In vivo genotoxicity assessment of N-(-9 acridinyl)-b-alanine hydrochloride (S-300) using a validated Pig-a mutagenesis assay.

BACKGROUND: N-(-9 acridinyl)-b-alanine hydrochloride (S-300) is the main byproduct of red blood cell (RBC) amustaline/glutathione(GSH) pathogen reduction, currently undergoing phase III US clinical trials following successful European studies(1-3). Phosphatidylinositol glycan, class A (Pig-a) X-linked gene mutagenesis is a validated mammalian in vivo mutation assay for genotoxicity, assessed as clonal loss of glycosylphosphatidylinositol-linked CD59 cell-surface molecules on reticulocytes (RETs) and RBCs. METHODS: Male Sprague-Dawley rats received continuous infusion of S-300 up to the maximum feasible dose (240 mg/kg/day-limited by solubility and volume) for 28 days. Positive controls received a known mutagen by oral gavage on Days 1-3. Plasma levels of S-300 were assessed by HPLC before, during and after infusion. CD59-negative RBCs and RETs were enumerated in pre-dose and Day 28 samples, using a flow cytometric method. Outcome was evaluated by predetermined criteria using concurrent and historical controls. Toxicity was assessed by laboratory measures and necropsy. RESULTS: S-300 reached maximum, dose-dependent levels (3-15 µmol/L) within 2-8 h that were sustained for 672 h and undetectable 2 h after infusion. Circulating RET levels indicated a lack of hematopoietic toxicity. Necropsy revealed minimal-mild observations related to poor S-300 solubility at high concentrations. Pig-a assessment met the preset acceptability criteria and revealed no increase in mutant RBCs or RETs. CONCLUSIONS: Maximum feasible S-300 exposure of rats by continuous infusion for 28 days was not genotoxic as assessed by an Organization for Economic Cooperation and Development-compliant, mammalian, in vivo Pig-a gene mutation assay that meets the requirements of International Conference on Harmonization (ICH) S2(R1) and FDA guidances on genotoxicity testing.

Preclinical safety assessment of pathogen reduced red blood cells treated with amustaline and glutathione.

BACKGROUND: The nucleic acid targeted pathogen reduction (PR) system utilizing amustaline (S-303) and glutathione (GSH) is designed to inactivate blood-borne pathogens and leukocytes in red blood cell concentrates (PR-RBCC). Inactivation is attained after amustaline intercalates and forms covalent nucleic acid adducts preventing replication, transcription, and translation. After pathogen inactivation, amustaline spontaneously hydrolyzes to S-300, the primary negatively charged reaction product; amustaline is below quantifiable levels in PR-RBCC. GSH quenches free unreacted amustaline. STUDY DESIGN AND METHODS: The genotoxic and carcinogenic potential of PR-RBCC, the reaction by-products, and S-300 were assessed in accordance with the International Conference on Harmonization (ICH) guidelines and performed in compliance with the Food and Drug Administration (FDA) good laboratory practice standards, 21 CFR Part 58. in vitro bacterial reverse mutagenicity and chromosomal aberration assays were performed with and without exogenous S9 metabolic activation, and in in vivo clastogenicity and carcinogenic assays using validated murine models. RESULTS: PR-RBCCs were not genotoxic in vitro and in vivo and were non-carcinogenic in p53+/- transgenic mice transfused over 26 weeks. Estimated safety margins for human exposure ranged from >90 to >36 fold for 2 to 5 PR-RBCCs per day, respectively. PR-RBCCs and S-300 did not induce chromosome aberration in the in vivo murine bone marrow micronucleus assay at systemically toxic doses. CONCLUSIONS: PR-RBCCs did not demonstrate genotoxicity in vitro or in vivo and were not carcinogenic in vivo. These studies support the safety of PR-RBCCs and suggest that there is no measurable genotoxic hazard associated with transfusion of PR-RBCCs.

Preclinical pharmacokinetic and toxicology assessment of red blood cells prepared with S-303 pathogen inactivation treatment.

BACKGROUND: A system has been developed to inactivate a wide spectrum of blood-borne pathogens in red blood cells (RBCs) before transfusion. The system utilizes S-303 to target nucleic acids of pathogens and white blood cells. The safety of pathogen inactivated RBC was assessed using S-303-treated RBCs (S-303 RBCs) and S-300, the primary degradation product of S-303. STUDY DESIGN AND METHODS: As part of a preclinical safety evaluation program, intravenous toxicity, safety pharmacology, toxicokinetic, and pharmacokinetic studies were conducted in rats and dogs with S-303 RBCs and S-300. RESULTS: Single and repeated transfusions of S-303 RBCs were well tolerated in rats and dogs at S-303 concentrations up to five times higher than that used to prepare RBCs for clinical use. For S-300, the doses ranged from the lowest level representative of a clinical exposure from transfusion of 1 unit (0.052 mg/kg/day) to up to the amount of S-300 that would result from exposure to more than 1900 units of RBCs (100 mg/kg/day). There were no related effects of S-303 RBCs or S-300 on mortality, clinical status, body weight, or clinical laboratory assessments and no evidence of organ toxicity. S-300 did not accumulate in the plasma of rats and dogs after repeated transfusions. For all the studies, plasma S-303 was consistently below the limit of quantitation. CONCLUSION: The level of residual S-303 and S-300 in the treated blood component is well below that at which no adverse effects were observed. These results support further clinical development of S-303 RBCs for prevention of transfusion-transmitted infections.