1. 病毒灭活血小板临床应用进展 邱艳 北京血液中心 2013 年 9 月 5 日淮安

2. Most transfusion recipients are battling serious disease and have weakened immune systems Screening limitations Gaps in current defenses exist, due to the window period and limited screening sensitivity Known pathogens Routine testing covers only a limited number Bacteria The most frequent transfusion-transmitted infection Leukocytes Residual cells and cytokines can cause harmful post- transfusion reactions New and emerging pathogens A risk that current safety measures cannot eliminate 为什么病毒灭活成分血？

3. Red – newly emerging diseases: Blue – re-emerging/resurging diseases Adapted from Morens DM. Nature. 2004;430:242–9. Emerging Infections Disease: continue to emerge worldwide

4. Timeline of the WNV epidemic in the USA 1999200020012002200320042005 First identification in humans, horses and birds in three states 66 human cases reported in 10 states 9,862 human cases, WNV Ab test approved First documented transmission by organ transplant and blood transfusion WNV activity in 47 continental states, 2,470 human cases WNV NAT implemented under IND; transmission confirmed or suspected in >40 blood recipients 199 WNV-(+) donors in 28 states PUBLIC HEALTH: WNV was recognized as a threat in 1999 BLOOD SAFETY: Experimental tests for WNV were not implemented until June 2003

5. 1. Hubálek Z, et al. Emerg Infect Dis. 1999;5:643–50. 2. Dauphin G, et al. Comp Immunol Microbiol Infect Dis. 2004;27:343–55. West Nile virus in Europe  Recent outbreaks of WNV in Europe reaffirmed that mosquito-borne viral diseases may occur on a mass scale, even in temperate climates 1  Frequent detection of equine, avian and human WNV in south France (2000 and 2003) suggests that some areas may be at increased risk of infection on a seasonal, rather than sporadic, basis 2 WNV isolation from mosquitoes or vertebrates, including humans Laboratory-confirmed human or equine cases of West Nile fever Presence of antibodies to WNV in vertebrates

6. Healthy blood donors can carry a range of asymptomatic herpesviridae infections Virus Potential symptoms in recipient* Prevalence Blood supply screening CMV Pneumonitis, retinitis, birth defects 50–85% by age 40 1 Tested as needed EBV Mononucleosis, pneumonitis, hepatitis, oncogenic potential ~95% by age 40 2 Not tested HHV-6 Pneumonitis, fever, encephalopathy, rash >90% in adults 3 Not tested HHV-7 Pneumonitis, fever, encephalopathy, rash >90% in adults 3 Not tested HHV-8Kaposi’s sarcomaVaries by geography**Not tested *Severe symptoms most likely in immunocompromised individuals. **Moderate prevalence in Mediterranean countries, low in rest of Europe. 1. http://www.cdc.gov/ncidod/diseases/cmv.htm 2. http://www.cdc.gov/ncidod/diseases/ebv.htm 3. Clark DA. Int J Hematol. 2002;76:246–52.

7. Studies have shown CMV transmission by CMV-negative and leukoreduced blood products Study reference Number of patients CMV infection CMV disease Type of blood product used Bowden, et al. (1995) 252 250 2/4* 3/6* 0/0* 3/6* CMV-negative Leukoreduced Ljungman, et al. (2002) 33 49 36360 Combination Leukoreduced Nichols, et al. (2003) 360 447 6 18Not given CMV-negative or leukoreduced Apheresis platelets Ronghe, et al. (2002) 9300 CMV-negative RBC and leukoreduced platelets Foot, et al. (1998) 11010CMV-negative *Primary and secondary endpoints (infections day 21–100 or 0–100 after SCT) Adapted from Ljungman P. Br J Haematol. 2004;125:107–16.

8. Availability of safety measures against transfusion- transmitted infections Syphilis HBsAgAnti-HBc, Anti-HTLV-I/II Pathogen Inactivation 1960198019851990199520002005197019751965 Anti-HIV-1 Anti-HCV Anti-HIV-2 HIV p24 Leuko- depletion NAT Adapted from Barbara, J. Transfus Med Hemother. 2004;31(suppl 1):1–10.

9. 血液制品病毒灭活的历史 IV TherapeuticsProcessAvailability IV solutionsSterilization1950’s Plasma fractions (FVIII, FIX, albumin) SD(one or more stages) 1980’s Plasma/FFPSD, methylene blue1990’s

10. 血浆成分血病毒灭活的现状 Trade Name Active Compound and Method Developer Approval/ Phase Octaplas® （ SD ） solvant :Tri (n) butyl phosphate (TnBP) détergent :Octoxynol-9 or Triton X 100 or Tween 80 or Chlolate de sodium B. HOROWITZ (NYBC,1985 – 1990)VI Technologies (Vitex) licensed in the US but is withdrawn and 20130117 approved for Blood-Clotting Disorders in US ARC OctaPharma 1992 approved in France 2006 EU Mark Mirasol 核黄素 +285- 365nmUVA+UVB 照射 Navigant / Caridian-BCT CE mark in 2008 Theraflex© MB-plasma 1 mM Methylene blue(MB) +180 J/cm 2 可见光照 ( 590 nm) Springer ，德国； Grifols, 西班牙 Use in 20 countries MacoPharma 2008 approved in France. Oct. 2011 withdrawn in France Intercept ® Amotosalen HCl （ S-59)+ UVA light Baxter/Cerus 2010 EU Mark regulatory review in US

11. Trade Name Active Compound And Method DeveloperApproval/Phase INTERCEPT Blood System™ Amotosalen HCl （ S-59 ） +3 Joules /cm 2 UV A light (320-400 nm) Baxter/Cerus 2002 CE Mark classe 3 2005 Afssaps France 2007 PEI German 2010 wissmedicSwis Phase III in US Mirasol® PRT Riboflavin 核黄素 + 6.2 J/cm 2 UV /visible light (265-370 nm) Navigant / CaridianBCT Phase I/II/III Theraflex© UV 0.3 J/cm 2 monochromatic UV light(200-275 nm ) CaridianBCT/ MacoPharma R&D 血小板成分血病毒灭活的现状

12. Trade Name Active Compound and Method Developer Approval/ Phase FRALE S-303 (bifunctional alkylator) Baxter/Cerus Phase I/II/III in US stopped for neoantigen Phase I in EU Phase I in US INACTINE PEN 110 (Ethyleneimine) VI TechnologiesPhase III Riboflavin Riboflavin(Vitamin B2) + 6.2 J/cm 2 UV (265- 370 nm) Navigant/ Caridian-BCT FDA approved clinical trials in the US 红细胞成分血病毒灭活的现状

13. WAVE LENGTH (nm) INTERCEPT High energy Low energy UVC UVB UVA VISIBLE UVC Mirasol 3 J/cm 2 6.2 J/mL 0.4 J/cm 2 RadioX, G Amotosalen 200 250 300 350 400 590 750 血小板成分血病毒灭活的机理

14. The INTERCEPT Blood System Introduction & Mechanism

15. Nucleic acid targeting using amotosalen HCl  Psoralen targets DNA and RNA (single- and double-stranded)  Crosslinking reaction is initiated by UVA light (320-400nm)  Replication of nucleic acid of pathogens and leukocytes is stopped  Platelets, plasma and RBCs do not require nucleic acid function for therapeutic effect Amotosalen O NH 2 O O O

16. Double-stranded DNA or RNA Single-stranded DNA or RNA Helical Regions Amotosalen crosslinks both single- and double-stranded nucleic acids

17. Amotosalen mechanism of action Targeting Amotosalen (S-59) IntercalationCrosslinking UVA Illumination Helical region of single- or double-stranded DNA or RNA Multiple crosslinks block strand separation and replication

18. The INTERCEPT Blood System for platelets: A broad spectrum of inactivation  Bacteria »Aerobic and anaerobic species »Gram-positive and Gram-negative species »Spirochetes (including Treponema pallidum and Borrelia burgdorferi)  Viruses »Enveloped and non-enveloped species »Species with either single- or double-stranded DNA or RNA  Protozoa »Including Plasmodium spp. (malaria), Trypanosoma cruzi (Chagas’ disease), and Leishmania spp. (leishmaniasis)  Residual donor leukocytes »Prevents replication and inhibits cytokine synthesis

19. Limitations of the INTERCEPT Blood System  Not effective against HAV »Highly infrequent labile component pathogen with few reported cases  Not effective against bacterial spores »Effective against vegetative state  Not effective against prions »Unique disease mechanism »Very low frequency »Preventable by other measures: livestock management

20. The INTERCEPT Blood System for Platelets Virus

21. The INTERCEPT Blood System for platelets: Viral inactivation overview Enveloped ss RNAHIV-1, HIV-2, HCV, HTLV-I, HTLV-II, BVDV, WNV ss/ds DNAHBV, DHBV ds DNACMV Non-enveloped ds RNABluetongue virus ss RNACalicivirus ds DNASimian virus 15 ss DNAParvovirus B19  Small non-enveloped picorna viruses (e.g., poliovirus, HAV) are resistant to inactivation

22.  灭活水平计算公式 : Log (Pre-treatment infectivity / Post-treatment infectivity) 或 Log (Pre-treatment infectivity) – Log (Post-treatment infectivity)  处理后没有存活病毒, 用 <1 infectious unit/volume assayed 表示  “>” 表示灭活效果的最低检测限  Log reduction was reported based on the volume assayed Measurement of pathogen inactivation: Log reduction

23. Examples of log reduction calculations Pre- treatment infectivity (IU/ml) Post- treatment results Post- treatment infectivity (IU/ml) Log reduction calculation Log reduction 10 6 5 organisms in 1 ml 5log10 6 – log55.3 10 6 5 organisms in 10 ml 0.5log10 6 – log0.56.3 10 6 No organisms in 1 ml <1> (log10 6 – log1)>6 10 6 No organisms in 10 ml <0.1> (log10 6 – log0.1)>7

24. *Approved product claims under CE Mark. “>” refers to below limit of detection for assay. INTERCEPT Blood System for platelets: Inactivation of routinely tested pathogens PathogenLog reduction* Hepatitis B HBV (human, MS-2 strain) DHBV (model virus for HBV) >5.5 >6.2 Hepatitis C HCV (human, Hutchison strain) BVDV (model virus for HCV) >4.5 >6.0 HIV Cell-free HIV-1 Cell-associated HIV-1 >6.2 >6.1 HTLV-I HTLV-II 4.7 5.1 Treponema pallidum (syphilis)>6.8 to <7.0

25. INTERCEPT Blood System for platelets: Inactivation of additional enveloped viruses PathogenLog reduction Cytomegalovirus>5.9* SARS-CoV>6.2 1 Vaccinia virus>4.7 2 West Nile virus>5.5 2 “>” refers to below limit of detection for assay. *Approved product claims under CE mark. 1. Pinna D, et al. Transfus Med. 2005;15:269–76. 2. Lin L, et al. Transfusion. 2005;45:580–90.

26. INTERCEPT Blood System for platelets: Inactivation of non-enveloped viruses PathogenLog reduction* Bluetongue virus, type 116.1–6.4* Parvovirus B19 1 standard protocol 30 min incubation † 2.0 3.9 Feline conjunctivitis virus (calicivirus)1.7–2.4* Simian adenovirus 150.7–2.3* Porcine parvovirus0–0.2* *Approved product claims under CE mark. † This incubation period was supplementary to the standard operating protocol. 1. Sawyer L. Manuscript in preparation.

27. Implementation & Data Management

28. INTERCEPT data management system (IDMS) IDMS server HOST system RegistrationAdd amotosalenIlluminationCAD incubationStorage

29. Macopharma Platelets

30. 谢谢 !