once/month for CD for 3 months Twice a month dosing Twice a month dosing Daily rFXIII dosing over 14 days in cynomolgus monkeys Daily rFXIII dosing over 14 days in cynomolgus monkeys  mg/kg rFXIII  NOAEL = 6.0 mg/kg Post-cardiopulmonary bypass study in progress Post-cardiopulmonary bypass study in progress Currently covered by toxicology studies Single dose or split dose within 24 hours Single dose or split dose within 24 hours Repeated dose once/month for CD for 3 months Repeated dose once/month for CD for 3 months Twice a month dosing Twice a month dosing Daily rFXIII dosing over 14 days in cynomolgus monkeys Daily rFXIII dosing over 14 days in cynomolgus monkeys  mg/kg rFXIII  NOAEL = 6.0 mg/kg Post-cardiopulmonary bypass study in progress Post-cardiopulmonary bypass study in progress

Z YMO G ENETICS Mechanism of Toxicity

Z YMO G ENETICS Hypothesis Free [A 2 ] 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 [A 2 ] 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…

Z YMO G ENETICS End of the story?

Z YMO G ENETICS Pharmacokinetics of rFXIII Jenn Visich Z YMO G ENETICS

cnA 2 cnB 2 cnB cnA 2 cnB 2 cnB cnA 2 cnB 2 rA 2 cnB 2 rA 2 cnB cnA 2 cnB 2 rA 2 cnB 2 rA 2 cnB rA 2 Circulating FXIII Species After Dosing Before Dosing

Z YMO G ENETICS FXIII Molecular Species Detected by ELISA and Activity Assays Assay FXIII Molecular Species Detected Total A 2 rFXIII [A 2 ] cnA 2 cnB 2 (endogenous cynomolgus FXIII) rA 2 cnB 2 (rFXIII complexed with cynomolgus FXIII-B subunit) rFXIII [A 2 ] cnA 2 cnB 2 (endogenous cynomolgus FXIII) rA 2 cnB 2 (rFXIII complexed with cynomolgus FXIII-B subunit) FXIII A 2 B 2 tetramer cnA 2 cnB 2 rA 2 cnB 2 cnA 2 cnB 2 rA 2 cnB 2 Free B cnB (free cynomolgus FXIII-B subunit) FXIII Activity (Berichrom®) FXIII Activity (Berichrom®) rA 2 cnA 2 cnB 2 rA 2 cnB 2 rA 2 cnA 2 cnB 2 rA 2 cnB 2 rA 2

Z YMO G ENETICS Total A 2 (rA 2 B 2 and rA 2 ) Plasma Concentration versus Time Profiles by Dose (One Intravenous Dose) Total A 2 (rA 2 B 2 and rA 2 ) Plasma Concentration versus Time Profiles by Dose (One Intravenous Dose)

Z YMO G ENETICS Plasma Concentration versus Time Profiles, All ELISAs (One Intravenous Dose rFXIII) Plasma Concentration versus Time Profiles, All ELISAs (One Intravenous Dose rFXIII) FXIII A 2 B 2 Total A 2 Free cnB

Z YMO G ENETICS Mean Free cnB Plasma Concentration versus Time Profiles (One intravenous dose) 0.5 mg/kg 5.0 mg/kg vehicle 1.0 mg/kg

Z YMO G ENETICS Parameter Units 0.5 mg/kg1.0 mg/kg5.0 mg/kg C0C0  g/mL (1.55) (3.99) (9.78) t 1/2,  z h (99.93) (70.22) (33.94) AUC INF h*  g/mL (201.22) (400.31) (781.02) AUC (0 to t) h*  g/mL (173.78) (356.18) (710.75) CL mL/h/kg 0.87 (0.41)1.15 (0.42)1.98 (0.43) V ss mL/kg (76.04) (88.64) (42.63) Total A 2 Noncompartmental PK Estimates Mean (Standard Deviation) Plasma concentration versus time data were corrected for individual pre-dose FXIII Total A 2 levels

Z YMO G ENETICS Pharmacokinetics of 125 I-rFXIII in Male and Female Cynos Iodinated rFXIII characterized Iodinated rFXIII characterized  Radiopurity  Activity  Structural Integrity  Ability to bind Free B subunit Bioanalytical Assay for analysis of plasma Bioanalytical Assay for analysis of plasma  SE-HPLC with radiodetection  Standard curve was created by spiking rA 2 into cyno plasma Iodinated rFXIII administered to cynos, plasma collected over 72 hours Iodinated rFXIII administered to cynos, plasma collected over 72 hours Iodinated rFXIII characterized Iodinated rFXIII characterized  Radiopurity  Activity  Structural Integrity  Ability to bind Free B subunit Bioanalytical Assay for analysis of plasma Bioanalytical Assay for analysis of plasma  SE-HPLC with radiodetection  Standard curve was created by spiking rA 2 into cyno plasma Iodinated rFXIII administered to cynos, plasma collected over 72 hours Iodinated rFXIII administered to cynos, plasma collected over 72 hours

Z YMO G ENETICS Mean Unbound rFXIII Plasma Concentrations vs.Time Single Dose, 5.0 mg/kg ( 125 I-) rFXIII SE-HPLC coupled with radiodetection rA rA cnB rA Elution time (minutes) 2 rA cnB Time (hours) Unbound rFXIII Concentration (µg/mL) t 1/2,λZ = 3.57 h t 1/2, z = 3.57 hr Time (hr) Unbound FXIII Concentration (  g/mL) Counts 0.25 hr 72 hr

Z YMO G ENETICS Toxicokinetics of rFXIII At high doses, Free B subunit is saturated At high doses, Free B subunit is saturated Uncomplexed rA 2 may be activated and cause cross-linking of fibrin(ogen) Uncomplexed rA 2 may be activated and cause cross-linking of fibrin(ogen) Kinetics of rA 2 vs. rA 2 B 2 Kinetics of rA 2 vs. rA 2 B 2  Circulating half-life of rA 2  Threshold concentration or duration of exposure of rA 2 At high doses, Free B subunit is saturated At high doses, Free B subunit is saturated Uncomplexed rA 2 may be activated and cause cross-linking of fibrin(ogen) Uncomplexed rA 2 may be activated and cause cross-linking of fibrin(ogen) Kinetics of rA 2 vs. rA 2 B 2 Kinetics of rA 2 vs. rA 2 B 2  Circulating half-life of rA 2  Threshold concentration or duration of exposure of rA 2

Z YMO G ENETICS Kinetics of rA 2 vs rA 2 B 2 rA 2 B 2 has a half-life of ~ 4-7 days rA 2 B 2 has a half-life of ~ 4-7 days Total A 2 – A 2 B 2  rA 2 Total A 2 – A 2 B 2  rA 2 Assay independent way to measure rA 2 kinetics Assay independent way to measure rA 2 kinetics rA 2 B 2 has a half-life of ~ 4-7 days rA 2 B 2 has a half-life of ~ 4-7 days Total A 2 – A 2 B 2  rA 2 Total A 2 – A 2 B 2  rA 2 Assay independent way to measure rA 2 kinetics Assay independent way to measure rA 2 kinetics