Donor Organ Machine Perfusion - Application of An Hemoglobin-based Oxygen Carrier - Robert J. Porte, MD, PhD Professor of Surgery Department of HPB Surgery and Liver Transplantation University Medical Center Groningen The Netherlands

Disclosure HBO2 Therapeutics LLC provided products for our research free of charge Not any other potential conflicts of interest

Donor Organ Shortage Shortage of suitable donor organs for transplantation Increased use of extended criteria donors (ECD) and donation after circulatory death (DCD) organs Conventional static cold storage does not provide optimal preservation of ECD and DCD donor organs Early graft dysfunction More graft-related complications Retransplantation Underutilization of the current donor pool Alternative strategy: machine perfusion www.organdonor.gov

The Current Principle of Organ Preservation - Based on Cooling- Static Cold Storage

What Do We Need for Machine Perfusion? Heart / pump  Lungs / gas exchange  Blood vessels / tubings 

Machine Preservation An Alternative For Static Cold Storage Advantages: Reduces ischemia / reperfusion injury Prolonged preservation times Better ex situ assessment of graft viability Potential of (pharmacological) preconditioning Potential to restore / regenerate damaged tissue Increase in numbers and quality of donor organs Disadvantages: More complex More expensive than static cold storage Oxygen carrier needed when performed at (sub)normthermia

Normothermic: 35-37 oC Hypothermic: < 12 oC Temperature (oC) Percentage of metabolism (%)

Hypothermic versus Normothermic Hypothermic Oxygenated Machine Perfusion Resuscitation of the mitochondria Restoration of cellular energy storage (ATP) Reduction of ischemia-reperfusion injury Relatively simple and safe Relatively low costs No oxygen carrier needed Normothermic Oxygenated Machine Perfusion The donor organ is metabolically active Enables ex situ assessment of organ function Potential: Therapeutic interventions and longer preservation time Potentially riskier? Expensive Oxygen carrier needed 0-12°C 35-37°C

Type and Timing of Machine Perfusion Procurement Pre-transport During Transport Pre-implantation Cold flush out Cold storage Cold transport Cold Hypothermic (oxygenated) MP Controlled oxygenated rewarming Normothermic MP Normothermic regional perfusion

Kidney and Lung Machine Perfusion Kidney Machine Perfusion

Liver Machine Perfusion Devices Hypothermic No active oxygenation Hypo- or normothermic (10 – 37 oC) Controlled rewarming Oxygenation Normothermic Normothermic

Restoration of energy content Hypothermic (Oxygenated) Machine Perfusion Procurement Pre-transport During Transport Pre-implantation Cold flush out Cold storage Cold transport Cold Normothermic Normothermicregional perfusion Hypothermic Oxygenated MP Cheap, simple, safe Restoration of energy content Reducing of I/R injury 12

First Human Study of End-ischemic Machine Perfusion Am J Transplant 2010 Non-oxygenated Cold Perfusion After Static Cold Storage and Transportation

Two Types of Normothermic Machine Perfusion Procurement Pre-transport During Transport Pre-implantation Cold flush out Normothermic MP Cold storage Cold transport 14

End-ischemic Normothermic Machine Perfusion Am J Transplant 2013 The first reported study on normothermic perfusion of human livers Proof of technical feasibility of NMP 6 hours of normothermic, oxygenated perfusion Perfusion fluid: RBC, FFP, nutrients, vitamins, trace elements, antibiotics Allows functional assessment of the liver before transplantation Potential: Therapeutic intervention and (re)conditioning

Normothermic Liver Machine Perfusion Using Two Different Oxygen Carriers Human Red Blood Cells HBOC-201 (Hemopure®)

HBOC-201 (Hemopure®) Bovine-derived cross linked free hemoglobin Smaller molecule, less viscous Shelf life of 3 years at room temperature No pro-inflammatory stimuli or infectious agents Compatible with all blood types Half-life of 19 hours Can be used at various temperatures First used for ex vivo organ machine perfusion by Paulo Fontes in Pittsburgh (Am J Transplant 2015) SA?

Comparison of RBC and Hemopure for Normothermic Liver Machine Perfusion Human donor livers declined for transplantation (n=24) During transportation to transplant center: static cold storage Normothermic machine perfusion (NMP) using pressure-controlled arterial and portal perfusion Study groups: 6 hr NMP with RBC + FFP (n=12) 6 hr NMP with Hemopure + FFP (n=6) 6 hr NMP with Hemopure + Gelofusine (n=6) Osmolarity, colloid, contents corrected

Comparison of RBC and Hemopure for Normothermic Liver Machine Perfusion Portal Vein Flow Hepatic Artery Flow Matton, Porte, et al. unpublished results

Comparison of RBC and Hemopure for Normothermic Liver Machine Perfusion Lactate in perfusate Glucose in perfusate Matton, Porte, et al. unpublished results

Cumulative Bile Production Comparison of RBC and Hemopure for Normothermic Liver Machine Perfusion Cellular energy / ATP Cumulative Bile Production Conclusion: Excellent results with HBOC-201 (Hemopure) as oxygen carrier during ex situ normothermic machine perfusion of donor livers Matton, Porte, et al. unpublished results

Hemopure-based Liver Machine Perfusion Phase I Clinical Trial Hemopure-based Liver Machine Perfusion End-ischemic ex vivo reconditioning and viability assessment of initially declined, suboptimal donor livers 1 hr of oxygenated hypothermic perfusion 10 oC Resuscitation of mitochondria Restoration of cellular ATP 1 hr of controlled oxygenated rewarming From 10 to 37 oC 2-6 hr of normothermic machine perfusion Viability assessment Transplantation if a liver meets viability criteria www.trialregister.nl (NTR5972)

Summary - I Machine preservation of donor organs is rapidly developing and entering the clinical arena Clinical experience with donor organ machine perfusion Hypothermic (oxygenated) machine perfusion Controlled oxygenated rewarming Normothermic machine perfusion (NMP) Oxygen carriers used during NMP Red blood cells Hemoglobin derived oxygen carriers (HBOC)

Summary – II Oxygen Carrier for Machine Perfusion RBC Most physiologic Human Buffering capacity Can only be used in the normothermic range Shortage of human RBC ABO matching HBOC Soluble Hb Bovine Limited buffering capacity Can be used a various temperatures Readily available No ABO cross matching

The Groningen Organ Preservation & Resuscitation Unit Central facility for: Back table preparation Simultaneous machine perfusion of: Lungs, liver, two kidneys