*To whom correspondence should be addressed at: American Red Cross Biomedical Services, Jerome H. Holland Laboratory for the Biomedical Sciences, 15601 Crabbs Branch Way, Rockville, MD 20855, U.S.A. Fax: 301-738-0704; e-mail: firstname.lastname@example.org
A series of four phenothiazine dyes, including methylene blue (MB), were previously tested for their ability to photoinactivate viruses in red cell suspensions. One of the dyes, 1,9-dimethyl-3-dimethylamino-7-dimethylaminophenothiazine (1,9-dimethylmethylene blue), exhibited good intracellular and extracellular virucidal activity for several RNA and DNA viruses under conditions that minimally affected red cell properties. In order to understand why the virucidal specificity of 1,9-dimethylmethylene blue was greater than other phenothiazines tested, the physical and chemical properties of the dye were compared to three other closely related analogues (MB, 1,9-dimethyl-3-diethylamino-7-dibutylaminophenothiazine [compound 4-140], 1,9-dimethyl-3-dimethylamino- 7-diethylaminophenothiazine [compound 6-136]). All compounds required light and oxygen for virucidal activity and had relatively high singlet oxygen yields (>0.5), but 1,9-dimethylmethylene blue had a singlet oxygen yield approximately 50% greater than that of MB. In addition, the hydrophobicity/hydophilicity of the compounds varied, with the partition coefficients (2-octanol : water) ranging from 0.11 for MB to 3560 for compound 4-140. The dyes had the following affinities for DNA: 1,9- dimethylmethylene blue > compound 6-136 > MB compound 4-140. This order was similar to the order of activities for photoinactivation of the nonenveloped bacteriophage, R17, by the four compounds. Results with the most hydrophobic compound, 4-140, contrasted with those obtained with 1,9-dimethylmethylene blue. Compound 4-140 had a high affinity for protein and a low affinity for DNA. Although compound 4-140 and light inactivated the nonenveloped bacteriophage R17 poorly, the dye readily photoinactivated enveloped viruses in buffer. However, unlike results with 1,9-dimethylmethylene blue, viral inactivation of enveloped viruses by compound 4-140 was completely inhibited by the presence of red cells and plasma. Thus, the high affinity of 1,9-di~methylmethylene blue for DNA and the dye's efficient singlet oxygen yield suggest viral nucleic acid as a potential target, which could explain the photosensitizer's ability to inactivate viruses without adversely affecting anucleate red cells.
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