Determination Of Residual 4'-Aminomethyl-4,5',8-Trimethylpsoralen And Mutagenicity Testing Following Psoralen Plus Uva Treatment Of Platelet Suspensions

Psoralens and UVA light have been used in the laboratory to study the inactivation of viruses that may be infrequently present in platelet concentrates that are prepared for transfusion. In order to evaluate safety aspects of the treatment of platelet suspensions with 4'-aminomethyl-4,5',8-trimethylpsoralen (AMT), we have investigated the residual levels and mutagenic potential of AMT after UVA phototreatment. 4'-aminomethyl-4,5',8-trimethylpsoralen, at a final concentration of 40 mug/mL, was added to platelet suspensions which contained 16% plasma and a synthetic medium. Platelet suspensions containing AMT were irradiated with up to 7.2 J/cm2 UVA light under normal oxygen levels. Residual levels of AMT were determined by HPLC and a bioassay based on bacteriophage phi6 inactivation. The photodestruction of AMT or its activity by UVA was characterized by a D37 Value of 0.6 and 0.3 J/cm2 with HPLC or bioassay, respectively. At 2.4 J/CM2 UVA, which results in approximately 5 log10 inactivation of vesicular stomatitis virus (VSV) and retention of platelet in vitro properties, 12% (HPLC) to 9% (bioassay) AMT remained.Like other psoralens, AMT was found to bind to serum proteins as shown by ultrafiltration. Results are consistent with approximately 36% of the initial drug load binding primarily to serum albumin. It was determined using H-3-AMT that 9 to 18% of radioactivity was bound to platelets in the absence of irradiation. Similar fractions (13 to 18%) of AMT were bound to platelets after 3.6 J/cm2 UVA irradiation, and 8 to 10% of total AMT was associated with saline-washed irradiated platelets and is presumably tightly bound.Mutagenicity testing (Ames test, in the absence of UVA) was also carried out on the UVA irradiated platelet samples. With Salmonella tester strains which detect primarily base substitution mutations (TA100, TA1535 and TA102), no increase from background mutagenesis levels was observed with any of the samples. However, tester strains which detect frameshift mutations (TA98, TA1537, and TA1538) displayed significant increases in histidine revertants over background levels for irradiated and non-irradiated AMT-containing samples tested in the presence of S9 microsomal enzymes. In the absence of S9 activation, a mutagenic response was observed only with tester strain TA 1 5 37. All frameshift tester strains exhibited decreased numbers of induced revertants with lower residual AMT concentrations (which correlated with higher UVA dose). Significant mutagenesis was still observed for platelet suspensions irradiated with virucidal levels of UVA which maintain platelet in vitro function (2.4 J/CM2). These results suggest that residual available AMT is mutagenic in the Ames test and that the observed frameshift mutations may be caused by binding of AMT or its metabolites to nucleic acids in the absence of UVA light.