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14. of the test population survives NOAEL no observable adverse effect level CHL Chinese hamster lunch fibroblast cell line Cmax maximum concentration in the bloodstream t 2 time to reduction in concentration by 50 agent for which testing was not readily available Since the appearance of WNV in the United States several cases of transfusion related WNV infections have occurred The low levels of WNV present in blood during a subject s subclinical viremia makes its detection difficult even with the most sensitive of the available nucleic acid testing technologies Pathogen reduction via a chemical or photo chemical process has also been used as a blood safety measure Methylene blue treated and sol vent detergent treated plasmas have been used as transfusion products such as fresh frozen plasma FFP and in the manufacture of fractionated and purified blood derived products such as factor VIII concentrate These treatments are effective against enveloped viruses and yield only slight reductions in protein quality Recently a technology using a psoralen compound and UV light has been developed for the reduction of pathogens as well as the inactivation of leukocytes in platelets and in plasma Similarly the Mirasol PRT System Gambro BCT Lakewood CO was developed to provide reduction of a wide variety of pathogens and inactivation of leukocytes This device uses UV light and riboflavin to reduce pathogens Ribofla
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19. Prowse CV Methylene blue treated fresh frozen plasma What is its contribution to blood safety Transfusion 43 1322 1339 2003 24 Corash L Lin L Novel processes for inactivation of leukocytes to prevent transfusion associated graft versus host disease Bone Marrow Transplant 33 1 7 2004 25 Lin L Dikeman R Molini B et al Photochemical treatment of platelet concentrates with amotosalen and long wavelength ultraviolet light inactivates a broad spectrum of pathogenic bacteria Transfusion 44 1496 1504 2004 26 Lin L Hanson CV Alter HJ et al Inactivation of viruses in platelet concentrates by photochemical treatment with amotosalen and long wavelength ultraviolet light Transfusion 45 580 590 2005 27 Unna K Greslin JG Studies on the toxicity and pharmacology of riboflavin J Pharmacol 76 75 80 1942 28 Hayashi M Kishi M Sofuni T et al Micronucleus tests in mice on 39 food additives and eight miscellaneous chemicals Food Chem Toxicol 26 487 500 1988 29 Kuhn R Boulanger P Uber die Giftigkeit der Flavine Hoppe Seylers Z Physiol Chem 241 233 238 1936 30 Studer A Zbinden G Uehlinger E Die pathologie der avitaminosen and hypervitaminosen in Buchner F Letterer E Roulet F eds Handbuch der Allgemeinen Pathologie Band II Berlin Springer Verlag 1962 pp 734 987 31 Williams Jr JR Grantham PH Yamamoto RS et al Effect of dietary riboflavin on azo dye reductase in liver and in bacteria of cecal contents of ra
20. Riboflavin based process retains functional activities of immumoglobulins Transfusion 43 Suppl 80A 2003 abstr 55 Perez Pujol S Tonda R Lozano M et al Effects of a new pathogen reduction technology Mirasol PRT on functional aspects of platelet concentrates Transfusion 45 911 919 2005 56 Goodrich RP Li J Pieters H et al Correlation of in vitro platelet quality measurements with in vivo platelet viability in human subjects Vox Sang 90 279 285 2006 57 AuBuchon JP Herschel L Roger J et al Efficacy of apheresis platelets treated with riboflavin and ultraviolet light for pathogen reduction Transfusion 45 1335 1341 2005 58 WHO Twenty fifth report of the Joint FAO WHO Expert Committee on Food Additives Evaluation of certain food additives Tech Rep Ser 669 1 48 59 EVM Expert Group on Vitamins and Minerals revised review of riboflavin 2002 1 48 60 Federation of American Societies for Experimental Biology Evaluation of the health aspects of riboflavin and riboflavin 5 phosphate as food ingredients SCOGS 114 Life Sciences Research Office Federation of American Societies for Experimental Biology Bethesda Maryland Prepared for US Food and Drug Administration Washington DC 1979 pp 1 25 61 Martindale W Sweetman SC eds The complete drug reference 33rd ed London UK Pharmaceutical Press 2002 p 1386 62 BIBRA Working Group Riboflavin and its derivatives toxicity profile Epsem Surrey UK BIBRA 1
21. europa eu int comm enterprise newapproach standardization harmstds reflist emc html eIVDO In vitro diagnostic medical devices Directive 98 79 EC http europa eu int comm enterprise newapproach standardization harmstds reflist invimedd html Low Voltage Directive http europa eu int comm enterprise electr_equipment lv index htm http europa eu int comm enterprise newapproach standardization harmstds reflist lvd html Machinery Directive 98 37 EC 000000 http europa eu int comm enterprise mechan equipment machinery index htm http europa eu int comm enterprise newapproach standardization harmstds reflist machines html Radio Telecommunications Terminal Equipment R amp TTE Directive 1999 5 EC http europa eu int comm enterprise newapproach standardization harmstds reflist radiotte html Medical Devise Directive 93 42 EEC http europa eu int comm enterprise medical_devices index htm http europa eu int comm enterprise newapproach standardization harmstds reflist meddevic html nun http europa eu int comm enterprise toys index en htm 39 393 000 000 000 0000 0000000000 2005 0 30 000000000000 0000000000 0000000 0 107 6006 000000 1 12 32 TEL 03 3582 6270 00000000 40
22. untreated human platelets 10 5 5 Test article Mirasol PRT treated platelets Low dose diluted in saline 1 3 5 5 0 Middle dose diluted in saline 1 1 5 5 0 High dose undiluted 10 5 5 Positive control cyclophosphamide 50 mg kg 5 5 0 Genotoxicity Studies Three different tests were performed to evaluate the genotoxicity of Mirasol PRT treated human platelet products an in vitro test for gene mutations in bacteria an in vitro test for clastogenicity in mammalian cells and an in vivo bone marrow micronucleus test Bacterial reverse mutation assay Ames test Mirasol PRT treated day 5 platelets with and without y irradiation and control untreated day 5 platelets were tested using Salmonella typhimurium tester strains TA98 TA100 TA1535 and TA1537 and Escherichia coli tester strain WP2 uvrA 1n the presence and absence of derived 59 prepared by the contract research organization The assay was performed in 2 phases using the plate incorporation method and normal saline as the diluent The first phase established the dose range for the confirmatory mutagenicity assay and provided a preliminary mutagenicity evaluation In the first phase the dose levels tested were 0 33 1 0 3 3 10 33 100 333 and 1000 uL per plate The second phase the confirmatory mutagenicity assay was used to evaluate and confirm the mutagenic potential of the test article with dose levels of 3 3 10 33 100 333 and 1000 uL per pla
23. 00000000000000000 0000000000000 36 390 EC Declaration of Conformity Black Monolith Inc 2001 Monolith Road Sea of Tranquility The Moon We declare under our solo responsibility that the product Product Super computer Model HAL 9000 to which this declaration relates complies with the provisions of following European Directives 73 23 EEC as amended by 93 68 EEC Directive on the harmonization of the laws of Member States relating to electrical equipment designed for use within certain voltage limits 89 336 EEC as amended by 92 31 EEC and 93 68 EEC Directive on the approximation of the laws of the Member States relating to electromagnetic compatibility Applied Harmonized Standards EN 60950 1 2001 Information technology equipment Safety Part 1 General requirements EN 55022 1998 1 2000 A2 2003 Limits and methods of measurement of radio disturbance characteristics of information technology equipment EN 61000 3 2 2000 Limits for harmonic current emissions equipment input current up to and including 16 A per phase EN 61000 3 3 1995 1 2001 Limitation of voltage changes voltage fluctuations and flicker in public low voltage supply systems for equipment with rated current gt 16 per phase and not subject to conditional connection Authorized Representative Black Monolith Europe GmbH 2010 Discovery Avenue Neverland Germany Signature Bowman David Bowman Technical Director B
24. 2 0 6 1 IU mL FIX activity 0 45 1 48 0 8 0 2 0 7 0 1 IU mL FX activity 0 68 1 48 0 8 1 0 8 0 1 IU mL FXI activity 0 42 1 44 0 8 2 0 6 1 IU mL FXII activity 0 40 1 52 3 0 8 1 IU mL Total protein 48 64 57 2 5313 9 1 Antithrombin 0 72 1 45 0 8 1 1 0 O 1 III IU mL PC IU mL 0 58 1 64 2 0 9 0 2 PS IU mL 0 56 1 68 0 8 1 2 PLG IU mL 0 68 1 44 0 8 0 2 0 9 0 2 A2A IU mL 0 72 1 32 1 02 0 9 0 1 HMWK IU mL 0 65 1 35 0 8 0 2 0 7 0 2 Values reported for Mirasol PRT treated plasma rected for the dilution due to the addition of riboflavin TOXICOLOGICAL REVIEW MIRASOL PRT Table 10 Complement Test Results in Treated and Untreated Control Platelet Preparations Sample Chemotaxis assay Enzyme release assay Reference range Positive Positive control 60 4 control 1 13 Negative Negative control 16 8 control 0 32 Control n 6 22 8 1 8 0 41 0 03 Treated n 6 24 2 5 0 0 45 0 14 P NS NS NOTE NS P gt 05 plasma proteins No evidence of neoantigenicity was observed with the Ouchterlony assay indicat ing that no new antigens were formed during treatment with the Mirasol PRT System Treatment with the Mirasol PRT System did not result in greater immunoglobulin G binding than what was observed in comparison with untreated controls when assessed with the Capture P assay In the tests of lumichrome cytotoxicity and of the cy
25. 2000 mg kg 27 Subacute 4 d Mouse sc 2 6 Mortalities at 1000 mg kg xd 1 28 Subchronic 4 6 wk Rat Oral Increased liver enzymes at 31 2 0 Chronic 22 mo Rat Oral Mild liver cell hypertrophy at 92 1 5 mg x kg x Chronic 140 d Rat Oral 50 mg x animal x d7 no adverse effects 27 Chronic 5 mo Dog Oral 25 mg kg x d no adverse effects 27 Developmental Rat Oral During pregnancy and lactation 33 NOAEL of 40 mg x kg x d Reproduction Rat Oral 3 Generations NOAEL of 27 50 mg x animal x d Carcinogenicity Rat Oral 1 5 mg x kg for 22 mo 32 no evidence of carcinogenicity Micronucleus Mouse IP Four doses at 1000 mg kg negative 28 Micronucleus Mouse Perioral 100 mg kg negative 34 Micronucleus oral mucosa Human Perioral 200 mg x kg x wk for 1 y negative 5 Clastogenesis CHL Preincubation Negative 36 Gene mutation E coli Preincubation Negative 37 Ames test TA1535 TA1537 TA1538 Preincubation 10 negative results 38 S typhimurium TA98 100 x S9 activation Ames test TA100 TA102 98 TA97A gt Preincubation 25 100 pg mL negative results 39 S typhimurium S9 activation cecal cell free extract Pharmacokinetics Human IV 11 6 mg subject in bolus 242 mL 40 Cmax 1209 nmol L Disposition 11 2 0 1006 h Renal clearance 11 2 0 7385 h Abbreviations SC subcutaneous IP intraperitoneal LDso lethal dose at which 50 of the test population survives lethal dose at which
26. Developmental Toxicity and Genotoxicity No developmental toxicity was observed in the embryo fetal development study All fetuses were REDDY ET AL examined for malformations and developmental variations Table 6 No mutagenicity was observed in the Ames test with and without S9 metabolic activating fraction for treated or control human platelets or for lumichrome The in vitro and in vivo tests for clastogenicity in mammalian cells chromosomal aberration in cultured CHO cells and micronucleus test in mouse bone marrow cells respectively were also performed with Mirasol PRT treated products Human platelets treated with the Mirasol PRT System gave negative results in all genotoxicity experiments Detailed results of the in vivo genotoxicity experiment are summarized in Table 7 Neoantigenicity and Cytotoxicity Results of studies using C labeled riboflavin and exposure of platelets and plasma to UV light did not demonstrate any detectable binding of riboflavin or its photoproducts to platelets or to Table 9 Clotting Factor Levels Mirasol PRT Treated FFP Mirasol FFP Treated assayed Reference postillumination Treated stored range n 27 for 1 y n 27 Fibrinogen 145 385 227 60 216 2 mg dL FVllla activity 0 52 1 55 0 6 0 2 0 9 z 0 2 IU mL FII activity 0 65 1 54 0 8 0 1 0 8 0 1 IU mL FV activity 0 54 1 45 0 7 0 1 0 6 1 IU mL FVII activity 0 62 1 65 0 9 50
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28. Embryo fetal development in rats exposed to Mirasol PRT treated products The developmen tal toxicity potential of rat plasma treated with the Mirasol PRT System was evaluated in female Sprague Dawley rats mean gestation interval 22 days Time mated pregnant animals in the test and control groups n 25 for control n 24 for test group were given IV PRT treated or untreated products respectively at a dose of 2 0 mL per animal 7 7 0 4 mL kg on the first day of dosing 6 0 0 3 mL kg on the last on gestation days 6 through 17 The animals were observed over the course of the study for clinical signs body weight and food consumption On gestation day 20 the animals were necropsied and the ovaries and uterus were investigated for the total number of implanta tions total number of corpora lutea early and late resorptions and viable and nonviable fetuses Individual body weights and sex of the fetuses were noted Gravid uterine weights were recorded and adjusted body weight changes were calculated All fetuses were given an external examination and processed for visceral or skeletal examination and malformations and developmental variations were recorded 140 REDDY ET AL Table 4 Mammalian Erythrocyte Micronucleus Test No of mice sex used for bone marrow collection after dose administration Dose 20 mL kg No of mice sex dosed 24h 48h Vehicle control 0 9 NaCl for injection 10 5 5 Negative control
29. Samples were then treated and stored frozen until analysis of factors in standard coagulation assays Results for all samples analyzed remained within historical reference ranges based on untreated historical and concur rent controls Results for Mirasol PRT treated FFP are representative of values obtained for plasma from Mirasol PRT treated platelets immediately posttreatment The plasma from Mirasol PRT treated human products contained significantly more C3a desarg Table 11 Results of Pharmacokinetic Study and Test for Leachables and Extractables Test Findings Pharmacokinetics of photolyzed C riboflavin in Mirasol PRT treated rat plasma Assessment of leachable and extractable compounds using standard medical device biocompatibility testing Assessment of leachable and extractable compounds in Mirasol PRT treated human platelets and untreated human platelets Approximately 9596 of total radioactivity administered to animals was eliminated within approximately 260 275 h postadministration Based upon observed areas under the curve tissues exhibiting highest to lowest overall exposure were liver gt kidneys gt large intestine gt small intestine gt spleen gt femur bone marrow gt lymphatics Observed mean 14 2 estimates determined from radioactivity excretion rate data in urine 55 3 h and determined from radioactivity excretion rate data in feces 55 1 h were in excellent agreement with 11 2 estimate 52 1
30. also make them at least temporarily deficient in riboflavin owing to overlap of the light absorption spectra of riboflavin and bilirubin 26 59 To avoid the risk of the potential 335 137 riboflavin photochemistry induced deficit ribofla vin 3 mg kg perioral is conventionally given to infants during the phototreatment regimen Concern was once expressed about the toxic potential of riboflavin with direct phototherapy of newborns based on theoretical consideration of its possible effect on DNA in the presence of light and on the results of experiments in simplified in vitro systems but there have been no reports of adverse effects in the clinical setting This includes results from large retrospective analyses which directly evaluated potential tumorigenic conse quences in more than 50000 infants undergoing the therapy for more than 10 years after photo therapy This type of therapy continues in wide clinical use today There are a few claims of uncertain quality in individual subjects of sensitization and possible enhancement of epileptiform seizures after several months of high daily doses of riboflavin eg 25 mg kg d for gt 6 months but those reports remain unsubstantiated In no other reports of high oral and parenteral doses of riboflavin in humans have toxic effects been described 27 probably because of its physiologic nature and the rapid excretion of an excessive dose There is an abundanc
31. appropriate plasma was regarded as an appropriate substitute for platelets and was used as the test article Treatment of plasma with the Mirasol PRT System 15 identical to the treatment of platelets Sample Size Determination Statistics and Data Analysis Sample size determination for studies was based on historical experience or published requirements where appropriate In all cases data were main tained in validated databases allowing archiving and analysis of data studies assays and data analysis were performed at accredited contract testing laboratories in accordance with Good Laboratory Practice GLP requirements except TOXICOLOGICAL REVIEW MIRASOL PRT where noted Comparisons of data between study groups were analyzed using Student test Wilcoxon distributions and other suitable analyses as appro priate for the particular study data being analyzed Systemic Toxicity Studies Acute toxicity Two acute toxicity studies 1 in rats and 1 in dogs evaluated the acute toxicity of Mirasol PRT treated species specific platelet pro ducts with or without y irradiation In those studies both males and females were used to reveal any sex dependent effects In addition an acute toxicity study of photolyzed riboflavin in saline was performed to test the toxicity of higher doses of riboflavin and its photoproducts than are attainable in a blood product The dose volume used in all rat studies 2 0 mL corresponde
32. h for plasma No significant differences before and after exposure to treatment conditions No polymeric material was detected by FTIR in either treated or untreated platelet extracts All metals detected in treated platelet extracts were present in untreated platelet extracts in similar amounts Abbreviation FTIR Fourier transform infrared spectroscopy 345 148 and 5 desarg 2 7 1 8 fold higher respec tively and significantly lower values for esterase 1 8 fold lower and 50 14 fold lower than in plasma from untreated controls Table 8 The desarg forms of C3a and C5a are biologically inactive forms of the anaphylatoxins produced in the common pathway of complement activation The increase in these inactive forms in treated products and decrease in the active compo nent CH50 total hemolytic complement are consistent Little to no complement derived activity was observed in plasma from either Mirasol treated platelets or untreated controls and there were no significant differences between treated and control results Table 10 Similar studies per formed on products after y irradiation yielded identical results data not shown Pharmacokinetics of Photolyzed C Riboflavin in Rats After a single IV administration of Mirasol treated plasma containing photolyzed C ribofla vin the radioactivity was well distributed from the whole blood to tissues selected for assay within the fi
33. light decreased reac tivity of the solutions was demonstrated The results clearly show that conversion of riboflavin from its native form into the photoproducts leads to decreased reactivity of the solution owing to decreases in the extinction coefficients of the resulting solution photobleaching MIRASOL PRT SYSTEM EFFICACY Pathogen reduction with the Mirasol PRT System has been tested with a wide variety of enveloped and 334 nonenveloped viruses as well as gram negative and gram positive bacteria Prevention of GVHD due to WBC inactivation by the Mirasol PRT System has been studied both in vitro and in vivo Parasite reduction studies have been conducted in collabora tion with the Walter Reed Army Institute of Research and are the subject of separate publica tions The results of these studies with bacteria viruses parasites and WBCs demonstrate broad efficacy and capacity for the process in the reduction of pathogenic agents in blood components Blood components treated with riboflavin and UV light exhibit satisfactory quality as assessed by in vitro measurements Initial studies on protein quality after treatment demonstrated ade quate retention of coagulation factors coagulation inhibitors and immunoglobulins all at levels required for therapeutic gt Some platelet quality indices were reduced in treated products in comparison with untreated controls but remained within acceptabl
34. quantitation of primary tissue and secondary microbial catabolites of Riboflavin that are excreted in mammalian rat urine J Nutr 117 468 475 Chastain J L and D B McCormick 1988 Characterization of a new flavin metabolite from human urine Biochim Biophys Acta 967 131 134 Chastain J L and D B McCormick 1987 Flavin catabolites identification and quantitation in human urine Am J Clin Nutr 46 830 834 Toxicity Testing of a Novel Riboflavin Based Technology for Pathogen Reduction and White Blood Cell Inactivation Heather L Reddy Anthony D Dayan Joy Cavagnaro Shayne Gad Junzhi Li and Raymond P Goodrich The Mirasol PRT System Gambro BCT Lakewood CO for platelets and plasma uses riboflavin and UV light to reduce pathogens and inactivate white blood cells in donated blood products An extensive toxicology program developed in accordance with International Organisation for Standardisation ISO 10993 guide lines was performed for the Mirasol PRT system Test and control articles for most of the reported studies were treated test or untreated control blood pro ducts For some studies pure lumichrome the major photoproduct of riboflavin or photolyzed riboflavin solution was used Systemic toxicity was evaluated with in vivo animal studies in the acute and subchronic HE COLLECTION SEPARATION and transfusion of red blood cells platelets plasma and fractionated plasma components are essen
35. with the pathogen reduction capacity of this system have the potential to greatly enhance the safety of blood products presently offered in routine clinical practice Acknowledgements The authors acknowledge support in part by the fol lowing Laboratories at Bonfils Inc 717 Yosemite Street Denver CO 80230 for the collection of apheresis platelets Gambro BCT 10811 West Collins Avenue Lakewood CO 80215 Dr Donald B McCor mick Ph D Fuller E Callaway Professor of Biochemistry Department of Biochemistry School of Medicine Emory University Atlanta GA 30322 3050 Kristin E Bjorgo Navigant Biotechnologies for help in preparing the manuscript REFERENCES 1 Curran J W and D N Lawrence 1984 AIDS associated with transfusion N Engl J Med 310 69 75 2 Warburg O and W Christian 1932 Uber das neue oxydationsfer ment Naturwissenschaften 20 980 3 Speck W T S Rosenkranz and H S Rosenkranz 1976 Further observations on the photooxidation of DNA in the presence of Riboflavin Biochim Biophys Acta 435 39 44 4 Kuratomi K and Y Kobayashi 1977 Studies on the interactions between DNA and flavins Biochim Biophys Acta 476 207 217 5 Olsen 7 H H Hertz S K Kjaer A L Mellemkjaer J D Boice Jr 1996 Childhood leukemia following phototherapy for neonatal hyperbilirubinemia Denmark Cancer Causes Control 7 411 414 6 Sisson T R N Kendall E Shaw and L Kecha
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55. 614 Christopher C Hardwick et al Table 5 RB and photoproducts in normal untreated blood Concentration nM Compound average range RB 23 9 8 6 79 6 LC 11 4 0 0 75 3 2KF 2 0 0 9 3 4 4KF 1 2 0 5 2 4 FMF 5 0 1 7 11 5 Hustad has reported a value of 6 9 nM 2 7 42 5 nM for RB concentration in plasma n 63 37 In a later report Hustad reported the average RB concentration in the plasma of 124 senior citizens as 15 3 nM 5 4 28 4 nM 25 Capo chichi reported in 2000 that the median concentration of RB in 10 infants and 10 adolescents to be 20 9 nM 12 7 53 4 and 18 5 nM 8 2 57 8 respectively 25 We now report for n 30 an average value of 23 9 nM 8 6 79 6 nM Fig 3 The higher mean value observed in this study might be a reflection of the fact that no additional protein precipitation or extraction step was required in this procedure re sulting in higher retention of starting levels of RB or might be due to partial leakage of erythrocytes still present in the buffy coat platelet products Photolysis of RB during the MIRASOL process results in the formation of four photoproducts 2KF 4KF FMF and LC These four photoproducts were found to be present in apheresis platelets that had not undergone any photochemical treatment although at a much lower concentration Table 5 Fig 4 It is also important to note in this context that the concentrations measured in the platelet products will be dilute
56. 990 pp 1 7 63 Direct Food Substances Affirmed as Generally Recog nized as Safe Riboflavin 21 CFR Sect 184 1695 2001 64 European Commission Health and Consumer Protection Directorate General Scientific Committee on Food Opinion of the Scientific Committee on Food on the tolerable upper intake level of vitamin B2 Brussels Belgium European Commission 2000 pp 1 10 65 Sisson TR Kendall N Shaw E et al Phototherapy of jaundice in the newborn infant Effect of various light intensities J Pediatr 81 35 38 1972 66 Sisson TR Photodegradation of riboflavin in neonates Fed Proc 46 1883 1885 1987 67 Bates CJ Human requirements for riboflavin Am J Clin Nutr 46 122 123 1987 68 Kostenbauder HB DeLuca PP Kowarski CR Photo binding and photoreactivity of riboflavin in the presence of macromolecules J Pharm Sci 54 1243 1251 1965 69 Yurdakok M Erdem G Tekinalp G Riboflavin in the treatment of neonatal hyperbilirubinemia Turk J Pediatr 30 159 161 1988 70 Olsen JH Hertz H Kjaer SK et al Childhood leukemia following phototherapy for neonatal hyperbilirubinemia Den mark Cancer Causes Control 7 411 414 1996 71 Speck WT Rosenkranz HS Phototherapy for neonatal hyperbilirubinemia A potential environmental health hazard 350 REDDY ET AL to newborn infants A review Environ Mutagen 1 321 336 1979 72 Maisels MA Kring EA Does intensive phototherapy produce hemolysis in newborns of 35 or more weeks
57. EN 62 000000000000000 30000000000000000000000000 0001000000000000 D Uu 00000000 e 000000000000 000 00000000 e 0000000000000000000000 e 00000000000000000000000000000000 e 0000000000060 000000 0000 25 379 EH ESSE FQ EZ ERIS ESI EE SEE LI GEM EN E E EI Er CES E Ez ESL EST SESS 0000000000000000000000000000000000 00000000000000000000000000000000 0 0000000000 1000000000000000000000000 0000000000000000000000000000000000000 0 0000000000000000000000000000000000 0000 000000000000000000000000000000 0000000000000000000 00 00 00 00 00 00 up WE PESE E 000000000000000000000000000000000000 0 000000000000000000000000000000000000 E 00000000000000000 0000000 00000000000000000 0000000000000000000000000000000000000000 0 000000000000000000000000000000000000000 000000000000000000000000000000000000000000 000000 1 00 00000000000000000000000000000 0000 0000000000000000000000000000000
58. Munoz A Reitz BA Transmission of retroviruses by transfusion of screened blood in patients under going cardiac surgery N Engl J Med 320 1172 1176 1989 16 Nelson KE Donahue JG Munoz A et al Transmission of retroviruses from seronegative donors by transfusion during cardiac surgery A multicenter study of HIV 1 and infections Ann Intern Med 117 554 559 1992 TOXICOLOGICAL REVIEW MIRASOL PRT 17 Grant IH Gold JW Wittner M et al Transfusion associated acute Chagas disease acquired in the United States Ann Intern Med 111 849 851 1989 18 Leiby DA Lenes BA Tibbals MA et al Prospective evaluation of a patient with Trypanosoma cruzi infection transmitted by transfusion N Engl J Med 341 1237 1239 1999 19 Wu DG Mason B Jong J et al Parvovirus BI9 transmission by a high purity factor VIII concentrate Transfu sion 45 1003 1010 2005 20 Biggerstaff BJ Petersen LR Estimated risk of transmis sion of the West Nile virus through blood transfusion in the US Transfusion 43 1007 1017 2002 21 Custer B Tomasulo PA Murphy EL et al Triggers for switching from minipool testing by nucleic acid technology to individual donation nucleic acid testing for West Nile virus Analysis of 2003 data to inform 2004 decision making Transfusion 44 1547 1554 2004 22 Hellstern P Solvent detergent treated plasma Compo sition efficacy and safety Curr Opin Hematol 11 346 350 2004 23 Williamson LM Cardigan R
59. NS Z0f6 4 6 lt 0 11 lt 0 11 PAI 1 IU mL 0 31 3 6 1 3 17 4 lt 05 PLG IU mL 0 68 1 44 1 04 0 10 0 88 0 08 lt 05 A2A IU mL 0 72 1 32 0 88 0 05 0 82 40 07 lt 05 D D ng mL 0 255 5 6 110 110 1 139 ng mL 0 3 61 1 5 0 2 4 8 0 9 lt 05 F1 2 0 4 1 8 0 5 0 1 0 0 1 05 nmol L TAT ng mL 0 0 5 0 30f6 lt 0 6 1 1 0 4 NA 0 7 0 1 C3a ng mL 0 940 5510 552 16594 lt 05 4939 C5a ng mL 4 7 9 5 8 1 16 3 lt 05 C1 esterase 8 0 19 51 11 2 741 lt 05 Inhibitor mg mL CH50 classic 176 2827 281 56 21 18 O05 pathway U mL Abbreviations PC protein C PS protein S A2A alpha 2 plasmin D D D dimer TAT thrombin antithrombin complexes Values reported for plasma from Mirasol PRT treated platelets are corrected for the dilution due to the addition of riboflavin Reference range specific to laboratory or assay kit P 05 5 Most data below detection limit of assay Table 6 In the repeated dose toxicity study the levels of riboflavin and lumichrome in blood samples from animals receiving Mirasol treated products were below the limits of quantifi cation as were the levels in blood samples from animals receiving untreated plasma These results were consistent with the observed rapid clearance of riboflavin after IV administration both in the literature and in the pharmacokinetic study with C riboflavin in Mirasol PRT treated products
60. OXICOLOGICAL REVIEW MIRASOL PRT blood A report of the U S Environmental Protection Agency Gene Tox Program Mutat Res 239 29 80 1990 90 Hayashi M Tice RR Macgregor JT et al In vivo rodent erythrocyte micronucleus assay Mutat Res 312 293 304 1994 91 Arndt PA Garratty Hill J et al Two cases of immune haemolytic anaemia associated with anti piperacillin detected by the immune complex method Vox Sang 83 273 278 2002 92 Creech HJ O Connell AP Immunochemistry of con jugates prepared from serum albumins and acridine nitrogen mustards ICR mutagens Cancer Res 41 3844 3851 1981 93 Dise CA Burch JW Goodman DB Direct interaction of mepacrine with erythrocyte and platelet membrane phospholipid J Biol Chem 257 4701 4704 1982 94 Chen RF Fluorescence of quinacrine mustard conju gated to proteins Arch Biochem Biophys 172 39 50 1976 95 Jusko WJ Levy Plasma protein binding of riboflavin and riboflavin 5 phosphate in man J Pharm Sci 58 58 62 1969 96 Tapia G Silva E Photo induced binding to the tryptophan residues of bovine and human serum albumin Radiat Environ Biol 30 131 138 1991 97 Diaz M Ines Becker M De Ioannes AE et al Development of monoclonal antibodies against a riboflavin tryptophan photoinduced adduct Reactivity to eye lens proteins J Photochem Photobiol 63 762 767 1996 98 Innis WSA McCormick DB Merrill AH Variations in riboflavin binding by human plasma Identification
61. P NF26 Edited by USP Compendial Committee pp 2439 2442 USP Rockville MD Hustad S P M Ueland and J Schneede 1999 Quantification of Riboflavin flavin mononucleotide and flavin adenine dinucleotide in human plasma by capillary electrophoresis and laser induced fluores cence detection Clin Chem 45 862 868 Piper J S Murphy R Schuyler R Haynes and A Wilson 2002 Assessment of the acute toxicity and genotoxicity risks associated with lumichrome the primary photoproduct of Riboflavin Vox Sang 83 192 Piper J T S E Murphy R S Schuyler R Haynes and A Wilson 2001 Evaluation of the acute toxicity and genotoxicity risks associated with the Riboflavin photoproduct lumichrome Transfusion 41 908 Piper J E Hansen M Woolum D Gampp L Goodrich and G Goodrich 2001 Evaluation of the genotoxicity and acute toxicity risks associated with a Riboflavin based pathogen inactivation process Int J Toxicol 20 404 Piergiorgio P and A Calatroni 1982 Hydrolysis of Riboflavin nucleotides in plasma monitored by high performance liquid chroma tography J Chromatogr 229 445 449 Baeckert P A H L Greene I Fritz D G Oelberg and E W Adcock 1988 Vitamin concentrations in very low birth weight infants given vitamins intravenously in a lipid emulsion measurement of vitamins A and and Riboflavin J Pediatr 113 1057 1065 Chastain J L and D B McCormick 1987 Clarification and
62. atelets were tested with the Capture P assay after 1 and 5 days of 142 storage This study was non GLP but was well documented performed in a respected clinical laboratory and included appropriate controls Neoantigenicity evaluated with the Ouchterlony assay The formation of new antigens was eval uated in vivo by comparing the immune response between animals given control untreated human platelets and animals given Mirasol PRT treated human platelets Rabbits were immunized with control article or with 1 of 2 test articles Mirasol PRT treated day 5 human platelets with or without y irradiation The immunizations occurred on days 0 21 and 42 of the in life portion of the study On day 49 sera from the immunized animals in the test and control groups were collected and tested for antibody production Antibodies were collected and used in the Ouchterlony assay to incubate with either control or test article Lines of immunoprecipitation were examined to determine reactions of identity partial identity or nonidentity Partial identity immunoprecipitation lines would indicate neoantigenicity if the test article contained new antigens arising as a result of the treatment Sensitivity of the assay was evaluated with chicken albumin and serum from rabbits immu nized with that compound The chicken albumin could be detected by weak precipitin lines at 0 15 ug 40 ug plasma protein and at 0 15 ug 200 ug plasma protein with antibody
63. ates that study was done with and without y irradiation of treated blood products activator inhibitor type 1 PAI 1 were assessed as assess physiologic relevance of any changes components of the fibrinolytic pathway 50 after treatment classical pathway C3a C5a and esterase 14 inhibitor were assessed as components of the Pharmacokinetics of Photolyzed C Riboflavin complement pathway and antithrombin III in Rats protein C PC and protein S PS were assessed The in vivo pharmacokinetics and elimination of as components of the coagulation pathway Mean riboflavin and its photoproducts in rat plasma values for test and control articles were com treated with the Mirasol PRT System were also pared using Student test In addition assays for studied Sterile filtered rat plasma was mixed with chemotaxis response to C5a and assays of 500 umol L V C riboflavin treated by the Mirasol myeloperoxidase release from cytochalasin PRT System and administered to male CD rats by treated human polymorphonuclear leukocytes IV injection in a single dose The study consisted of response to C3a and C5a were used to further 2 treatment groups of male Crl CD SD IGS BR 342 TOXICOLOGICAL REVIEW MIRASOL PRT 145 Table 7 Summary of the Results for the Mammalian Erythrocyte Micronucleus Study Test Findings Test for clastogenicity in mammalian cells in vivo mouse bone marrow micronucleus test rats A
64. container used to collect and store platelet products obtained with the Trima automated blood collection device Gambro BCT The sterile riboflavin solution contains riboflavin 500 umol L in 0 9 sodium chloride NaCl solution with pH adjusted with hydrochloric acid to be in the range of 4 0 to 5 0 A volume of 35 gt 5 mL of this solution 15 added to a blood component product to produce a final concentration of approximately 50 umol L The illuminator delivers the necessary dose of UV light 6 2 J mL to the contents of the illumination bag based on product volume derived from weight and measured flux rate Robustness studies demonstrated that the system is suitable for use with initial platelet 333 or plasma product volumes ranging from 170 to 365 mL and platelet product cell concentrations ranging from 1100 to 2100 x 10 mL The Mirasol PRT System reduces the infectivity of pathogens by 3 combined mechanisms The first is the direct damage of nucleic acids of pathogens with the UV light source used in this system The second is the damage of pathogen nucleic acids proteins and membranes by reactive oxygen species generated when riboflavin absorbs light and interacts with dissolved oxygen in solution The Mirasol process was designed to minimize the contributions of this mechanism because of its non specific nature The third is the damage of pathogen nucleic acid by the interaction of riboflavin with nucleic acids Exposu
65. d by a factor of 16 to 20 fold upon infusion of the products into a patient s blood stream thus lowering the difference between concentrations determined in these products and the levels naturally circulating in blood The demonstration of the existence of these agents in naturally occurring blood products suggests that the introduction of a RB based PRT process will not introduce new agents into the blood supply which are not already present to some extent The consequence of increased levels of these agents in blood products resulting from this process is being evaluated in separate toxicology studies which include short term and long term exposure to each of the agents described and Overlay Endogenous RB Photoproducts vs Illuminated RB Riboflavin Endogenous Lumichrome asss Illuminated RB 2 Ketoriboflavin Fluorescence Intensity Retention Time minutes Figure 4 HPLC photochemical profile generated during treatment of 50 uM RB in apheresis platelet concentrates MIRASOL treated apheresis platelet concentrates overlaying the photochemical profile generated by direct measurement of a sample of untreated apheresis platelets characterized in this study 38 45 The presence of these agents in our blood the ubiquitous nature of RB exposure its presence in our diets and our ability to metabolize it and manage its inherent photochemistry suggests a low risk profile for this product These features in combination
66. d to a range of doses per body weight mean dose of 9 0 0 7 mL kg for males and 11 5 gt 1 3 mL kg for females in the study of treated rat platelets and 9 4 0 3 mL kg for the male rats in the study of photolyzed riboflavin The dose volume used in the acute dog studies 40 mL yielded mean doses per body weight of 3 9 0 3 mL kg for males and 5 1 0 4 mL kg for females Table 3 provides a summary of the number of animals and of the dose frequency route and material for the acute toxicity studies Each study was performed in accordance with ISO 1099350 and International Conference on Harmonization guidelines These studies evaluated any immedi ate systemic toxicity due to administration of Mirasol PRT treated platelet products by observa tions of mortality clinical signs of toxicity hematol clinical chemistry food consumption general behavior and weight gain Complete necropsies were conducted for all animals and macroscopic observations were made In the study performed with dogs electrocardiographic examinations were con ducted before randomization at the conclusion of dosing and before necropsy The studies provided Observations of effects on major physiologic sys tems such as functioning of the nervous cardiovas cular and respiratory systems to exclude any pharmacologic effects of test article administration Subchronic toxicity repeated dose study for subchronic toxicity was done in the dog to
67. e are some infectious agents for which screening tests are not routinely used notably Parvovirus B19 cytomega lovirus and the causative agents of parasitic diseases such as Chagas disease malaria babesio sis and leishmaniasis The recent entry of West Nile virus WNV into the blood supply in North America is an example of the emergence of a new From Navigant Biotechnologies Inc Lakewood CO Access BIO Boyce VA and Gad Consulting Services Cary NC A D D is an Independent Consultant in London Current affiliation for J L Thermogenesis Sacramento CA Address reprint requests to Raymond Goodrich PhD Navigant Biotechnologies Inc 1215 Quail Street Lakewood CO 80215 Current affiliation for J L Thermogenisis Sacramento CA E mail ray goodrich navbio com 0887 7963 08 see front matter O 2008 Elsevier Inc All rights reserved doi 10 1016 j tmrv 2007 12 003 Transfusion Medicine Reviews Vol 22 No 2 April 2008 pp 133 153 133 134 REDDY AL Table 1 Reported Toxicity Findings After Riboflavin Administration Study type Species Route Results Ref Acute Mouse SC 1200 mg kg slight reduction in 27 body temperature 2400 mg kg neuromuscular effects and mortalities Acute Mouse IP 1086 340 mg kg 28 29 LD 750 mg kg Acute Rat IP LDso 560 mg kg 27 Acute Rat sc LDo gt 5000 mg kg 27 Acute Rat Oral LDso gt 10000 mg kg 27 Acute Mouse IV LDso gt 50 mg kg 30 Acute Dog Oral NOAEL of
68. e of references on the toxicity of riboflavin administered by various routes These data support its use in total parenteral nutrition regimens Table 1 summarizes representative pub lished toxicity data for oral subcutaneous perioral intraperitoneal and IV exposures to riboflavin Although the safety of riboflavin has been extensively studied there were no reports that directly supported its use in the Mirasol PRT System Therefore Navigant Biotechnologies Lakewood Colo conducted a comprehensive preclinical safety evaluation program in support of the Mirasol PRT System designed to investigate all potential sources of concern The work was done in compliance with International Organisation for Standardisation ISO 10993 7 METHODS Test Article Definition To establish the safety of Mirasol PRT treated blood products in vivo animal toxicity studies were performed with treated platelets and in some instances with treated plasma as well as with pure lumichrome and with photolyzed riboflavin in 138 Table 3 Studies of Systemic Toxicity REDDY ET AL Acute toxicity Subchronic toxicity Mirasol PRT treated rat platelets Mirasol PRT treated dog platelets Photolyzed riboflavin solution Mirasol PRT treated dog plasma No of animals group Duration of study Dose route Group 1 Dose material Dose frequency Group 2 Dose material Dose frequency Group 3 Dose material Dose frequency Grou
69. e transfusion limits These studies were repeated after y irradiation of the products and showed no significant additional effect C Hardwick and R Goodrich unpublished data 2003 Despite the differences in in vitro measurements Mirasol PRT treated platelets exhibited normal function in models of adhesion aggregation and thrombus formation Data from a clinical trial in healthy human subjects illustrated that Mirasol PRT treated plate lets exhibited decreased recovery and survival time in comparison with untreated platelets Never theless recovery values and cell quality indices for Mirasol PRT treated platelets remained well TOXICOLOGICAL REVIEW MIRASOL PRT within the acceptable range for clinical utility of platelet products currently licensed and in routine use Additional studies involving evaluation of the efficacy of treated platelet products in the clinical setting with thrombocytopenic patients are in progress THERAPEUTIC USE AND TOXICITY OF RIBOFLAVIN IN HUMANS Riboflavin was chosen as the photosensitizing agent for this pathogen reduction process because it can be photoactivated to damage pathogens it has a substantial history of safe clinical use and it is both a naturally occurring vitamin B5 and an essential dietary nutrient The latter features afforded it the advantage of a well known and well characterized safety profile on which to base its evaluation in transfusion medicine The me
70. een very important to confirm that prediction and to extend it to include the special cells and plasma proteins also involved in the Mirasol System To this should be added the evidence of retention of acceptable platelet function and survival after 347 149 treatment which has been the subject of additional peer reviewed publications 6 57 Thus there 15 evidence of the safety of this process in a broad range of laboratory tests from which a qualitative extrapolation of safety in patients can be made Quantitative prediction of safety or at least of the margin of safety represented by the difference between the relative exposures of man and animals and the observed effect level in the laboratory must be more cautious but there are relevant data on which to base some realistic predictions The likely exposure of humans to riboflavin and its photoproducts is 0 077 mg kg for each unit of product transfused Likely exposure of recipients in a clinical setting was calculated on the assumption of a mean recipient weight of 70 kg nominal riboflavin solution concentration of 500 mean riboflavin photoconversion of 18 and nominal riboflavin solution volume of 35 mL This exposure level can be compared with the lethal dose at which 50 of the population survives reported for IV riboflavin in mice 50 100 mg kg yielding a safety factor of at least 50 0 077 649 However this safety margin calculation is some what il
71. effects on other system components comprising the medical grade plastic connectors and bags used in this process The results obtained have been compared with extensive literature on the effects of riboflavin and its photoproducts in animals and humans arising from its medical use as a vitamin and more widely as a food colorant or additive The methods used have been those recommended by current official guidelines inter preted according to the special nature and proper ties of platelets and plasma and designed to provide as much information as possible from focused in vitro and in vivo studies The difference in size between human patients and in vivo animal TOXICOLOGICAL REVIEW MIRASOL PRT test systems has restricted application of the standard toxicological strategy of administering a range of doses far exceeding the human dose to reveal possible toxic hazards In studying Mirasol PRT treated blood products the largest volumes or doses have been given consistent with main taining the welfare of any animals used and to separate effects due to toxicities of the components from toxicities due to volume overload alone The results of the in vivo and in vitro toxicity tests have reproduced previously reported findings Table 1 for riboflavin and its photoproducts in animals and humans under relevant conditions despite the special circumstances of the presence of blood cells and proteins and the use of various medical grade plastic c
72. elet concentrates using Riboflavin and light Transfusion 44 877 885 15 Treadwell G E W L Cairns and D E Metzler 1968 Photochemical degradation of flavins V Chromatographic studies of the products of photolysis of Riboflavin J Chromatogr 35 376 388 16 Cairns W L and D E Metzler 1971 Photochemical degradation of flavins VI A new photoproduct and its use in studying photolytic mechanism J Am Chem Soc 93 2772 2777 17 Oka M and D B McCormick 1985 Urinary lumichrome level catabolites of Riboflavin are due to microbial and photochemical events and not rat tissue enzymatic cleavage of the ribityl side chain J Nutr 115 496 499 18 Smith E C and D E Metzler 1963 The photochemical degradation of Riboflavin J Am Chem Soc 85 3285 3288 329 20 21 22 23 24 25 26 27 28 29 30 31 32 Song P S and D E Metzler 1967 Photochemical degradation of flavins IV Studies of the anaerobic photolysis of Riboflavin Photo chem Photobiol 6 691 709 Halwer M 1951 The photochemistry of Riboflavin and related compounds Dissertation 73 4870 4874 Speek A J F van Schaik J Schrijver and W H Schreurs 1982 Determination of the B2 vitamer flavin adenine dinucleotide in whole blood by high performance liquid chromatography with fluorometric detection J Chromatogr 228 311 316 Ichinose N K Adachi and G Schwedt 1985 Determinatio
73. ervations did not yield any test article related effects in dogs of either sex Test article related findings for decreased body weight increased eosinophils increased chloride and decrease thymus weights with associated microscopic findings were noted however these findings were minor and were not considered to be toxicologically significant Reproductive toxicity Embryo fetal development in rats exposed to Mirasol No test article related clinical signs of toxicity were PRT treated plasma observed during treatment period no dam had all fetuses resorbed and no deaths occurred during study Treated group exhibited significant increase in gestation body weight on gestation day 18 and body weight changes were significantly increased on gestation day intervals 12 15 6 18 and 0 20 Increase correlated with significantly increased food consumption in treated group animals No significant differences were observed in fetal weight g gravid uterine weight adjusted weight change postimplantation loss nonviable fetuses litter size early and late resorptions mean no of corpora lutea litter no of implantation sites preimplantation loss no of viable fetuses letter and sex ratio 96 males litter No external or test article related visceral abnormalities were reported in any of fetuses examined No significant difference in skeletal malformation and variations were observed in control and treated groups Indic
74. ic analysis was based on toxicity of the test article the lowest dose with at least 50 reduction in cell growth and 2 lower doses in all harvests Mammalian erythrocyte micronucleus test Mirasol PRT treated platelets and control untreated platelets were tested to assess clastogeni city in vivo with the mammalian erythrocyte micronucleus 850 An increase in the frequency of micronucleated polychromatic erythrocytes is expected in the bone marrow of animals exposed to genotoxic compounds The assay was performed in 2 phases In the pilot toxicity study neat test article was administered intraperitoneally to 5 male and 5 female mice at a volume of 20 mL kg the highest optimal single dose volume No mortality was observed during the course of the study All mice were observed for clinical signs of toxicity after dose administration and daily for 3 days afterward Body weights were recorded before dose adminis tration 1 day after and 3 days after dose administration Results from the pilot study were TOXICOLOGICAL REVIEW MIRASOL PRT used to assess toxicity of the test article and to set dose levels for the definitive study In the definitive micronucleus study 1 intraper itoneal injection was administered to the male and female mice in each group The animals were observed for clinical signs of toxicity after dose administration Table 4 describes the study groups the number of animals per group and test or control a
75. ie J et al Creutz feldt Jakob disease and blood transfusion Results of the UK Transfusion Medicine Epidemiological Review study Vox Sang 91 221 230 2006 7 Wroe SJ Pal S Siddique D et al Clinical presentation and pre mortem diagnosis of variant Creutzfeldt Jakob disease associated with blood transfusion A case report Lancet 368 2061 2067 2006 8 Peden AH Head MW Ritchie DL et al Preclinical vCJD after blood transfusion in a PRNP codon 129 heterozygous patient Lancet 364 527 529 2004 9 Blajchman MA Bacterial contamination of cellular blood components Risks sources and control Vox Sang 87 Suppl 98 103 2004 10 US Food and Drug Administration Minutes of 36th Meeting of the Blood Product Advisory Committee 1992 May 28 29 Bethesda Maryland Washington DC USFDA 1992 11 Yomtovian R Lazarus HM Goodnough LT et al A prospective microbiologic surveillance program to detect and prevent the transfusion of bacterially contaminated platelets Transfusion 33 902 909 1993 12 Schroeder ML Transfusion associated graft versus host disease Br J Haematol 117 275 287 2002 13 Saunders C Herbert P Rowe G et al In vitro evaluation of the PALL Leukotrap Affinity Prion Reduction Filter as a secondary device following primary leucoreduction Vox Sang 89 220 228 2005 14 Blajchman MA Goldman Baeza Improving the bacteriological safety of platelet transfusions Transfus Med Rev 18 11 24 2004 15 Cohen ND
76. ion was also given to minimizing the need for animal experi mentation as far as permitted by the information required and the need for reliable scientific experiments The system design and the experi mental program were intended to answer potential scientific and medical concerns and to permit a comprehensive safety assessment of the Mirasol PRT System The design of the preclinical safety studies was also intended to conform to specific regulatory requirements in different countries adapted to the nature of this innovative device The official guidelines used for the study design included ISO 109937 for medical devices and the general toxicity and pharmacokinetic guidelines for drugs of the International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use 95 The nonclinical toxicity program was based throughout on studying preparations that had undergone Mirasol PRT treatment a process shown to be effective in reducing levels of several types of infectious 46 49 50 106 107 The testing of treated blood products has examined the potential for toxicity resulting from the entire process from addition of riboflavin and controlled illumination in the Mirasol illumination bag and has examined the potential for harm to the blood products themselves Assessments included effects on the cells and plasma proteins the toxicity of riboflavin itself and its photopro ducts and the
77. lack Monolith Inc Date of Issue 1 April 2005 0000000000 37 391 9 CEJ 1 LU LUI LI L 0000000000000000000000000 000000000000000 0 0000000000000000000000000 6000000000000 0 0 00000000000 000000000000000000000 000000000000000000000000000000000000000000 00000000000 000000 0 00000000000000000000000000000000000000 0 000000000000 0000000000000000000000000 0000000005 20000000000000000000000000000000 000000000000000000000000000000000000000000 0000000000000000 0 0 0000000000 000000000000000000000000 000000000000000000000000000000000000000 0000000000000 000000000000000 38 392 0000000000000 0000000000 http www newapproach org http www newapproach org ProductFamilies Default asp The New Approach to technical harmonisation and standardisation Harmonised Standards http europa eu int comm enterprise newapproach standardization harmstds index en html 00000000000004000000 Active Implantable Medical Devices Directive 90 385 EEC http europa eu int comm enterprise newapproach standardization harmstds reflist implmedd html Directive 89 336 EEC http europa eu int comm enterprise electr equipment emc index htm http
78. li G et al Mirasol PRT treatment of donor white blood cells prevents the development of xenogeneic graft versus host disease Rag 2 double knockout mice Transfusion 46 1553 1560 2006 48 Fast LD DiLeone G Li J et al Functional inactivation of white blood cells by Mirasol treatment Transfusion 46 642 648 2006 49 Cardo LJ Rentas FJ Ketchum L et al Pathogen inactivation of Leishmania donovani infantum in plasma and platelet concentrates using riboflavin and ultraviolet light Transfusion 90 85 91 2006 50 Rentas FJ Harman R Salata J et al Inactivation of Orientia tsutsugamushi in RBCs plasma and platelets using riboflavin and light as demonstrated in an animal model Transfusion 47 240 247 2007 51 Li J de Korte D Woolum MD et al Pathogen reduction of buffy coat platelet concentrates using riboflavin and light Comparisons with pathogen reduction technology treated apher esis platelet products Vox Sang 87 82 90 2004 152 52 Li J Lockerbie O de Korte D et al Evaluation of platelet mitochondria integrity after treatment with Mirasol pathogen reduction technology Transfusion 45 920 926 2005 53 Kumar V Motheral T Luzniak G et al Mirasol TM plasma pathogen reduction technology Riboflavin based pro cess conserves protein C protein S and antithrombin activities Transfusion 43 Suppl 80A 2003 abstr 54 Kumar V Motheral T Luzniak G et al Mirasol TM pathogen reduction technology
79. lusory because there is no reliable indication from the clinical literature or from appropriate animal experiments of the toxic dose of riboflavin in man and so the upper bound of the toxic effect level remains unknown It is reasonable only to conclude that there is no reason to predict toxicity in man due to parenteral exposure to riboflavin and its photoproducts in Mirasol treated products until much higher exposures are reached than would ever be feasible from platelet or plasma transfusions For similar reasons compar ison of exposure from treated platelets with the ADI for riboflavin of 0 to 0 54 mg x kg x d is no less misleading because the ADI refers to life long daily exposure to the substance in the diet and is not an appropriate comparator for a short term par enteral exposure even if the latter is repeated up to several times per week at least for a few months In these circumstances which are not unusual for physiologically essential substances the best that the toxicologist can offer is the prediction that riboflavin and its photoproducts have low experi mental toxicity in vivo Published work suggests that administration of the agent in the extreme may result in renal damage due to crystalluria as the most prominent toxic effect in rodents This work 18 150 derived from studies in which animals were given very large IV doses and the harmful effect is very probably a consequence of the limited a
80. n could be seen as foreign in the bloodstream and result in an immune response Immune responses to antibiotics bound to the surface of red blood cells have been observed Quinacrine mustard which binds to proteins and blood cells also results in the production of antibodies If Mirasol PRT treatment were to lead to chemical modification of blood constituents in vitro the in vivo generation of an immune response might be possible Prior studies have identified the potential for binding of 339 141 riboflavin to albumin Reports of additional studies carried out in nonphysiologic conditions including the use of artificial media preparations anaerobic conditions and nonphysiologic pH values claimed to demonstrate the formation of riboflavin protein adducts via tryptophan residues in the protein This work also extended further to show the formation of possible antibodies against such complexes Other studies with human plasma samples were unable to reproduce these results and instead showed riboflavin interactions to occur primarily with immunoglobulins which act as natural carriers for the compound in vivo and are a potential source of claims for antibody genera tion The studies of C riboflavin binding before and after photolysis were thus undertaken to assess the potential for adduct formation under the specific conditions used for Mirasol treatment of blood products To determine the distribution of riboflavin a
81. n of B2 vitamers in serum of fish using high performance liquid chromatogra phy with fluorescence detection Analyst 110 1505 1508 Zempleni J J R Galloway and D B McCormick 1996 The identification and kinetics of 7 alpha hydroxyriboflavin 7 hydroxyme thylriboflavin in blood plasma from humans following oral adminis tration of Riboflavin supplements Int J Vitam Nutr Res 66 151 157 Zempleni J 1995 Determination of Riboflavin and flavocoenzymes in human blood plasma by high performance liquid chromatography Ann Nutr Metab 39 224 226 Hustad S M C McKinley H McNulty J Schneede J J Strain J M Scott and P M Ueland 2002 Riboflavin flavin mononucleotide and flavin adenine dinucleotide in human plasma and erythrocytes at baseline and after low dose Riboflavin supplementation Clin Chem 48 1571 1577 Lopez Anaya A and M Mayersohn 1987 Quantification of Riboflavin Riboflavin 5 phosphate and flavin adenine dinucleotide in plasma and urine by high performance liquid chromatography J Chromatogr 423 105 113 Capo chichi C D J L Gueant F Feillet F Namour and M Vidailhet 2000 Analysis of Riboflavin and Riboflavin cofactor levels in plasma by high performance liquid chromatography J Chromatogr B Biomed Sci Appl 739 219 224 Floridi A C A Palmerini C Fini M Pupita and F Fidanza 1985 High performance liquid chromatographic analysis of flavin adenine dinucle
82. nd its photoproducts after Mirasol PRT treatment a known amount of radiolabeled riboflavin C was added to unlabeled riboflavin solution and the resulting solution was used to treat platelet products from 6 different donors with the Mirasol PRT System Preillumination samples from each platelet product were used as controls and test articles were postillumination samples Cell concentrations pro tein associated radioactivity and protein concen trations were determined for all samples The method was sensitive enough to determine differ ences of less than 1 in binding of C labeled compound at micromolar concentrations per milli gram of protein if such effects were occurring Given prior reports of the levels and extent of photochemical modification of proteins under specific conditions of treatment if such modifica tions were occurring with Mirasol treatment this method would be able to detect it 9 Evaluation of nonimmunologic alterations To assess the potential for nonimmunologic alterations to cells that could subsequently induce an immu nologic response another test the Capture P assay Immucor Inc Norcross GA was used to evaluate the binding of immunoglobulin G to Mirasol treated platelets and to untreated platelets after exposure to allogeneic and autologous plasma Platelet products and the associated plasma from 2 different donors were used to provide the test and control articles for this study Pl
83. o prepare test articles were always within the range verified for clinical use of the device Wherever possible Mirasol PRT treated plate lets in autologous plasma were used as the test article so that any toxicity from the individual components and any interaction between them storage bag riboflavin light blood product would be detected The doses used in the in vivo studies were designed to maximize exposure while minimizing volume associated effects and discom fort to the animals In some cases pure com pounds were used to yield higher doses of the compounds of interest in a study such as lumichrome and photolyzed riboflavin In certain animal studies such as embryo fetal development in rats and subchronic repeated dose test in dogs the volume of animal platelets required to prepare test and control articles for the study would have been very large and would have been required over many weeks To obtain a consistent test article in as humane a fashion as possible for those studies species specific plasma was used rather 336 than platelets The photochemistry of riboflavin yields equivalent photoproduct profiles in plasma products and in platelet products which consist mainly of plasma The absence of platelets eliminates the possibility of detecting toxic altera tions to the platelet surface however that issue was addressed in the neoantigenicity and riboflavin binding studies Therefore when neces sary and
84. of gestation J Perinatol 26 498 500 2006 73 Stein G Sperschneider H Koppe S Vitamin levels in chronic renal failure and need for supplementation Blood Purif 3 52 62 1985 74 Allman MA Truswell AS Tiller DJ et al Vitamin supplementation of patients receiving haemodialysis Med J Aust 150 130 133 1989 75 Gentile MG Porrini M Manna GM et al Water and fat soluble vitamin status in chronic renal insufficiency patients Contrib Nephrol 98 89 97 1992 76 Fouty B Frerman F Reves R Riboflavin to treat nucleoside analogue induced lactic acidosis Lancet 352 291 292 1998 77 Schoenen J Jacquy J Lenaerts M Effectiveness of high dose riboflavin in migraine prophylaxis Neurology 50 466 470 1998 78 Luzzati R Del Bravo P Di Perri G et al Riboflavine and severe lactic acidosis Lancet 353 901 902 1999 79 International Organisation for Standardisation Interna tional Standard ISO 10993 biological evaluation of medical devices Part 1 Evaluation and testing Geneva Switzerland International Organisation for Standardisation 1993 80 International Organisation for Standardisation Interna tional Standard ISO 10993 biological evaluation of medical devices Part 11 Tests for systemic toxicity Geneva Switzer land International Organisation for Standardisation 1993 81 International Organisation for Standardisation Interna tional Standard ISO 10993 biological evaluation of medical devices Part 10 Tests fo
85. of immu noglobulins as the major proteins responsible Biochem Med 34 151 156 1985 99 Merrill AH Innis Whitehouse WSA McCormick DB Characterization of human riboflavin binding immunoglobulins in Edmondson DE McCormick DB eds Flavins and flavoproteins Berlin Walter de Gruyter amp Co 1987 pp 445 448 351 153 100 International Organisation for Standardisation Interna tional Standard ISO 10993 biological evaluation of medical devices Part 5 Tests for in vitro cytotoxicity Geneva Switzerland International Organisation for Standardisation 1999 101 International Organisation for Standardisation Interna tional Standard ISO 10993 biological evaluation of medical devices Part 4 Selection of tests for interactions with blood Geneva Switzerland International Organisation for Standardi sation 2002 102 Material Sciences Toxicology Laboratories Autian method ATTP I Memphis TN University of Tennessee Center for the Health Sciences 1977 103 Hemolysis Rabbit blood evaluation of hemodialyzers and dialysis membranes 77 1294 213 1977 DHEW Publication NIH 104 Hellstern P Sachse H Schwinn H et al Manufacture and in vitro characterization of a solvent detergent treated human plasma Vox Sang 63 178 185 1992 105 International Conference on Harmonisation of Technical Requirements for Pharmaceuticals ICH S3B Pharmacoki netics Guidance for Repeat Dose Tissue Distribution Studies Geneva Swi
86. one marrow tissues were determined from mean concentration time data in the test animal No mortality occurred at any dose level during course of micronucleus study Clinical signs noted on days after dose administration included lethargy and piloerection in males and females dosed with untreated platelets and in all animals dosed with test article neat and diluted In addition hunched position was noticed in animals at middle 1 1 dilution and high neat dose All other mice treated with vehicle or positive control articles appeared normal during course of study No appreciable reduction in ratio of polychromatic erythrocytes to total erythrocytes was observed in groups treated with negative control or test article relative to vehicle control groups This suggests that negative control and test article did not inhibit erythropoiesis No significant increase in incidence of micronucleated polychromatic erythrocytes in negative control or test article treated groups relative to respective vehicle control groups was observed regardless of sex dose or bone marrow collection time Appropriate validated software was used to calculate these parameters Measurement of Leachable and Extractable Compounds Mirasol PRT Treated Products In addition to standard medical device testing for plastic components several novel methods were applied to evaluate the levels of leachable and extractable compounds in human blood produc
87. onnectors and storage con tainers There has been no indication even after repeated administration ofthe maximum achievable exposure to Mirasol PRT treated products 6 times per week for 13 weeks in the subchronic test in the dog of any untoward effect on the functions of the major physiologic systems or in producing any target organ toxicity Furthermore a variety of special tests have shown that no detectable new antigens are present Mirasol PRT System treated cells or proteins and that such preparations did not stimulate antibody formation The wide range of toxicity tests has also excluded any genotoxic risk and any toxic effect on pregnant animals and on embryo fetal development Local tolerance at the site of IV infusion has been shown to be good The treated platelet preparations have not shown any cytotoxicity nor did they leach unwanted substances from the containers and connectors used in the Mirasol system These results indicate the lack of specific toxicity of Mirasol PRT treated products and because of the experiments done they give a strong indication that the same medical accept ability 15 likely also to apply to both human platelets and to human plasma treated in the same way The absence of toxicity was considered likely given the extensive prior experience in humans and the laboratory experience of the safety of the chemicals involved riboflavin lumichrome and the other riboflavin photoproducts but it has b
88. otide in whole blood nt J Vitam Nutr Res 55 187 191 Johansen K and P O Edlund 1990 Determination of water soluble vitamins in blood and plasma by coupled column liquid chromatogra phy J Chromatogr 506 471 479 Roughead Z K and D B McCormick 1990 Flavin composition of human milk Am J Clin Nutr 52 854 857 Roughead Z K and D B McCormick 1990 Qualitative and quantitative assessment of flavins in cow s milk J Nutr 120 382 388 Oka M and D B McCormick 1985 Urinary lumichrome level catabolites of Riboflavin are due to microbial and photochemical events and not rat tissue enzymatic cleavage of the ribityl chain J Nutr 115 496 499 330 33 34 35 36 37 38 39 40 41 42 43 44 45 Photochemistry and Photobiology 2004 80 615 Zempleni J Link and W Kubler 1992 The transport of thiamine Riboflavin and pyridoxal 5 phosphate by human placenta Int J Vitam Nutr Res 62 165 172 Zempleni J J R Galloway and D B McCormick 1996 Pharmacokinetics of orally and intravenously administered Riboflavin in healthy humans J Clin Nutr 63 54 66 Fall H F and H G Petering 1956 Metabolite inhibitors I 6 7 dimethyl 9 formylmethylisoalloxazine 6 7 deimethyl 9 2 hydrox yethyl isoalloxazine and derivatives J Am Chem Soc 78 377 380 USP United States Pharmacopeia 2003 MD Validation of Compendial Methods In US
89. ow right and left at 540 to 545 nm Test and control articles were tested in triplicate Platelet function studies Platelet function and the thrombogenicity of Mirasol PRT treated platelets were directly evaluated in a series of non GLP experiments The hemostatic activity and thrombogenicity of treated platelets were mea sured in an ex vivo model in which Mirasol PRT treated human platelets were mixed with whole blood previously depleted of platelets The details of the experimental system are described by Perez Pujol et al In brief 8 Trima apheresis platelet collections were performed to collect more than 340 mL at a concentration between 1180 and 2160 x 106 cells mL Half of the product volume was treated with the Mirasol PRT System The other half was used as the paired control Treated and control platelets were stored in a platelet storage incubator at 22 2 C for 5 days Samples were tested using flow cytometric and perfusion methods on days 0 3 and 5 of storage The flow cytometric analysis used commer cially available monoclonal antibodies tagged with fluorescein or phycoerythrin to detect glycoprotein GP IIb IIIa GPIb GPIV P selec tin the 53 kd lysosomal membrane protein and coagulation factor V The exposure of aminopho spholipids on the outer leaflet of the platelet membrane was also quantified using annexin V The binding of fibrinogen and von Willebrand factor was detected with the corresponding pol
90. p 4 Dose material Dose frequency 10 Male rats 10 Female rats 14 d dosing on d 1 IV injection via tail vein Untreated rat platelets Three 2 0 mL injections 2 h apart Mirasol PRT treated rat platelets Three 2 0 mL injections 2 h apart Mirasol PRT treated y irradiated rat platelets 2 0 mL injections Mirasol PRT treated y irradiated rat platelets Three 2 0 mL injections 2 h apart 4 Male dogs 4 Female dogs 14 d dosing on day 1 IV infusion via catheter Untreated dog platelets Three 40 mL infusions 2 h apart Mirasol PRT treated dog platelets Three 40 mL infusions 2 h apart Mirasol PRT treated y irradiated dog platelets 40 mL infusions Mirasol PRT treated y irradiated dog platelets Three 40 mL infusions 2 h apart 10 Male rats 9 d dosing on day 1 IV injection via tail vein Sterile 0 996 Three 2 0 mL injections 2 h apart Photolyzed riboflavin solution Three 2 0 mL injections 2 h apart Photolyzed riboflavin solution 2 0 mL injections NA 6 Male dogs 2 in recovery 6 Female dogs 2 recovery 13 wk with 4 wk for recovery group dosing daily 6 d wk IV infusion via catheter Untreated dog plasma Daily 40 mL injections via catheter Mirasol PRT treated dog plasma Daily 40 mL injections via catheter NA NA Abbreviation NA not applicable the absence of platelets and plasma The Mirasol PRT System parameters used t
91. present at 10 15 20 or 25 ug per well in the assay plate Cytotoxicity Studies Agar diffusion test Mirasol PRT treated human platelets and control untreated human platelets were tested to assess cytotoxicity with the agar diffusion assay The indicator cells for this assay were mouse fibroblast L929 cells which are classically used for cytotoxicity studies because they demonstrate sensitivity to leachable cytotoxic compounds 09 Cultures of L929 cells were used in the assay after replacement of the liquid medium with a serum supplemented medium agar mixture that is stained with a vital dye neutral red The culture was protected from light for the duration of the assay to prevent cell damage elicited by photoactivation of the stain The test article and control article were applied directly to a filter paper disc surface area 100 mm at a volume of 100 mL and placed on the surface of the agar All plates were incubated for 48 hours at 37 C 19 in a humidified REDDY ET AL atmosphere containing 5 1 carbon dioxide The negative control article negative control plastic and the positive control article natural rubber were prepared and exposed similarly to the test and control articles The extent of decoloriza tion was evaluated at times 0 24 and 48 hours Minimal essential medium elution study The minimal essential medium elution study was conducted to evaluate the potential cytotoxicity of Mirasol PRT trea
92. queous solubility of riboflavin This is far removed from any circumstance associated with administration of Mirasol PRT treated products in the transfusion setting There is no realistic indication that chemical toxicity will be a limiting factor in any likely course of treatment with Mirasol PRT treated products Results from all genotoxicity studies with the Mirasol PRT System have been negative This has included evaluation of all photoproducts and photolyzed platelets and plasma at the maximum tolerable or maximum soluble limits for these assays The absence of any indication of potential tumorigenic effects in the subchronic and other tests the lack of genotoxicity the chemical nature of riboflavin and its photoproducts and the lack of suspicion arising from the physiologic role and human experience of this vitamin obviated the need for further carcinogenicity testing Prior carcino genicity testing of riboflavin in historical studies Table 1 is consistent with these findings and also supportive of the lack of carcinogenic potential of this compound REDDY ET AL The Mirasol PRT System has been evaluated in an extensive preclinical safety evaluation program The historical literature available on riboflavin and its photoproducts as well as the results of this work support its safety profile in man The results obtained from the studies conducted and reported here are consistent with these data and indicate that the risk of toxici
93. r irritation and sensitization Geneva Switzerland International Organisation for Standardisation 1995 82 International Conference on Harmonisation of Technical Requirements for Pharmaceuticals ICH M3 R1 nonclinical safety studies for the conduct of human clinical trials for pharmaceuticals Geneva Switzerland ICH 1997 83 Ames B McCann J Yamasaki E Methods for detecting carcinogens and mutagens with the Salmonella mammalian microsome mutagenicity test Mutat Res 31 347 364 1975 84 Green MHL Muriel WJ Mutagen testing using trp reversion in Escherichia coli Mutat Res 38 3 32 1976 85 Evans HJ Cytological methods for detecting chemical mutagens in Hollaender A ed Chemical mutagens principles and methods for their detection vol 4 New York NY Plenum Press 1976 86 Swierenga SHH Heddle JA Sigal EA et al Recom mended protocols based on a survey of current practice in genotoxicity testing laboratories IV Chromosome aberration and sister chromatid exchange in Chinese hamster ovary V79 Chinese lung and human lymphocyte cultures Mutat Res 246 301 322 1991 87 Galloway SM Ardema AMI Ishidate MJ et al Report from working group on in vitro tests for chromosomal aberrations Mutat Res 312 241 261 1994 88 Heddle JA A rapid in vivo test for chromosomal damage Mutat Res 18 187 190 1973 89 Mavournin KH Blakey DH Cimino et al The in vivo micronucleus assay in mammalian bone marrow and peripheral T
94. re of riboflavin to light when it 15 associated with or in proximity to nucleic acids leads to a Type I photochemical reaction that can induce oxidation of guanine residues and strand breaks The result of this chemistry is irreversible damage to the nucleic acid MIRASOL PRT PHOTOCHEMISTRY Photolysis of riboflavin during treatment results in the formation of 4 major quantifiable photo products 2 ketoriboflavin 4 ketoriboflavin for mylmethylflavin and lumichrome Several minor components are present in negligible quantities and 136 REDDY ET AL Table 2 Concentration of Riboflavin and Photoproducts in Treated and Untreated Platelets Mirasol PRT platelets n 30 Untreated platelets n 30 Pretreatment umol L Posttreatment umol L Day 5 treated umol L nmol L Riboflavin 48 33 1 13 47 1 50 4 38 68 0 68 38 0 39 9 39 65 1 22 37 0 41 3 23 97 8 6 79 6 Lumichrome 0 23 0 08 0 1 0 3 2 90 0 30 2 5 3 0 3 60 0 30 3 2 4 1 11 4 0 0 75 3 2 Ketoflavin ND 1 27 0 17 1 10 1 49 0 71 0 12 0 59 0 90 2 0 0 9 3 4 4 Ketoflavin ND 0 53 0 03 0 48 0 58 0 16 0 04 0 11 0 22 1 2 0 5 2 4 Formylmethylflavin ND 1 99 0 24 1 63 2 32 1 72 0 22 1 42 2 07 5 0 1 7 11 5 Abbreviation ND not detected All studies for treated products were performed with and without y irradiation No significant difference Student t test P gt 05 was observed Platelet p
95. rikumar P Nair PM et al Assessment of the genotoxic potential of riboflavin and lumiflavin Mutat Res 298 9 16 40 Zempleni J Galloway JR McCormick DB Pharmaco kinetics of orally and intravenously administered riboflavin in healthy humans Am J Clin Nutr 63 54 66 1996 4 Kumar V Lockerbie O Keil SD et al Riboflavin and UV light based pathogen reduction Extent and consequence of DNA damage at the molecular level J Photochem Photobiol 80 15 21 2004 42 Hardwick CC Herivel TR Hernandez SC et al Separation identification and quantification of riboflavin and its photoproducts in blood products using high performance liquid chromatography with fluorescence detection a method to support pathogen reduction technology J Photochem Photobiol 80 609 615 2004 43 Holmstrom B Spectral studies of the photobleaching of riboflavin phosphate Arkiv for Kemi 22 281 301 1964 44 Dekker RH Srinivasan BN Huber JR et al Photo chemistry of flavins I Conventional and laser flash photolysis study of alloxazines J Photochem Photobiol 18 457 466 1973 45 Koziol J Studies of flavins in organic solvents L Spectral characteristics of riboflavin riboflavin tetrabutyrate and Iumichrome J Photochem Photobiol 5 41 54 1966 46 Ruane PH Edrich R Gampp D et al Photochemical inactivation of selected viruses and bacteria in platelet concentrates using riboflavin and light Transfusion 44 877 885 2004 47 Fast LD DiLeone G Cardarel
96. roduct with Mirasol riboflavin solution before UV light exposure Platelet product with Mirasol riboflavin solution immediately after UV light exposure Platelet product with Mirasol riboflavin solution and UV light exposure stored for 5 days at room temperature with agitation Mean 1 SD T Range of measured concentrations Mean represent less than 1 of the total photoproduct peak areas These minor components have been identified as isomers of the major components by analysis using high pressure liquid chromatography followed by 2 stages of mass spectroscopy Each of these 4 major photoproducts is a normal metabolite of riboflavin and each has been detected untreated apheresis platelets Table 2 although at much lower concentrations than observed in Mirasol PRT treated platelets The concentra tions measured in Mirasol PRT treated blood products are diluted by a factor of 16 to 20 fold upon infusion of the products into a patient s blood stream based on 250 300 mL infused into a subject of 5600 mL blood volume on average thus reducing the concentrations toward the levels naturally circulating in the bloodstream The existence ofthese photoproducts in freshly donated nonilluminated blood products shows that a ribo flavin based PRT system will not introduce new chemicals into the blood supply These photoproducts have been examined in earlier studies of riboflavin photolysis in various solutions After exposure to
97. rst hour postdose Most of the excreted urinary radioactivity was recovered by 12 hours postdose and more than half of all radioactivity was excreted in urine Blood levels of radioactivity declined rapidly post dose as expected from studies of riboflavin metabolism and excretion in humans Measurements of the radioactivity associated with the C riboflavin treated plasma indicated rapid initial apparent distribution and or clearance from the systemic circulation that appeared to be complete within the first 8 to 48 hours postdose Table 11 contains a summary of the key study results Leachables and Extractables The leachables and extractables analyses detected no polymeric material in either test or control platelet products Table 11 The Mirasol illumination storage bag does not contain the plasticizer di 2 ethylhexyl pthalate DEHP and testing verified that this plasticizer was present in treated and stored products No toxicologically relevant concentrations of metals were found These results correlate with those from the biocompatibility testing of the Mirasol illumination storage bag elements all elements are biocompatible REDDY ET AL DISCUSSION The design of the Mirasol PRT device the strategy of the investigative work and the specific experiments that were conducted to evaluate toxicity were based first on scientific considera tions and then on the need to comply with regulatory requirements Considerat
98. rticles given to each group as well as the sacrifice times Immediately after sacrifice bone marrow was aspirated from the femurs into a syringe Two slides of bone marrow suspension were prepared and fixed for each mouse The slides were analyzed for the presence of micronuclei in bone marrow cells polychromatic erythrocytes and normochro matic erythrocytes Neoantigenicity Studies The potential for Mirasol PRT treated platelets to exhibit neoantigenicity the production of new antigenic determinants due to the treatment was evaluated with 2 in vitro and lin vivo test Because the photoproducts associated with the Mirasol PRT System are the same as metabolites catabolites photoproducts of riboflavin nor mally present in humans under routine conditions there was no reason to consider that they might themselves affect the immune system and there were no indications of any such effect in the toxicity tests C riboflavin binding evaluation The poten tial for neoantigen formation was first assessed in vitro by measuring the binding of radiolabeled riboflavin to platelets and plasma proteins after Mirasol PRT treatment and comparing the results with those obtained with untreated controls This method assesses binding of a radiolabeled compound to proteins and cellular membranes The presence of covalently bound compounds on cellular membranes or proteins indicates that chemical modification has occurred This modifica tio
99. support more prolonged and repeated administra tion of Mirasol PRT treated platelets to patients some of whom may require intermittent transfu sions over several weeks Parameters evaluated in the repeated dose toxicity study included morbidity 337 139 and mortality outward signs of toxicity behavioral and physical examinations clinical chemistry of blood and urine hematologic indices body weights and food consumption and pathologic and histo pathologic evaluation of tissues Test and control article administration is described in Table 3 The daily dose 40 mL was equivalent to the human dose the range of doses relative to body weight was 4 7 0 3 mL kg in the first week of dosing and 4 1 0 3 mL kg in the last week of dosing for the males and 5 6 0 5 mL kg in the first week of dosing and 4 8 0 4 mL kg in the last week of dosing for the females On days 0 and 86 of the study blood samples were removed from the animals to measure riboflavin and lumichrome and thereby assess any accumulation of riboflavin or photoproduct during the course of this experiment Two animals per sex per group were observed for a 4 week recovery period after the 13 week dosing regimen At the end of the terminal and recovery periods complete necropsies were performed Organ weights were recorded and select tissues were microscopically examined for the recovery group animals Reproductive Toxicity Study
100. tabolic role of ribo flavin has been well characterized principally as a source of the essential coenzymes flavin adenine mono and dinucleotides The recommended daily intake of riboflavin is about 1 3 mg d for the average adult and up to 1 6 mg d for the lactating woman Riboflavin is administered in medical practice by the oral intramuscular and intravenous IV routes to treat deficiency states and it is widely taken orally as a dietary supple ment Recommended therapeutic doses by any route are up to 30 mg d for an adult but there are records of people taking up to 200 mg d or more orally for 6 months with no adverse effect 59 62 Riboflavin is very widely used as a permitted food coloring in the United States where it has generally regarded as safe status and in Europe where it has been approved by the Scientific Committee on Food The Joint Food Agriculture Organization World Health Organization FAO WHO Expert Committee on Food Additives has reviewed its use in food and it has been given a value for acceptable daily intake ADI of 0 to 0 5 mg kg d 1 which is a maximum ADI of 35 mg d for a subject of average weight 70 Riboflavin is given to newborn babies under going phototherapy for neonatal 95 66 Administration of supplemental riboflavin to these vulnerable patients has become standard practice because the treatment accelerated clearance of bilirubin via phototherapy could
101. te The in vitro test for gene mutations in bacteria Ames test was also performed with lumichrome the major product of riboflavin photolysis The in vitro mutagenicity of lumichrome was evaluated in the dark to test its mutagenic potential in the absence of light activation The maximum concentration tested was at the limit of solubility of lumichrome Chromosomal aberration Chinese hamster ovary cells Mirasol PRT treated platelet pro ducts and control untreated platelet products were tested to assess potential clastogenicity chromo some breakage in vitro with the mammalian 338 chromosome aberration test The assay was per formed using Chinese hamster ovary CHO 85587 Based on the findings from the pre liminary toxicity assay the doses chosen for the chromosome aberration assay using the test article Mirasol PRT treated platelets and negative con trol untreated platelets ranged from 0 005 to 0 1 mL mL for all 3 exposure groups The test and control articles were soluble in saline at a concentration of 1 mL mL the maximum concen tration used Both test and control article were tested in the absence and the presence of an Aroclor induced S9 activation system The CHO cells were exposed to test or control article for 4 and 20 hours in the nonactivated test system and for 4 hours in the S9 activated test system All cells were harvested 20 hours after treatment initiation Selection of doses for micro scop
102. ted human platelets The test was designed for the evaluation of test article extracts with assessment of biologic reactivity of a mammalian cell culture L929 Aliquots of test article Mirasol PRT treated human platelets and control article untreated human platelets were centrifuged after which the resulting platelet pellets were mixed with the extraction medium minimal essential medium and incubated at 37 C 19 for 24 2 hours After extraction the medium was removed and applied to an L929 cell culture The L929 cultures were incubated for 48 hours at 37 C 1 C and then evaluated for the response of the cell monolayer Biologic reactivity cellular degeneration and malforma tion was rated on a scale of 0 to 4 In addition the primary product of riboflavin photolysis lumichrome was tested for in vitro cytotoxicity by direct exposure to the indicator The lumichrome solution at the maximum possible lumichrome concentration was exposed to the indicator cells Hemocompatibility Studies Hemolysis test Mirasol PRT treated human platelets and control untreated human platelets were assessed for hemolytic activity in direct contact with human blood 9 Fresh whole human blood type matched to be compatible with test and control articles was collected into EDTA coated Vacutainer tubes The positive control article solution was obtained by adding 10 mL of sterile water for injection per vial The test and con
103. tial medical practices often required for the preservation of life and for the treatment of disease Although the transfusion of these components is a vital therapy transfusions are still associated with some risk for transmission of disease to the patient Bacteria viruses and parasites are all potential sources of infection that have been transmitted by allogeneic transfusions Residual donor white blood cells WBCs can cause a series of severe immune responses in a transfusion recipient result ing in the failure of the transfusion or other complications as a result Prion transmitted dis eases are also a recent and growing concern owing to the potential transfusion mediated transmission of variant Creutzfeldt Jakob disease vCJD A number of safety measures have been taken to reduce the risk of disease transmission They include increased diligence in preparing the donor s arm before donation and diversion of the first portion of blood from the donor to decrease the risks of bacterial contamination of the blood product Decreasing the risk of bacteria related morbidity and mortality in recipients was also the driver for the reduction in storage time for platelets from 7 to 5 days Additional approaches to prevent disease transmission include the use of questionnaires to screen donors and thereby mini mize the risks of collecting blood with viral or other diseases the use of antibody testing and nucleic acid testing to detect vir
104. totoxicity of Mirasol PRT treated products no cytotoxicity was observed Hemocompatibility In tests of hemocompatibility no hemolysis was observed In functional assessments when mixed with thrombocytopenic whole blood the function of Mirasol PRT treated platelets was well pre served in comparison with controls Treated platelets displayed no evidence of hyperactivation or hypercoagulability evaluation of plasma 147 removed from platelet products stored for 5 days statistically significant changes in PRT treated samples were observed in FXI FXII PK PC PS PAI 1 A2A and F1 2 The activities of FXII PK PC PAI 1 and F142 were within the reference range The activities of the remaining proteins FXI PS A24 HMWK and were outside the reference range but the changes were not deemed to be clinically relevant compared with the levels in normal untreated blood Table 8 Similar studies on platelets after irradiation yielded similar findings Because ana lysis of plasma protein levels in platelet products stored at room temperature for extended periods shows degradation in protein quality due to storage alone Mirasol PRT treated FFP was assessed for factor activity Results are presented in Table 9 for samples held for 8 hours at room temperature after collection and before treatment with Mirasol PRT to simulate worst case conditions for routine treat ment of plasma products
105. treated group of 3 rats group 1 and a treated group of 15 rats group 2 each received the radiolabeled test article at a target radiolabeled riboflavin dose level of 0 48 mg kg with approxi mately 555 000 Bq per animal mean actual dose levels of approximately 0 38 mg kg and 547 600 Bq per animal The dose volume for both groups was approximately 4 mL per animal animals were observed twice daily for morbidity mortality injury and the availability of food and water Urine and feces were collected from group 1 animals predosing and at intervals through 144 hours postdosing In addition whole blood was collected and plasma was prepared predosing and at the end of the experiment The tissues collected for postmortem study were residual carcass pelt and tail skin as well as small intestine large intestine spleen kidneys liver lymphatics and right and left femur bone marrow The concentration of radioactivity in whole blood plasma urine feces tissues and cage residues was determined and analyses ofthe kinetics of C labeled compound excretion were performed The plasma and tissue concentrations obtained were for radioactivity associated with riboflavin and its photoproducts rather than for individual com pounds Table 5 summarizes the samples analyzed for each group The pharmacokinetic parameters of riboflavin and photoproduct in plasma small intes tine large intestine spleen kidneys liver lymphatic and femur b
106. trol article solutions were obtained by adding 5 of material to 10 mL of sterile 0 9 NaCl vials were incubated 37 C 2 C water bath for 30 minutes followed by addition of 0 2 mL of diluted human blood and incubation in a 37 2 C water bath for 60 minutes After incubation the vials were centrifuged for 5 minutes at approximately 1500g and the absorbance of each supernatant was determined against an NaCl blank TOXICOLOGICAL REVIEW OF MIRASOL PRT 143 Table 5 Summary of Samples for Pharmacokinetic Study Group no Urine Feces Cage rinse Cage wash and wipe Blood Tissues 1 Intact Predose 0 12 h Predose 24 h 24 h intervals 144h 144h 144 12 24 intervals to 144 h to 120 h 24 h intervals to 144 2 Intact NA NA NA NA 1h 8h 24 h 1h 8h 24 h 48 h 96h 48h 96h Urine and feces collected on wet ice Urine assayed directly by liquid scintillation chromatography LSC Remaining urine saved for analysis of metabolites Blood collected from all animals in groups 1 and 2 predose and at designated time points Prep and assay plasma by LSC Remaining plasma saved for analysis of metabolites Tissues collected include the following group 1 residual carcass pelt hair and skin with underlying fat layer tail skin group 2 small intestine large intestine spleen kidneys liver lymphatics lymph nodes includes splenic inguinal and popliteal and femur bone marr
107. ts Biochem Pharmacol 19 2523 2525 1970 32 Scotto JM Stralin HG Lageron A et al Influence of carbon tetrachloride or riboflavin on liver carcinogenesis with a single dose of aflatoxin 01 Br J Exp Pathol 56 133 138 1975 33 Leclerc J Influence of thiamine riboflavin and vitamin B6 content in food on tissue content of these vitamins in the female lactating rat and young rats Ann Nutr Aliment 28 11 20 1974 34 Lim Sylianco C Genotoxic studies of street soils in Quezon City Philippines in Sumino K ed Environmental and occupational chemical hazards no 8 Proceedings of the Asia Pacific Symposium in Environmental and Occupational Tox icology 1987 Oct 4 10 Singapore Kobe Japan Kobe University School of Medicine 349 151 35 Munoz N Hayashi M Bang LJ et al Effect of riboflavin retinol and zinc on micronuclei of buccal mucosa and of esophagus A randomized double blind intervention study in China J Natl Cancer Inst 79 687 691 1987 36 Sugiyama M Ando A Nakao K et al Influence of vitamin B2 on formation of chromium V alkali labile sites and lethality of sodium chromate VI in Chinese hamster V 79 cells Cancer Res 49 6180 6184 1989 37 Haveland Smith RB Evaluation of the genotoxicity of some natural food colours using bacterial assays Mutat Res 91 285 290 1981 38 De Flora S Study of 106 organic and inorganic compounds in the Salmonella microsome test Carcinogenesis 2 283 298 1981 39 Kale H Ha
108. ts after treatment with the Mirasol PRT System This non GLP analysis was performed to identify and quantify compounds that might migrate directly into the platelet product as a result of treatment with the Mirasol PRT System Untreated and Mirasol treated products were analyzed by gas chromato graphy and mass spectrometry for volatile and semivolatile organic compounds by inductively coupled plasma atomic emission spectroscopy ICP for metals and by Fourier transform infrared spectroscopy FTIR for polymeric materials RESULTS In all studies of toxicity y irradiated Mirasol PRT treated products yielded the same results as non irradiated Mirasol PRT treated products Systemic Toxicity No toxicologically significant findings were observed in any of the studies of acute toxicity Table 6 No toxicologically significant findings were observed in the repeated dose toxicity study 343 146 Table 8 Clotting Factor Levels in Treated and Untreated Control Platelet Preparations After 5 Days of Storage Reference Control Treated range n 6 6 FXI IU mL 0 42 1 44 0 86 0 13 0 59 0 12 lt 5 FXII IU mL 0 40 1 52 1 40 2 0 1 07 0 23 5 PK IU mL 0 65 1 35 1 11 0 06 0 73 0 10 lt 05 HMWK 0 65 1 35 0 78 0 12 0 68 0 29 NS IU mL Antithrombin 0 72 1 45 0 82 0 01 0 84 0 11 NS III IU mL PC IU mL 0 58 1 64 1 02 0 19 0 75 40 8 lt 5 PS IU mL 0 56 1 68 0 16 0 08 0 13 0 03
109. ty due to the use of this system in the transfusion setting should be low ACKNOWLEDGMENTS We would like to thank Mr Jon White Dr Ali Faqi Dr Mary Sherman Ms Sheryl Loux Ms Leigh Bergeron Mr Devaki Sadhu Dr Florence Burleson and Dr Ramadevi Gudi for conducting the toxicology research work described in this review article We also would like to acknowledge the work of Dr George Garratty s and Dr Patsy Giclas laboratories for their analysis of platelet immunologic properties We are grateful to Deanna Gampp Nick Hovenga Suzann Doane Denise Gilmour Shawn Keil and Christopher Hardwick for their laboratory support work for Navigant s toxicology program REFERENCES 1 Busch MP Kleinman SH Nemo GJ Current and emerging infectious risks of blood transfusions JAMA 289 959 962 2003 2 Vamvakas EC Blajchman MA Deleterious clinical effects of transfusion associated immunomodulation Fact or fiction Blood 97 1180 1195 2001 3 Lee JH Klein HG From leukocyte reduction to leukocyte transfusion The immunological effects of transfused leukocytes Baillieres Best Pract Res Clin Haematol 13 585 600 2000 4 Gould DS Auchincloss HJ Direct and indirect recognition The role of MHC antigens in graft rejection Immunol Today 20 77 82 1999 5 Llewelyn CA Hewitt PE Knight RS et al Possible transmission of variant Creutzfeldt Jakob disease by blood transfusions Lancet 363 417 421 2004 6 Hewitt PE Llewelyn CA Mackenz
110. tzerland ICH 1995 106 Goodrich RP Edrich RA Li J et al The Mirasol PRT System for pathogen reduction of platelets and plasma An overview of current status and future trends Transfus Apheresis Sci 35 5 17 2006 107 Goodrich RP Edrich RA Goodrich L et al The antiviral and antibacterial properties of riboflavin and light Applications to blood safety and transfusion medicine in Silva E Edwards AM eds Flavins Photochemistry and photo biology Cambridge UK The Royal Society of Chemistry 2006 pp 83 113 0 000000 00000 2005 00 00000 ARS 6 8 352 PLACING ON THE MARKET AND PUTTING INTO SERVICE ESSENTIAL REQUIREMENTS 0 CONFORMITY ASSESSMENT jx iAP NOTIFIED BODIES HARMONIZED STANDARD
111. uc EC XM 575 L 72 AR WRU A LATLEE 0123 AZER 4 12638812108 427 BLU Annex VI 0000000000000 0000000000000000000 15013485 00 00000 0 000 0 0 0000000 U II 000000000000000000000 00 0000000 0000000000000000000000000000000000000 Annex 000000000 0 000000000000000000000000000000000000000000 0000000000000000000000000000000000000000000 AnnexIV 000000000 000000000000000000000000000000000000000 0000 0 000000000000000 Annex VII 0000 00000000 100000 5 0000000000000000000000 0000000000000000000000000000 00000 19 373 O00 Class 0000000000000000000 1 00000000000000 000000 0000000 0000000000000000000 000000000000000000000000000000000 543 000000000000 0 000000000000000000 000000000000000000 0000000 00000000000000000000000000000000 0000000000000000000000000000000000 00000000000000000000000000000 00000000000000000000000000000000 00000000 000000000000000000000000
112. uses and the use of culture techniques to detect bacteria y Irradiation has settings Developmental toxicity was evaluated with an in vivo animal study Genotoxicity and neoantigenicity were evaluated with in vitro and in vivo tests Hemocompatibility and cytotoxicity were assessed with standard in vitro assays The pharmacokinteics excre tion and tissue distribution of and its photoproducts was evaluated with an in vivo animal study The possible presence of leachable or extractable compounds from the disposable set was evaluated with novel assays for measuring these compounds in blood No treatment related toxicity was observed in any of the studies 2008 Elsevier Inc All rights reserved been used to reduce the possibility of transfusion associated graft vs host disease GVHD in sus ceptible patient populations Leukoreduction has also been adopted in some parts of the world to reduce the possibility of prion transmission Despite the use of the various approaches as outlined transfusion transmitted infection from agents such as HIV hepatitis B virus hepatitis C virus and bacteria continue In part these events arise from the inability of available tests to detect agents in blood at low levels which are nonetheless infectious or during a latent period Risks related to transfusion associated GVHD remain primarily because y irradiation of blood components is not universally used Ther
113. varz Oliai 1972 Phototherapy of jaundice in the newborn infant II Effect of various light intensities J Pediatr 81 35 38 7 Sisson R 1987 Photodegradation of Riboflavin in neonates Fed Proc 46 1883 1885 8 Sisson T R Slaven and P B Hamilton 1976 Effect of broad and narrow spectrum fluorescent light on blood constituents Birth Defects Orig Artic Ser 12 122 133 9 Boadi W Y L Thaire D Kerem and S Yannai 1991 Effects of dietary supplementation with vitamin E Riboflavin and selenium on central nervous system oxygen toxicity Pharmacol Toxicol 68 77 82 10 Webster R P M D Gawde and R K Bhattacharya 1996 Modulation of carcinogen induced DNA damage and repair enzyme activity by dietary Riboflavin Cancer Lett 98 129 135 11 Bates C J 1987 Human Riboflavin requirements and metabolic consequences of deficiency in man and animals World Rev Nutr Diet 50 215 265 12 Corbin F III 2002 Pathogen inactivation of blood components current status and introduction of an approach using Riboflavin as photosensitizer Int J Hematol 76 Suppl 2 253 257 13 Goodrich R and M S Platz 1997 The design and development of selective photoactivated drugs for sterilaization of blood products Drugs Future 22 159 171 14 Ruane P H R Edrich D Gampp S D Keil R L Leonard and R P Goodrich 2004 Photochemical inactivation of selected viruses and bacteria in plat
114. vin was chosen as a photochemical sensitizer because of its well documented safety profile as evidenced by 332 TOXICOLOGICAL REVIEW MIRASOL PRT 1 Sterile connect collection bag and transfer 135 Broad spectrum UV light 6 2 J mL 500 uM riboflavin 2 Add riboflavin Illumination and storage in one container Fig 1 3 Illuminate for 6 10 minutes 4 Ready to transfuse o Diagram of the steps involved with the use of the Mirasol PRT System Platelets or plasma product in an illumination storage bag is combined with riboflavin solution to achieve approximately 50 umol L then exposed to 6 2 J mL UV light The treated product is then ready for clinical use or blood bank storage Riboflavin remains in the treated blood product no removal device is used the reports referenced in Table 1 It is an essential nutrient for all living organisms including humans The novel nature ofthis pathogen reduction treatment PRT required comprehensive toxicological evalua tion to prove that use ofthis technology to treat blood components would not introduce new hazards The strategy nature and results of these toxicological studies are the subject of this review MIRASOL PRT SYSTEM DESCRIPTION The Mirasol PRT System consists of an illumi nator the illumination storage bag and riboflavin solution Fig 1 The Illumination Storage bag 18 made of biocompatible materials and is adapted from the
115. way the fibrinolytic pathway the complement pathway and the coagulation pathway Factor XI factor XII high molecular weight kininogen prekallik rein PK and activated factor XII FXIIa were assessed as components of the contact pathway Prothrombin fragments 1 and 2 F1 2 thrombin antithrombin complex and D dimer were assessed as components of the thrombin pathway Plasmi nogen o antiplasmin A2A 144 REDDY ET AL Table 6 Summary of the Results of the Systemic and the Reproductive Toxicity Studies Test Findings Systemic toxicity Acute toxicity of photolyzed riboflavin in rats No test article related changes in mortality clinical signs body weight food consumption clinical pathology organ weights or macroscopic pathology evaluations were observed Acute toxicity of Mirasol PRT treated platelets in rats No test article related mortality clinical observations body weight changes food consumption changes or macroscopic changes were observed Acute toxicity of Mirasol PRT treated platelets in dogs No test article related mortality clinical observations body weight changes food consumption changes or macroscopic changes were observed Subchronic toxicity of Mirasol PRT treated plasma in dogs No toxicologically significant findings were observed in clinical physical electrocardiographic or ophthalmascopic examinations Macroscopic obs
116. yclonal antibodies and nonspecific membrane immunofluorescence was evaluated Blood for perfusions was obtained from healthy volunteers and depleted of platelets and leukocytes by filtration Samples taken from the test and 341 control articles at different days were incorporated into the thrombocytopenic blood at volumes aimed to increase the platelet count in the perfusate to 150 x 10 L with a final volume of 20 mL Perfusions were performed at 37 C in annular chambers with enzymatically denuded New Zeal and rabbit aorta segments Blood was recirculated through the chamber for 10 minutes at a shear rate of 800 s At the end of the perfusion the segments were rinsed with 20 mL of PBS removed from the chamber and sliced off and washed with a fixing buffer Platelets interacting with the subendothelium were evaluated and classified as either adhesion contact platelets that are attached but not spread and groups of platelets that form aggregates of less than 5 in height or thrombus platelets that form aggregates of 5 um or more in height Coagulation proteins and complement path way Mirasol PRT treated human platelets and control untreated human platelets were stored at 22 C for 5 days after which samples were removed and centrifuged to isolate the plasma fraction The plasma fraction was analyzed with function based assays for key protein components of each hemostatic pathway the contact pathway the thrombin path

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