Ehud Ben-Hur

Treatment of viral infections with 5-aminolevulinic acid and light

When 5‐aminolevulinic acid (ALA) is exogenously supplied, protoporphyrin IX (PpIX) is accumulated in various cells and makes them light sensitive. The possibility of using such an approach for the treatment of viral infections was studied in this work.

New phthalocyanines for photodynamic virus inactivation in red blood cell concentrates.

Abstract Cationic phthalocyanines with either aluminum or silicon as the central metal were evaluated for their ability to inactivate viruses in red blood cell concentrates (RBCC) photodynamically. In addition, the virucidal potential of a substituted anionic phthalocyanine, aluminum dibenzodisulfophthalocyanine hydroxide (AlN2SB2POH) was evaluated and compared with that of the much studied anionic aluminum tetrasulfophthalocyanine hydroxide (AIPcS4OH). Based on the rate of inactivation of the lipid‐enveloped vesicular stomatitisvirus (VSV), the viruci dal potential of these phthalocyanines was: HOSiPCOSi(CH3)2(CH2)3N+(CH3)3I‐ (Pc 5) = SiPC[OSi(CH3)2‐(CH2)3N+(CH3)3I‐]2 (Pc 6) > AIPCOSi(CH,)2(CH2)˜N+(CH3)2(CH˜)11CH3I‐ (Pc 21) = A1N2SB2POH = AlPcS4 > HOSiPc[OSi(CH3)2(CH2)3N+(CH3)2(CH2)11CH31–]2(Pc 14) > AIPcOSi(CH3)2(CH2)3N+(CHS)3I‐ (Pc 2). Phthalocy anine ligand 14 and Pc 21 are new phthalocyanines, made by quaternizing known amino analogues. Compared to VSV, the rate of inactivation of Sindbis virus (another model lipid‐enveloped virus) was identical when treated in red blood cells (RBC) with Pc 5 and slightly higher when treated with Pc 6 and AlPcS4OH. Treatment of RBCC containing cell‐free human immunodeficiency virus (HIV‐1) with Pc 5 or AlPcS4OH required 15 min of irradiation to inactivate (>5 log10 reduction) the virus. The extent of HIV‐1 inactivation with AlN2SB2POH was 3.7 log10 after 60 min of red light exposure. The RBC integrity after photosensitization was measured by the ability of the cells to bind to plates coated with poly‐L‐lysine, (which reflects the retention of the RBC surface negative charges) and hemolysis of the cells over a 7 day storage period. The RBC damage using these criteria was most pronounced with Pc 5 and Pc 6 but could be reduced when treatment was in plasma instead of buffer. These data indicate that lipid‐enveloped viruses differ in their sensitivity to phthalocyanine photosensitization. Therefore, for virus sterilization of RBCC for transfusion the ability to inactivate human pathogenic viruses completely will have to be evaluated for each virus. The cationic Pc 5 appears to be a potentially useful virucidal agent.

Virus inactivation in red cell concentrates by photosensitization with phthalocyanines : protection of red cells but not of vesicular stomatitis virus with a water-soluble analogue of vitamin E.

BACKGROUND: Photodynamic treatment of red cells (RBCs) with phthalocyanines and red light inactivates lipid‐enveloped viruses, such as vesicular stomatitis virus (VSV) and human immunodeficiency virus. To protect RBCs from photodynamic damage, type I free radical quenchers, such as mannitol, which did not affect virus inactivation, were added.

Inactivation by phthalocyanine photosensitization of multiple forms of human immunodeficiency virus in red cell concentrates.

BACKGROUND: The use of phthalocyanines in conjunction with red light has been shown to inactivate model lipid‐enveloped viruses in red cell concentrates. The ability of this treatment to inactivate multiple forms of human immunodeficiency virus (HIV) was evaluated in this study. STUDY DESIGN AND METHODS: The phthalocyanines used were aluminum phthalocyanine tetrasulfonate (AIPcS4) and the silicon phthalocyanines HOSiPcOSi(CH3)2(CH2)3 N(CH3)2 (Pc 4), and HOSiPcOSi(CH3)2 (CH2)3N+(CH3)3I‐(Pc 5). HIV was studied in a cell‐free form, in an actively replicating form, in latently infected cells, and in blood from HIV‐positive patients. RESULTS: All three phthalocyanines inactivate > or = 10(5) infectious doses of cell‐free HIV. However, only Pc 4 effectively inactivated actively replicating HIV and latently infected cells. The latter was about four times as sensitive to inactivation as was actively replicating HIV. Increasing the hematocrit of red cells during treatment decreased the rate of inactivation, especially at lower light doses. Under treatment conditions that completely inactivated the laboratory isolates of HIV, cell‐associated HIV in blood from HIV‐positive patients was also completely inactivated. The polymerase chain reaction signal from the gag gene of HIV was not affected on treatment of cell‐free virus, but it was reduced after treatment of cell‐associated HIV, particularly in some latently infected cell lines. CONCLUSION: Pc 4 and red light are effective in eliminating the infectivity of HIV in red cell concentrates. The usefulness of this approach for blood banking depends on future demonstration of the preservation of red cell circulatory survival and function in vivo.

THE EFFECT OF IRRADIANCE ON VIRUS STERILIZATION AND PHOTODYNAMIC DAMAGE IN RED BLOOD CELLS SENSITIZED BY PHTHALOCYANINES

Abstract— Phthalocyanines are being studied as photosensitizers for virus sterilization of red blood cells (RBC). During optimization of the reaction conditions, we observed a marked effect of the irradiance on production of RBC damage. Using a broad‐band light source (600–700 nm) between 5 and 80 mW/ cm2, there was an inverse relationship between irradiance and rate of photohemolysis. This effect was observed with aluminum sulfonated phthalocyanine (AlPcSn) and cationic silicon (HOSiPc‐OSi[CH3]2 [CH2]3N+[CH3]3I‐ phthalocyanine (Pc5) photosensitizers. The same effect occurred when the reduction of RBC negative surface charges was used as an endpoint. Under the same treatment conditions, vesicular stomatitis virus inactivation rate was unaffected by changes in the irradiance. Reduction in oxygen availability for the photochemical reaction at high irradiance could explain the effect. However, theoretical estimates suggest that oxygen depletion is minimal under our conditions. In addition, because the rate of photohemolysis at 80 mW/cm2 was not increased when irradiations were carried out under an oxygen atmosphere this seems unlikely. Likewise, formation of singlet oxygen dimoles at high irradiances does not appear to be involved because the effect was unchanged when light exposure was in D2O. While there is no ready explanation for this irradiance effect, it could be used to increase the safety margin of RBC virucidal treatment by employing exposure at high irradiance, thus minimizing the damage to RBC.

Protecting Fibrinogen with Rutin during UVC Irradiation for Viral Inactivation

Abstract— Fibrinogen solutions were irradiated with UVC (254 nm) to inactivate contaminating viruses. In order to protect fibrinogen during UVC irradiation, 0.5 mM rutin was added prior to UVC exposure and subsequently removed during processing. Viral kill by 0.1 J/cm2 UVC resulted in the following inactivation values (log 10): non‐lipid‐enveloped viruses: Parvo 5.5; encephalomyocarditis virus 6.5; hepatitis A virus 6.5: lipid‐enveloped viruses: human immunodeficiency virus 5.7; vesicular stomatitis virus 5.7. Fibrinogen irradiated with 0.5 mM rutin did not significantly differ from unirradiated material in terms of clot time and breaking strength. In the absence of rutin, UVC irradiation of fibrinogen at similar fluence led to loss of solubility, increased clot time and the cleavage of fibrino‐peptides that reacted with dinitro‐phenyl hydrazine as a test for ketonic carbonyl groups. High‐performance liquid chromatography and mass spectrometry data showed that rutin exposed to UVC formed numerous breakdown, oxidation and combinational products. Experiments with 3H‐rutin showed that after UVC irradiation, subsequent processing by a C18 resin and alcohol precipitation removed >99% rutin, representing <10 ppm rutin in the final fibrinogen preparations. Residual 3H‐rutin was not covalently bonded to the fibrinogen. Immunochemical studies with rabbit antisera to UVC irradiated (with rutin) fibrinogen showed the absence of neoimmungens. By all measures, rutin prevents fibrinogen degradation during virucidal UVC irradiation.

Photodynamic Treatment of Red Blood Cell Concentrates For Virus Inactivation Enhances Red Blood Cell Aggregation: Protection with Antioxidants

Abstract— Photodynamic treatment (PDT) using phthalocyanines and red light appears to be a promising procedure for decontamination of red blood cell (RBC) concentrates for transfusion. A possible complication of this treatment may be induced aggregation of RBC. The production of RBC aggregates was measured with a novel computerized cell flow properties analyzer (CFA). The PDT of RBC concentrates with sulfonated aluminum phthalocy‐anine (AIPcS4) and the silicon phthalocyanine Pc 4 under virucidal conditions markedly enhanced RBC aggregation and higher shear stress was required to disperse these aggregates. The clusters of cells were huge and abnormally shaped, unlike the rouleaux formed by untreated RBC. This aggregation was prevented when a mixture of antioxidants was included during PDT. Addition of the antioxidants after PDT reduced aggregation only partially. It is concluded that inclusion of antioxidants during PDT of RBC concentrates prior to transfusion may reduce or eliminate the hemodynamic risk that the virucidal treatment may present to the recipient.

Elimination of potential mutagenicity in platelet concentrates that are virally inactivated with psoralens and ultraviolet A light

BACKGROUND: For virus sterilization of platelet concentrates (PCs), treatment with aminomethyltrimethyl psoralen (AMT) and long‐wavelength ultraviolet A light (UVA) has shown efficacy. It has been found that treatment with 50 micrograms per mL of AMT and 38 J per cm2 of UVA in the presence of 0.35‐mM rutin efficiently kills viruses while maintaining platelet integrity. There is, however, concern about the mutagenic potential of psoralens and UVA (PUVA)‐treated PCs.

Inactivation of Trypanosoma cruzi trypomastigote forms in blood components by photodynamic treatment with phthalocyanines.

Abstract— ‐Three phthalocyanine dyes HOSiPcOSi(CH3)2(CH2)3N(CH3)2 (Pc 4), HOSiPc‐OSi(CH3)2(CH2)3N+(CH3)3I‐ (Pc 5) and aluminum tetrasulfophthalocyanine hydroxide (AlOHPcS4) were evaluated for their ability to inactivate the trypomastigote form of Trypanosoma cruzi in fresh frozen plasma (FFP) and red blood cell concentrates (RBCC). The compound Pc 4 was found to be highly effective in killing T. cruzi, Pc 5 less effective and AlOHPcS4 ineffective. With FFP as the medium, a complete loss of parasite infectivity in vitro (≥5 log10) was found to occur with 2 μM Pc 4 after irradiation with red light (>600 nm) at a fiuence of 7.5 J/cm2, while with RBCC as the medium, a complete loss was found to occur at a fiuence of 15 J/cm2. Even without illumination, Pc 4 at 2 μM also killed about 3.7‐4.1 log10 of T. cruzi in FFP during 30 min. Observed differences in T. cruzi killing by the various phthalocyanines may relate to differences in binding; Pc 4 binds to the parasites about twice as much as Pc 5. Ultrastructural analysis of treated parasites suggests that mitochondria are a primary target of this photodynamic treatment. The data indicate that Pc 4 combined with exposure to red light could be used to eliminate bloodborne T. cruzi parasites from blood components intended for transfusion. The inactivation of T. cruzi by Pc 4 in the dark suggests a possible therapeutic application.

Quencher-enhanced specificity of psoralen-photosensitized virus inactivation in platelet concentrates

BACKGROUND: Treatment with psoralens and UVA (PUVA) has been shown to be efficacious in eliminating the risk of virus transmission by platelet concentrates (PCs). It has previously been demonstrated that, during the inactivation of cell‐free vesicular stomatitis virus (VSV) by aminomethyltrimethylpsoralen (AMT) and UVA in PCs, platelet function could be protected either by oxygen removal before irradiation or by inclusion of a type I free radical quencher, such as mannitol.