pathogen inactivation of cellular components.pptx

Chemical inactivation
Photo-inactivation
Solvent-detergent Plasma

Photosensitizers

Methylene Blue light treatment
Psoralen Ultraviolet Light Treatment
Riboflavin Light Treatment
INTERCEPT System
Mirasol system
Platelet and plasma Pathogen Inactivation
FRALE and azridine compounds

1. Pathogen inactivation technologies for cellular components Presented by- Dr. Shiny Moderator- Dr. Anshul Gupta

2. Introduction Two interchangeable terms, pathogen inactivation (PI) and pathogen reduction (PR) have been used for a proactive approach in reducing contaminating pathogens in blood and blood components. But by definition, PI refers to the complete prevention of infectivity by a pathogen, whereas PR refers to decreasing the amount of an infectious pathogen

3. Properties of ideal PI agents a. Should inactivate or remove all types of infectious agents. b. Should not add neoantigens in the cellular blood components. c. Should have no effect on the function or life span of a blood component. d. Should not have any residual toxic effects. e. Should not involve risk greater than any TTI risk to the patient.

4. T wo basic mechanisms of PI methods C hemical inactivation Photo-inactivation The chemical inactivation- based on the addition of heterocyclic chemical compounds, which interact with nucleic acid (both DNA and RNA). P hotometric excitation with UV or visible light- leads to an excited electronic state which reacts directly with the nucleic acid (photochemical reaction) or secondary activation to form free oxygen radicals or reactive oxygen in a photodynamic reaction (e.g., methylene blue).

5. Plasma Pathogen Inactivation Solvent-detergent Plasma Photosensitizers Methylene Blue light treatment Psoralen Ultraviolet Light Treatment Riboflavin Light Treatment

6. 1. Solvent/Detergent Treatment SD treatment- most extensively used and best validated PI technology 50 to 60 million doses of SD-treated pooled fractionated plasma proteins have been given without the reported transmission of enveloped viruses. However, the method does not inactivate nonenveloped viruses , and nano-filtration or heat treatment has been introduced as a second PI step for many fractionated plasma proteins. SD treatment was first licensed for clotting factors in 1985 and has been in use for the treatment of plasma since 1991.

7. SD Treatment- Method M ost frequently used : Combination of 1% tri-(N-butyl)-phosphate (TNBP) and 1% polyoxyethylene -p-t- octylphenol (Triton X-100) for 4 hours at 30 to 31ºC. TNBP : acts as an organic solvent and removes lipids from the membranes; it is used alone in some protocols for PI. Triton X-100 : a non-ionic detergent that disrupts lipid bilayers for easier extraction of lipids; it also stabilizes TNBP. Inactivation is below virus detection levels (6 log) within 15 minutes SD plasma is prepared from a pool of plasma from many donors (630-1520) that is additionally tested for non-enveloped viruses parvovirus B19 DNA and hepatitis E virus.

8. Advantages of SD Treatment A major advantage of SD treatment is that the inactivation reagents in general do not affect proteins and protein activity in plasma. Pooling also dilutes and in some instances neutralizes antibodies and allergens , and allows the production of ABO-independent universal Plasma Particularly important is that there have been no reports of transfusion-related acute lung injury ( TRALI ) after transfusion of over 5 million units of SDPP in Europe Hemovigilance data indicate that febrile, allergic, or anaphylactic reactions are reduced by 70% to 80% with SDPP

9. An improved version of SDPP is ABO-universal plasma that eliminates the need for ABO group-specific plasma. It is based on the principle that anti-A and anti-B can be neutralized by soluble A and B antigens in plasma. Since 1996, a lyophilized ABO-universal SDPP, Bioplasma FDP (National Bioproducts Institute, Pinetown, South Africa), has been prepared from low titered plasma pools in South Africa

10. 2. Photosensitizers 2. a. Methylene Blue Light Treatment Methylene blue (MB) : a positively charged phenothiazine dye with high affinity for negatively charged compounds, such as guanine, proteins, and some lipids . MB-treated plasma is the second most frequently used pathogen-inactivated FFP M ore than 4 million units have been used clinically in European countries .

11. Advantage- MB light treatment is effective against enveloped and some non enveloped viruses. Disadvantage- MB does not effectively permeate plasma membranes; thus intracellular pathogens and leukocytes may not be fully inactivated. Therefore, the original protocol developed by Institute Springe introduced a freeze-thaw step to destroy residual leukocytes.

12. METHOD Methylene blue (MB) is added to thawed FFP, followed by its activation using visible light. MB intercalates with nucleic acids or binds to lipids, and following activation results in nucleic acid strand breakage or lipid peroxidation, with subsequent modification of surrounding membrane proteins. Plasma is refrozen after the removal of MB-using the filter. MB treated plasma contains 15-20% less factor VIII and fibrinogen than untreated plasma.

13. 2.b. Psoralen Ultraviolet Light Treatment Furocoumarins, which include psoralens, are active compounds isolated from plants- known as photosensitizers since ancient times. Amotosalen hydrochloric acid (also known as S-59 ) : is a synthetic psoralen specially selected by Cerus (Concord, CA) for PI of blood components because it crosses plasma membranes efficiently and demonstrates excellent protection against pathogens

14. T his treatment is called INTERCEPT System – Amotosalen and UV light are passed through the component which irreversibly damages the nucleic acids of various viruses, bacteria and protozoans. Fibrinogen, Factor V, Factor VII, Factor VIII, and Factor X are reduced by 17% to 30%, while coagulation factor inhibitor activity is less a ffected The INTERCEPT Blood System device developed by Cerus uses S-59, UVA illumination, and a Compound adsorption device (CAD) for the removal of residual S-59 and metabolites

15. 2.c. Riboflavin Light Treatment In spite of phototoxic targeting of nucleic acids, vitamin B2 (riboflavin) is generally recognized as safe by the US FDA. Navigant Biotechnologies (Lakewood, CO) has developed the Mirasol system for riboflavin treatment of blood components. Mirasol has been adapted for PI of platelets and plasma, while treatment of red cells and whole blood still is under development. Advantage- Extensive PI of selected viruses (including parvovirus), bacteria, and protozoa in addition to leukocyte inactivation has been reported in platelet concentrates

16. Platelet Pathogen Inactivation US FDA has recently cleared Amotosalen and UVA light for pathogen inactivation of apheresis platelets suspended in platelet additive solution. Psoralen UV Light Treatment Riboflavin-Treated Platelets Thionine Light Treatment

17. 1. Psoralen UV Light Treatment The INTERCEPT Blood Systems device for PI of platelets is similar to the one for plasma INTERCEPT treatment also inactivates mitochondrial DNA and inhibits cytokine synthesis, while platelet metabolic functions seem retained. T he system has been used for the production of more than 50,000 doses of PI platelets for routine clinical use in 14 European countries, and is reported to have replaced bacterial testing, gamma irradiation, and the use of cytomegalovirus-negative platelets.

18. 2. Riboflavin-Treated Platelets similar to the one for plasma. It is less complex than the INTERCEPT system in that removal of reagents is not performed After a Phase III study was completed in Europe in 2007, the Mirasol system for platelet PI was granted CE marking for application under GMP conditions in blood centers.

19. 3. Thionine Light Treatment The phenothiazine dye thionine and UV light combined with strong agitation is effective in the PI of platelets, and development of a protocol is being pursued by Blood Centers of the German Red Cross and MacoPharma . However, strong agitation could pose problems with irreversible activation of platelets.

20. Red Cell Pathogen Inactivation Alkylating agents Frale compounds Aziridine compounds Photosensitizers Riboflavin light treatment

21. 1. Alkylating Agents 1.a. FRALE Compounds Frangible anchor linker effector (FRALE) compounds. Common- S-303 specifically inactivate DNA/RNA via covalent adducts to the nucleic acid METHOD- Treatment is performed at room temperature for 12 hours followed by incubation with a CAD in the final storage container for 8 hours in order to remove residual S-303/ S-300 After satisfactory results with in-vitro studies and genotoxicity tests, successful Phase I and II trials were performed, followed by Phas e III trials in cardiac surgery and sickle cell/thalassemia patients.

22. 1.b. Aziridine Compounds- Common- PEN 110 Compounds such as ethylenimine, have been used to inactivate vaccines for the past 30 years. The method inactivates both enveloped and nonenveloped viruses , including members of the Parvoviridae family, which is often difficult to accomplish. METHOD- The treatment is performed at room temperature for 6 to 24 hours, followed by washing with unbuffered saline to levels below the limit of detection of PEN110

23. 2. Photosensitizers PI of red cells with photosensitizers poses problems because of light absorption by hemoglobin and the high number of red cells, which requires complex technical adjustments to secure uniform distribution of light for the treatment of RBC units B ecause of hemolysis following PI of red cells with these aromatic dyes, the methods was abandoned. A new approach was opened by the discovery of flexible dyes that can act as photosensitizers when rigidly bound to substrate but do not generate reactive oxygen species when in solution

24. A protocol for PI of red cells is designed with TP and DP- Thiopyrylium (TP) is a flexible photosensitizer that intercalates with nucleic acids and also binds to red cells. Dipyramidole (DP) ( vasodilatator , antioxidant, red cell band 3 ligand) acts as a competitive inhibitor of TP binding to red cells This method inactivated model viruses including intracellular HIV 6 log, and six bacterial species 5 log reduction Hemolysis increases within acceptable levels and most, but not all, red cell properties are retained after 42 days of storage

25. 3. Riboflavin Light Treatment Pathogen activation of microorganisms and leukocytes is similar to Mirasol treatment of platelets and plasma, while hemolysis and in-vitro red cell properties are within acceptable limits The Mirasol system has now also been adapted for PI of whole blood, which afterwards can be separated into red cells, platelets, and plasma

26. PRION ELIMINATION None of the previously mentioned PI technologies eliminate prions. However, a significant reduction in infectivity occurs during plasma fractionation, resulting in relatively low risk for prion transmission with fractionated plasma proteins Several companies are developing prion reduction filters for red cell components and interesting studies are in progress with ligand chromatography, which may reduce prion infectivity of plasma significantly

27. Advantages of PI This technique has shown great efficacy in reducing the infectivity of a number of pathogens harmful to oncology patients including CMV, parvovirus B19 , and T. cruzi . Prevents major transfusion and hemolytic reactions like TRALI Prevention of transfusion associated Chickengunya , West Nile Virus, Lymphocytic Choriomeningitis Virus Prevention of protozoan infections like leishmaniasis, babesiosis and toxoplasmosis

28. Toxic effects of PI agents INTERCEPT treatment of plasma decreases fibrinogen, Factor V and Factor VIII activity by approximately 17% to 30% and thrombin generation capacity is impaired in plasma. • In vitro testing of INTERCEPT treated platelets showed increased CD61 microparticle formation, higher metabolic rate, accelerated metabolic changes and reduced agonist induced aggregation responses, but no neo-antigenicity was observed. • Riboflavin treated platelets also leads to some platelet inactivation as expressed by increased metabolic activity and P-selectin expression.

29. SUMMARY CE Mark already obtained and under wide use in hospitals- Solvent detergent treatment of FFP- most extensively used MB treatment of FFP P soralen light treatment of platelets as well as FFP Riboflavin light treatment of whole blood, which subsequently could be separated into plasma, platelets, and red cells. Under trial- Alkylating agents treatment of red cells R iboflavin light treatment of FFP

30. References DGHS. Tranfusion Medicine Technical Manual 3 rd edition Rossi’s Principles of Transfusion Medicine, Edition 4 AABB Technical manual 18 th edition THANKYOU