Hepatic Inflammation and Cellular Therapy

Transcription

1 Hepatic Inflammation and Cellular Therapy Lee K. Landeen, Jessica Van Allen, Patricia W. Bedard Vital Therapies, Inc., San Diego, CA, USA

2 Alcoholic Hepatitis: Role of Inflammation Inflammatory Mediators (PAMPs, DAMPs) Leaky Gut Activation Cytokine Storm Immune Dysfunction Infections Secondary Organ Failure 2

3 Anti-inflammation Therapies for Alcoholic Hepatitis Anti-tumor necrosis factor alpha (TNF ) (infliximab) Double-blind randomized controlled trial (prednisolone infliximab) Naveau S, et al Hepatology Did not demonstrate clinical benefit (higher infection rates, death rates, lower ex vivo stimulation capacity of neutrophils in treatment group) Thalidomide Pilot study to reduce TNF N , DH Adams (Queen Elizabeth Hospital, Birmingham, UK), Study abandoned Steroids (prednisolone, pentaxifylline) STOPAH Thursz MR, 2015 N Eng J Med Did not demonstrate medium- to long-term clinical benefit IL-1ra (anakinra) NCT , anakinra + pentoxifylline + zinc sulfate vs. methylprednisolone On-going 3

4 The Case for Cell-based Liver Therapies/Treatments Porcine Hepatocytes HepatAssist (Circe Biomedical >Arbios Systems) SRBAL (Mayo Clinic) Human Hepatoblastoma-derived C3A Cell Line ELAD (Vitagen>Hepatix> Vital Therapies) Allogeneic Human Hepatocytes Human Hepatocellular Carcinomaderived HepaRG Cell Line MELS (Charite Virchow Clinic) (BALANCE, Netherlands) Cells are capable of producing multiple factors Cells can respond dynamically over time Cells can potentially respond to individual patients 4

5 The ELAD System VTL C3A Cells 5

6 Mechanism of Action: Soluble Factors Previous emphasis was on factors that ELAD C3A cells could remove: Bilirubin, ammonia, toxins, etc. Current focus is on what ELAD C3A cells can contribute: Anti-inflammation, regeneration, coagulation, transport, anti-oxidation, etc. 6

7 VTL C3A Cells Secrete Many Potentially Useful Proteins 148 proteins identified to date via immunoassay; 62 proteins via LC/MS 39 proteins secreted above 1 mg/day/cartridge 15 proteins secreted 10x above normal serum levels Inflammation: Alpha-1-Antitrypsin Complement C3 Ferritin Gelsolin Haptoglobin Intercellular Adhesion Molecule 1 Interleukin-1 Receptor Antagonist Interleukin-8 Tumor Necrosis Factor Alpha Angiogenesis: Angiopoietin-2 Placental Growth Factor Vascular Endothelial Growth Factor Vascular Endothelial Growth Factor-C Regeneration: Amphiregulin Growth/differentiation factor 15 Heat-Shock protein 70 Heparin-Binding EGF-like Growth Factor Hepatocyte Growth Factor Platelet-Derived Growth Factor-BB Tissue Inhibitor of Metalloproteinases 1 Tissue Inhibitor of Metalloproteinases 2 Tissue Inhibitor of Metalloproteinases 3 Transforming Growth Factor Alpha Hematopoiesis: Erythropoietin Transport: Albumin Alpha-Fetoprotein Apolipoprotein A-I Apoliproprotein A-II Apolipoprotein A-IV Apolipoprotein B Apoliproprotein C-I Apolipoprotein C-II Apoliproprotein C-III Apolipoprotein E Apoliproprotein H Fatty Acid-Binding Protein, liver Serotransferrin Oxidative Stress: Peroxiredoxin-4 Fatty Acid-Binding Protein, liver *Proteins may have multiple roles depending on concentration and targeted cell type 7

8 Working Model of ELAD Therapeutic Effect Recovery/ Regeneration Stabilization Immune Function Improved Bile Flow Reduced inflammation Metabolic Support Regeneration Reduced Apoptosis ELAD Treatment C3A Cell Proteins Multi-organ failure: Alcohol Consumption Leaky Gut Toxic Injury Alcoholic Hepatitis (Severe Inflammation/ Dysfunction) Cholestasis Immune Dysfunction Apoptosis/Necrosis Liver Failure Kidney Failure Coagulopathy Respiratory Failure Death Immune/Sepsis 8

9 Are ELAD C3A Cells Capable of an Acute-Phase Response? Inflammatory Factors: IL-1, IL-6, TNF, LPS? ELAD C3A Cell Cartridge 9

10 VTL C3A Experimental Model VTL C3A Cells IL-1β, IL-6, &/or TNFα or LPS Supernatants Collected 24, 48, 54h? Protein Secretion (immunoassay) 10

11 A l b u m i n ( g / m L ) Fibrinogen (ng/ml) VTL C3A Cells Exhibit an Acute-Phase Response IL-1β + IL-6 C3A 24 hr Decreased Albumin Increased Fibrinogen p = C o n t r o l I L - 1 x I L

12 I L - 1 R a ( n g / m L ) A A T ( n g / m L ) VTL C3A Cells Produce Anti-Inflammatory Proteins IL-1β + IL-6 C3A 24 hr Increased IL-1Ra No Change AAT 3 0 IL-1Ra AAT 2 0 p = p = C o n t r o l I L I L C o n t r o l I L I L

13 IL -1 R a (p g / m L ) A A T (n g / m L ) VTL C3A Cells Produce Anti-Inflammatory Proteins LPS C3A 24 hr Increased IL-1Ra Increased AAT L P S (E U / m L ) L P S (E U / m L ) 13

14 FITC Will Healthy Neutrophils Respond to ELAD-Treated AH Plasma? Healthy peripheral blood neutrophils Exposed to plasma from Normal individuals or ELAD-Treated subjects D0=before treatment 24 h after treatment initiated EOT=end of treatment 30 d after treatment initiated Measure fluorescent signal of neutrophils actively phagocytizing FITC-labeled E. coli Phagocytosis Normal T0 D0 24h T1 EOT T2 30d T3 Plasma Increasing phagocytic capacity with Days on ELAD Treatment suggests restored function R Jalan, RP Mookerjee, NA Davies (University College London) 14

15 Will Macrophages Respond to ELAD C3A Factors? PMA 24-hrs Time (Days) Rest 5-Days Polarize 24-hrs +/- ELAD C3A Cell Conditioned Medium IFN-γ M1 7 THP-1 monocytic cells Adherent macrophages + LPS Pro-Inflammatory Phenotype 8 Collect Supernatants 24-well plate 1x10 6 cells/well Cytokine Immunoassays 15

16 I L - 1 ( p g / m L ) M1 Macrophages Reduce Pro-Inflammatory IL-1 When Treated with ELAD Conditioned I LMedium *** M 1 M 1 + C M 1 M 1 + C M *** <0.001, Student s T-Test, n=6 biological replicates tested in duplicate

17 Pro-Inflammatory Cytokines Decrease During ELAD Treatment of AH Subjects 200 TNF TNFa 1000 IL pg/ml 100 pg/ml Day 0 Day 1 Day 0 Day 1 day 0 day 1 0 Day 0 Day IL-6 40 IL-1 IL1b pg/ml pg/ml Day 0 Day 1 Day 0 Day 1 Day 0 Day 1 0 Day 0 Day 1 R Jalan, RP Mookerjee, NA Davies (University College London)

18 VTL C3A Cells Produce IL-8 In Response to IL-1 But not in response to IL-6 or LPS IL-8 is elevated in patients with chronic alcoholic liver disease Swiatkowska-Stodulska 2006 Med Sci Monit IL-8 can rescue human hepatocytes from TNF -induced apoptosis, and induce DNA synthesis Osawa 2002 Infect Immun LPS 18

19 Is There Evidence of an Anti-Inflammatory Response in ELAD-Treated AH Subjects? Case Study Plasma collected prior to, during, and after ELAD Treatment Immunoassayed for IL-1Ra 19

20 Concentration (ng/ml) IL-1Ra Levels Increased During ELAD Treatment of AH Subject IL-1Ra Study Day 20

21 Translational Biomarkers for Survey of VTI-208 Samples Inflammation: IL-1Ra - anti-inflammatory protein expressed by VTL C3A cells AAT - anti-inflammatory protein expressed by VTL C3A cells IL-1β - Inflammatory protein in AH IL-6 - Inflammatory protein in AH TNFα - Inflammatory protein in AH C-reactive protein - increased in AH with Systemic Inflammatory Response Syndrome (SIRS) Pro-calcitonin marker of SIRS Anti-Apoptosis: Cytokeratin 18 (CK18) - a marker of caspase-mediated apoptosis Amphiregulin - EGFR ligand most highly expressed by VTL C3A cells Soluble Fas - blocks Fas-ligand, expressed by VTL C3A cells VEGF- reduces HAEC apoptosis, expressed by VTL C3A cells

22 Conclusions Single-drug therapies have not proved successful in treating AH ELAD cell-based treatment may provide improved clinical benefit by reducing inflammation, among other mechanisms of action Recovery/ Regeneration Stabilization Immune Function Improved Bile Flow Reduced inflammation Metabolic Support Regeneration Reduced Apoptosis ELAD Treatment C3A Cell Proteins Multi-organ failure: Kidney Failure Alcohol Consumption Leaky Gut Toxic Injury Alcoholic Hepatitis (Severe Inflammation/ Dysfunction) Cholestasis Immune Dysfunction Apoptosis/Necrosis Liver Failure Coagulopathy Respiratory Failure Death Immune/Sepsis 22

23 Acknowledgements Neutrophil studies were completed by the Liver Failure Group, University College London Institute for Liver and Digestive Health, Royal Free Hospital (London, UK) Rajiv Jalan, MD Rajeshwar P Mookerjee, MBBS, PhD Nathan A Davies, PhD VTL Scientific Advisory Board Cliff Steer, M.D. (University of Minnesota) Charles Dinarello, M.D. (University Colorado Denver) George Michalopoulos, M.D. (University of Pittsburgh School of Medicine) Alan Hofmann, M.D. (emeritus) (University of California, San Diego ) Fernando Camargo, Ph.D. (Harvard and Boston Children s Hospital) Nikolaos Pyrsopoulos, M.D., M.B.A. (Rutgers New Jersey Medical School) Mike Millis, M.D. (University of Chicago Medicine) 23

24 Please Visit our Poster for More Information/Discussion Hepatic Inflammation and Cellular Therapy Lee K. Landeen, PhD, Jessica Van Allen, BS, and Patricia W. Bedard, PhD 24