Update on THERAFLEX UV-Platelets: a novel system for pathogen reduction of platelets

Transcription

1 Update on THERAFLEX UV-Platelets: a novel system for pathogen reduction of platelets Axel Seltsam, MD, MHBA German Red Cross Blood Service NSTOB, Springe Fribourg 2011

2 THERAFLEX UV-Platelets Efficient UVC penetration through mixing and a thin layer of platelets Formation of 6-4 photoproducts UVC UVC UVC Platelet concentrate UVC Formation of cyclobutane pyrimidine dimers (Thymine dimers) DNA Optimal UVC exposure UVC-induced before iradiation under agitation DNA damage

3 Procedure

4 Virus inactivation Virus Model for THERAFLEX UV-Platelets HIV-1 1 West Nile virus (WNV) Sindbis virus HCV 5.3 Encephalomyocarditis virus (EMCV) HAV 4.0 Porcine parvovirus (PPV) Parvovirus B Vesicular stomatitis virus (VSV) 6.3 Suid herpes virus (SHV-1) CMV 2.8 Hepatitis C (HCV) 5.0 Influenza, H3N2 5.3 Hepatitis A (HAV) 4.1

5 Species Bacteria inactivation (Spiking) Strain Aerobic/ anaerobic Gram stain Spore former Reduction factor (log 10 ) Bacillus cereus PEI-B aerobic ± 0.81 Clostridium perfringens PEI-B anaerobic Escherichia coli PEI-B-19* fac. anaerobic Enterobacter cloacae DSMZ fac. anaerobic Klebsiella pneumoniae PEI-B-08* fac. anaerobic ± 0.28 Pseudomonas aeruginosa ATCC aerobic Propionibacterium acnes ATCC 6919 anaerobic ± 1.13 Serratia marcescens ATCC fac. anaerobic Staphylococcus aureus PEI-B A aerobic ATCC aerobic 4.93 Staphylococcus epidermidis PEI-B-06* aerobic ± 0.51 Pseudomonas fluorescens ATCC aerobic Listeria monocytogenes ATCC fac. anaerobic Streptococcus pyogenes ATCC BAA-1064 fac. anaerobic Streptococcus pyogenes Isolat aus TK fac. anaerobic ± 0.26 Acinetobacter baumannii ATCC aerobic Fac.= facultative, WHO Repository Transfusion-Relevant Bacteria Reference Strains (WHO RTRBRS)

6 Bacteria inactivation (Sterility) Species 0.83 x standard dose BacT/ALERT results after 7 days* Standard dose 1.33 x standard dose Bacillus cereus 10 sterile, 2 unsterile 12 sterile 11 sterile, 1 unsterile Enterobacter cloacae 12 sterile 12 sterile - Escherichia coli 12 sterile 12 sterile 12 sterile Klebsiella pneumoniae 12 sterile 12 sterile 12 sterile Propionibacterium acnes 12 sterile 12 sterile 12 sterile Pseudomonas aeruginosa 12 sterile 12 sterile - Staphylococcus aureus 10 sterile, 2 unsterile 12 sterile 12 sterile Staphylococcus epidermidis 12 sterile 12 sterile 12 sterile *Spiking concentration 100 CFU/ml Aerobic as well as anaerobic culture bottles were used Mohr et al., Transfusion 2009

7 Parasite inactivation Parasites tested Disease Log 10 reduction Trypanosoma cruzi Chagas` disease >2.8 - >4.2 Leishmania infantum leishmaniosis >1.4 * Plasmodium falciparum malaria 4-5 Babesia divergens babesiosis >5 * due to assay limitations only a small dynamic range was observed quantitative PCR assay Castro et al., Transfusion 2014

8 Inactivation of leukocytes PBMC spike Spiked Pool PC (1 x 10 6 PBMCs /ml PC) 350 ml 350 ml 350 ml γ-irradiation untreated UVC-irradiation PBMC removal γ 30 Gy 0 J/cm J/cm J/cm 2 PBMC: peripheral blood mononuclear cells In vitro assays n=5

9 Inhibition of T cells - Limiting Dilution Assay - T cell Frequency Gamma Log 10 reduction of viable T cells* UVC Donor f control f 30Gy f 0.1 UVC f 0.2 UVC 30 Gy 0.1 J/cm J/cm 2 1 1/171 < 1/1.0 x 10 7 < 1/1.0 x 10 7 < 1/1.0 x 10 7 > 4.8 > 4.8 > /202 1/5.2 x /5.3 x 10 5 <1/1.0 x > /81 < 1/2.1 x 10 7 < 1/2.1 x 10 7 < 1/2.1 x 10 7 > 5.4 > 5.4 > /149 < 1/2.1 x /3.7 x 10 5 < 1/2.1 x 10 7 > > / x /3.6 x 10 6 < 1/1.1 x > 5.1 Mean 1/138 1/1.1x10 7 1/8.1 x 10 6 < 1/1.5 x > 5.0 SD ±1/ /1.0 x /8.0 x /5.9 x * the log 10 reduction is calculated as log 10 (f control / f treated )

10 Prevention of GVHD in a xenogeneic model Treatment group No. of deaths Survial days Human T- cells dectable Histopatholgical signs of GvHD untreated (N=12) Gamma (N=12) UVC (N=12) 12/12 < 30 12/12 12/12 0/12 61* 2/12 0/12 0/12 61* 0/12 0/12 *observation period Injection dose: 2 x 10 7 PBMCs/mouse i.p.

11 Parameter In vitro platelet quality Days after UVC irradiation Day 2 Day 5 Day 7 control UVC control UVC control UVC PLT concentration [x 10 9 /ml] 1.19 ± ± ± ± ± ± 0.05 ph 7.17 ± ± ± ± ± ± 0.04 Glucose [mmol/l] 5.9 ± ± ± ± ± ± 0.8 Lactate [mmol/l] 8.5 ± ± ± ± ± ± 1.3 Hypotonic shock response [%] 79 ± 3 68 ± 8 62 ± 0 56 ± 7 57 ± 3 51 ± 1 Collagen-induced aggregation [%] 10 µg/ml collagen Collagen-induced aggregation [%] 2 µg/ml collagen 93 ± 4 93 ± 3 88 ± 4 89 ± 6 68 ± ± 9 33 ± ± 7 23 ± ± 12 7 ± 2 16 ± 7 CD62 [%] 44 ± ± 3 38 ± ± 2 47 ± 4 53 ± 4 Annexin V [%] 4 ± 1 3 ± 1 6 ± 2 6 ± 2 7 ± 2 9 ± 2 PAC-1 binding [Geo mean] 571 ± ± ± ± ± ± 220 significant at p<0.05 (paired t-test) Seltsam & Müller, Transfus Med Haemother 2011

12 Survival and recovery apheresis platelets McMaster University, Hamilton, Canada Split design Stored for 3 days N=21 Recovery (%) Survival (d) Recovery Survival Mean ± SD Mean ± SD UVC/Control (%) UVC/Control (%) UVC 40.1 ± ± 1.2 Control 50.6 ± ±

13 Survival and recovery buffy coat platelets NHSBT, Cambridge, U.K. 2 study arms BEST protocol 5 days storage N=6 per group Recovery (%) Recovery Survival (d) Survival (d) Fresh Stored % stored vs. fresh Fresh Stored % stored vs. fresh Control group UVC group % reduction treated vs. control N/A Bashir et al., Transfusion 2012

14 Tolerability (preclinical study) No addition and/or removal of any photoactive reagent photoreagent-related adverse events excluded Animal model (Beagle dog): No acute or subchronic toxicity No evidence for immunogenicity Pohler et al., Transfusion 2012

15 Clinical phase I study Monocentric 11 healthy volunteers UVC-treated apheresis platelet concentrates Primary endpoint: safety and tolerance Secundary endpoints: CCI, UVC-specific platelet antibodies

16 1.Series 2 days 2 Weeks Study plan 2.Series 2 days 2 Weeks 3.Series Apheresis Sequential Retransfusion 2 days Apheresis Complete Retransfusion Double Apheresis Double Retransfusion

17 Results phase I study No transfusion-related adverse events No UVC-specific platelet antibodies Good CCIs

18 Summary-THERAFLEX UV-Platelets Simple and fast process A one-step inactivation process (<60 sec) Complete treatment of platelet concentrates < 10 min Improved inventory and logistics Minimal platelet loss Only two simple transfer steps No platelet loss due to compound removal Platelet viability and efficacy demonstrated In vitro parameter up to 7 days Good recovery and survival

19 Summary-THERAFLEX UV-Platelets Inactivation / reduction of pathogens Highly effective against bacteria responsible for critical transfusion-related infections Effective against a broad range of viruses and parasites Alternative to gamma irradiation Effectively inactivates white blood cells Prevention of GvHD (demonstrated by xenogeneic mouse model) Safety and tolerability No chemical compound is added No evidence of acute or subchronic toxicity nor immunogenicity Low plasma content reducing adverse reactions