the photodecontamination of cellular blood components: mechanisms and use of photosensitization in...

The Photodecontamination of Cellular Blood Components: Mechanisms and Use of Photosensit ization in

Transfusion Medicine

E. Ben-Hur, A.C.E. Moor, H. Margolis-Nunno, R Gottlieb, M.M. Zuk, S. Lustigman, B. Horowitz, A. Brand, J. Van Steveninck, and T.M.A.R. Dubbelman

p HOTOSENSITIZATION is a term that covers many phenomena, but a general definition is

"the action of a component (photosensitizer) of a system that causes another component of the system to react to light." The photosensitizer in its ground state serves as a chromophore (light ab- sorber). On light absorption, the sensitizer becomes electronically excited to a higher energy level. The excited sensitizer molecule can react directly with a substrate or with some other molecule (frequently oxygen) in the reaction mixture, giving products that, in turn, can react with the substrate. Thus, photosensitized processes typically have an initial "light step" followed by one or more "dark steps."

The photosensitizer molecules in the dark are almost always in the singlet state, ~ in which the molecule has no unpaired electron spins. Absorp- tion of a photon (hv) promotes an electron to a higher molecular orbital without a change in its spin. Thus, the first excited state is also a singlet, tS. Few photosensitized reactions are directly mediated by this state because of its short lifetime (1 to 100 ns). Excited singlet states can decay to the ground state, emitting heat or light (fluorescence). In the case of an effective photosensitizer, there is a fast spin inversion of the high-energy electron, resulting in a metastable triplet state, 3S, which has two unpaired electrons and a much longer lifetime (1 to 1000 ps). The sensitizer in its excited triplet state can undergo many collisions with other molecules during its lifetime and, as a result, can mediate photosensitized reactions with high efficiency. These processes are shown schematically.

hv ~ ---' 1S ---, ~S ---, photosensitized reactions

PHOTODYNAMIC REACTIONS

In most photosensitized reactions, the triplet sensitizer ultimately returns to the ground state and can absorb another photon. In a few cases, the sensitizer is consumed stoichiometrically in the reaction. Oxygen is involved in many photosensitized reactions, which are termed photodynamic.

The involvement of oxygen is mediated by two pathways, as described subsequently.

Type 1 Mechanism

This mechanism involves the sensitizer in its excited triplet state undergoing its primary reaction with molecules in its vicinity by an electron or hydrogen transfer process. The result is the produc- tion of semireduced sensitizer, S" or SH', and a semioxidized substrate. These free radicals are reactive chemically. In most cases, the resultant substrate radicals react with oxygen to give oxidized products of various types (eg, peroxides), which can react further to initiate free-radical autooxidation processes. The semireduced sensitizer can react with ground state oxygen, 302 , to give a ground state sensitizer and the superoxide radical anion

S" + 302--~ ~ + 02"

which, in tuna, can react with biomolecules 1 or undergo dismutation to produce H20> The latter reaction is catalyzed by the enzyme superoxide dismutase (SOD). Superoxide can also be produced with low efficiency by the transfer of an electron from the sensitizer, in its excited triplet state, to oxygen:

3 S --1- 302 ~ S + -}- 0 2 "

The resulting semioxidized sensitizer can react with a reducing substrate to give ground state sensitizer and semioxidized substrate, which can react further.

Type I photosensitized processes are most effi-

From the New York Blood Center, New York, NY; Sylvius Laboratory, Leiden, The Netherlands; and The Blood Bank, Academic Hospital, Leiden, The Netherlands.

Supported in part by award No. RO1-HL41221 from the National Heart, Lung and Blood Institute and by grant No. 28-2414from Praeventiefonds.

Address reprint requests to E. Ben-Hur, New York Blood Center, 310 East 67th St, New York, NY 10021.

Copyright �9 1996 by W.B. Saunters Company 0887- 7963/96/1001-000253.00/0

Transfusion Medicfne Reviews, Vot X, No 1 (January), 1996: pp 15-22 15

16 BEN-HUR ET AL

cient at high substrate and low oxygen concentration, because oxygen competes with substrate for interaction with triplet sensitizer to produce singlet oxygen (see later). The formation of noncovalent sensitizer-substrate complexes before illumination increases the probability of type I reactions because of the proximity of reactants.

Type H Mechanism

This mechanism involves the triplet sensitizer interacting with ground state oxygen by energy transfer, resulting in ground state sensitizer and an electronically excited singlet state of oxygen, 102:

38 ~- 302----r 0 S + 102

Because both the sensitizer and ground state oxygen are triplets (ground state oxygen is one of a few stable triplet molecules), their interaction does not require a change in electron spin direction and is efficient. Oxygen can exist in two singlet excited states; the longer lived form, IAg, with an excess energy of 23 Kcal/mole (corresponding to a photon energy of about 1 eV = 1,270 nm) is the principal species involved in photodynamic reactions. Its lifetime in water is 4 ps. However, in the presence of biological molecules that can react with it, its lifetime can be much shorter. 2 The reaction of singlet oxygen with organic compounds is not spin forbidden, as it is with ground state oxygen. Furthermore, singlet oxygen is more electrophilic than 302 and, therefore, can react rapidly with the electron-rich regions of many biomolecules to give oxidized species. Some of the major reaction pathways are the following3:

1. Addition of 102 to olefins with allelic hydrogen atoms (such as unsaturated fatty acids and cholesterol) to give hydroperoxides

2. Addition of 102 to diene systems in heterocy- clics (such as histidine) to give endoperoxides

3. Reaction of 102 with organic sulfides (such as methionine) to form the corresponding sulfox- ides

ANAEROBIC PHOTOSENSITIZED REACTIONS

In some cases, light-absorbing molecules sensi- tize the photoalteration of biomolecules in the absence of oxygen. The most important group of these sensitizers are the psoralens (furocuomarins), which target nucleic acids and are described in detail later. Another group are ketones, such as

acetone, whose triplet excited states can be generated by light of 300 to 360 nm. In the dark the triplet state of ketones can be generated by oxida- tion with certain enzymes or by decomposition of lipid peroxides. 4 The triplet energy of many ketones is higher than that of DNA bases; therefore, excited ketones can react with DNA by triplet energy transfer, resulting in ultraviolet (UV)-type DNA damage, (viz the formation of cyclobutane pyrimidine dimers). 5,6

STERILIZATION OF CELLULAR COMPONENTS OF BLOOD

Transfusion of red blood cell concentrates (RBCC) and platelet concentrates (PC) involves a certain risk of transmission of pathogenic viruses despite serological screening and donor self- exclusion protocols. The risk of hepatitis B virus, hepatitis C virus (HCV), and human immunodeficiency virus (HIV) transmission in the United States with a single blood unit has been estimated at 0.0005%, 0.03%, and 0.0005%, respectively. 7 Patients who receive a large number of RBCC or PC units are at a much higher risk for virus infection. Other infectious agents of concern include cytomegalovirus and parvovirus in patients with compromised immune systems. In addition, transmission of parasitic infections, although rare in the United States, can be significant in develop- ing countries. These include Chagas disease, caused by Trypanosoma cruzi and malaria, caused by various Plasmodium sp. The former is endemic to Latin America, where 16 to 18 million people are infected, whereas the latter is widespread in Africa and Southeast Asia. In the United States, T. cruzi infection among immigrants from Latin America is about 5% 8 (ie, 100,000 to 150,000 persons). De- spite the fact that donated blood is not screened for T. cruzi in the United States, only two cases of transfusion-transmitted Chagas disease have been reported. 9 In both cases the disease was severe because the patients were immunosuppressed. Many more unrecognized cases are probably occurring in immunocompetent persons whose symptoms may be mild; nonspecific; and, therefore, not recognized as acute Chagas disease.

In the case of PC, an additional problem is bacterial contamination. PC are stored for several days at room temperature, conditions under which pathogenic bacteria can proliferate.l~ Indeed, transfusion of contaminated platelets may result in

PHOTOCHEMICAL DECONTAMINATION OF BLOOD

significant morbidity and mortality, lj,12 This is the main reason the storage of PC is limited to 5 days.

Sterilization appears to be the best way to ensure a high level of safety applicable to transfusion of blood and its components. Implementation of sterilization methods would avoid the necessity of introducing screening tests for rarely occurring viruses or parasites, public confidence in the blood supply would be enhanced, and the overall management of the blood supply would be improved. Indeed, before virus sterilization, almost every vial of a therapeutic coagulation factor concentrate contained HCV, and many vials contained HIV. With virus sterilization, these products have achieved an admirable safety record. ~3

Currently, all plasma-derived blood products are available in sterilized forms. Sterilization of cellular blood components presents a unique challenge because cell structure and function are disrupted more easily than those of individual proteins. Various approaches have been taken for virus sterilization of RBCC and PC.~4 With the exception of photochemical methods, none of these is able to inactivate sufficient virus under conditions where blood cell structure and function are maintained. As a result, almost all the efforts are now focused on the use of various photosensitizers in conjunction with the appropriate wavelength of light.

PSORALENS AND ULTRAVIOLET LIGHT FOR DECONTAMINATION OF PLATELET

CONCENTRATES

Psoralen (Fig 1) and its derivatives are heterocy- clic, planar compounds that are found in many plants. Psoralens bind to nucleic acids in the dark by intercalation between the bases. On exposure to UVA light (320 to 400 nm), the excited psoralen reacts via its 3,4-double-bond with a pyrimidine to form a cyclobutane monoadduct. The monoadducts can further react, via the 4', 5'-furan-side double-

8 1

[I. 4 r J ~

5 4 Fig 1. The molecular structure of psoralen.

17

bond, on absorption of a second photon with a pyrimidine on the complementary DNA strand to form cross-links. The latter photoproducts are more lethal than monoadducts because they prevent strand separation and are more difficult to repair. For a review of the photochemistry and photobiology of psoralens see reference 15.

The ability of psoralens to target nucleic acids is an obvious advantage for decontamination of platelets, which lack a nucleus. However, most psoralens can produce singlet oxygen when activated by UVA. 16 In addition, psoralens can form covalent adducts with unsaturated fatty acids. ~7 As a result, at psoralen-UVA (PUVA) doses required for virucidal action there is platelet damage that impairs their ability to aggregate in response to ago- nists.18,19 This problem has been dealt with initially by removing the oxygen from the platelet concentrate during irradiation. 18-20 Because oxygen depletion is inconvenient and potentially harmful for platelets, and difficult to standardize, the authors employed quenchers of reactive oxygen species to eliminate photodynamic reactions. 19,21 All of these studies employed either 8-methoxypsoralen (8- MOP) or 4'-aminomethyl-4',5',8-trimethylpsor- alen (AMT). The advantages of the latter are (1) it is water soluble; (2) because it is positively charged, it binds to nucleic acids in the dark more tightly and is particularly effective for the photoinactivation of single-stranded RNA viruses. 22 The disadvantage of AMT is that it is mutagenic in the dark in the presence of liver microsomal enzymes, with some of the Ames tester strains. 23 To try to circumvent this potential problem when using AMT clinically, the authors developed a procedure to remove free AMT following light exposure) 4 The method em- ploys C18 resin, which is effective in reducing AMT mutagenicity, as measured with the Ames test, below detection level without affecting platelet aggregation.

To quench the photodynamic action of AMT, mannitol, a scavenger of hydroxyl radicals, has been used. 19 However, mannitol has been found not to be sufficiently effective when higher AMT and UVA light doses (50 ~ag/mL and 38 J/cm 2) were used to inactivate cell-associated vesicular stomatitis virus (VSV). 21 Under these conditions plant flavonoids, which can quench both type I and type II reactive oxygen species, have been found to be effective. Among the flavonoids, rutin at a concentration of 0.35 mM gave the most favorable results

18

(ie, maximal protection against platelet damage with minimal reduction of VSV inactivation). Un- der these conditions, AMT-UVA also inactivated >6 logl0 of HIV, both cell free and cell associated (the authors' unpublished results) and >6 log10 T. cruzi in PC. 25 Because at the chronic stage of infection the number of parasites in the blood is small (<100/mL), this should yield a sufficient margin for T. cruzi sterilization. These treatment conditions resulted in fully functional platelets when assayed for their ability to correct bleeding time in a thrombocytopenic rabbit model 26 (the authors' unpublished data).

Another approach has been to use brominated psoralens, which are claimed to have improved selectivity toward virus inactivation. 27,28 More studies are needed, however, to demonstrate the valid- ity of this claim. Still other psoralens with undis- closed structure are reported to be highly virucidal, nonmutagenic, and not requiring oxygen removal for preservation of platelet function. 29 The lack of published data make it difficult to evaluate the latter claims.

The photochemical inactivation of pathogenic bacteria in PC using 8-MOP and UVA has been recently reported. 3~ Because the bacterial genome presents a much larger target to PUVA than viruses, bacteria are more sensitive to killing by this treatment. As a result, much lower 8-MOP concen- trations are required to inactivate >5 log~0 of five gram-positive and six gram-negative pathogenic bacteria and 4.1 log~0 of the most resistant gram- negative organism (Pseudomonas aeruginosa). Un- der these mild treatment conditions, oxygen depletion of the PC was not required to preserve platelet function in vitro.

RED LIGHT-ABSORBING PHOTOSENSITIZERS FOR THE PHOTODECONTAMINATION OF RED

BLOOD CELLS

In the sterilization of red blood cell concentrates (RBCC), it is important that the sensitizer absorbs maximally at h >600 nm, to avoid absorption of light by hemoglobin. Therefore, psoralens are not useful for this purpose. The sensitizers that are studied for use in RBCC exert their virucidal action mainly via photodynamic mechanisms (ie, 02 is required). In addition to the sensitizers described subsequently, several other sensitizers have been shown to inactivate viruses, such as hypericin, 31 rose bengal, 32 tertiophenes, 33 sapphyrins, 34 and tetraphenylporphyrins. 35 Even though their usefulness

BEN-HUR ET AL

in the sterilization of RBCC has not been estab- lished, extensive studies have been performed using hypericin and its derivatives, 36 some of which are highly active against cell-free HIV.

Sensitizers with porphyrin-like structures have been used in the treatment of different malignan- cies. 37,38 Most of these photosensitizers are amphi- philic and localize in membranes, suggesting a preference to inactivate lipid enveloped, rather than nonenveloped viruses. 39 Hematoporphyrin derivative (HPD) has proved to be efficient in the inactivation of different enveloped viruses in tissue culture medium and whole blood. 4~ Nonenvel- oped viruses were not inactivated by the treatment with these sensitizers and light, indicating that the major target for the photodynamic effect is the virus envelope. The benzoporphyrin derivative monoacid A (BPD-MA) has a strong absorption peak at 692 nm, thereby making it a more suitable sensitizer for use in RBCC than HPD, which absorbs weakly at 630 rim. BPD-MA has been shown to inactivate the enveloped virus VSV in whole blood (maximal >-7 lOgl0 kill). The RBC damage was determined (hemolysis) and was minimal 48 hours after treatment. 42,43 Recently it has been shown that free and cell-associated HIV-1 could be inactivated in blood by treatment with BPD-MA and red light. It was suggested that this approach could be a beneficial treatment modality for the management of blood-borne viral infections, such as HIV-1, either alone or in combination with other treatments. 44

Merocyanine 540 is a photosensitizing dye that has been tested for the purging of leukemia and lymphoma cells from autologous bone marrow transplants. A wide range of enveloped viruses have been shown to be highly susceptible for MC540 photoinactivation, when treated in a suspen- sion with a low concentration of erythrocytes (<15%). 45,46 The treated erythrocytes showed 4% to 20% more hemolysis than the untreated control and a decrease of 30% to 70% in ATP content after 42 days' storage, depending on erythrocyte concentration, light fluence, and dye concentration. A major drawback of MC540 for use in the sterilization of RBCC is that its absorption peak is located at 540 nm, which overlaps with that of hemoglobin. Derivatives of MC540, which absorb at higher wavelengths, could be useful in this respect, 47 but the effect of these derivatives on RBC has not been studied.

Methylene blue (hm,x = 665 nm) is used for the

PHOTOCHEMICAL DECONTAMINAT ION OF BLOOD

sterilization of plasma in Germany and Switzer- land. 4s It is effective in the inactivation of different enveloped viruses in RBCC, but the viruses display different sensitivities to the treatment. Intracellular HIV-1 is not sensitive to such treatment, indicating that it would be necessary to use a high-efficiency white cell filter or another inactivation step in combination with methylene blue treatment. 49 At a dose where efficient virus-kill is achieved, RBC surface is altered by the photodynamic treatment, resulting in specific IgG binding and K § leakage. 5~ Lower light fluences cause less RBC damage but also reduced virus kill. Methylene blue, therefore, does not appear to be useful for virus sterilization of RBC. In addition, methylene blue has a geno- toxic potential, increasing the mutation frequency in mouse lymphoma cells (at the thymidine kinase locus) approximately fourfold. 51

A more promising group of photosensitizers are the phthalocyanines (Fig 2). These are metal containing porphyrinlike ring structures, in which the methylene bridges connecting the pyrrole rings are replaced by nitrogen atoms, and benzene rings are fused onto the pyrroles. Phthalocyanines absorb strongly in the red ( h m a x = 675 nm) and are being studied as second-generation sensitizers for photodynamic treatment of cancer. 52,53

Aluminum phthalocyanine (AIPc) and its sulfonated derivatives AIPcS2 and AIPcS4 are effective in sensitizing the killing of VSV 54,55 added to whole blood or RBCC as well as cell-free and cell- associated HIV-1. However, the nonenveloped en- cephalomyocarditis virus was not inactivated, indicating that a major target in virus killing is the

H2Pc

OH O--Si N - - I - o ~ s _ I I

OH

AWcS4OH Pc 5

Fig 2. The structures of H2Pc, phthalocyanine; AIPcS4OH, a

phthalocyanine with ring substituents and a central metal carrying an axial ligand; and Pc 5, a phthalocyanine with a central metalloid carrying two axial ligands.

19

envelope of the virus. This is further supported by direct electron microscopic observations. 56 Red blood cell damage is minimal, as indicated by the low hemolysis after treatment. In further experi- ments with AIPcS4, it was shown that following virucidal treatment hemolysis was lower than 2%, the osmotic fragility of the RBC was only slightly increased, and autologous RBC had a normal recovery and circulation survival in baboon. 55 Us- ing the retrovirus N2, Ben-Hut et al57 studied the effects of sulfonation and metal ligand on virus inactivation in growth medium by phthalocyanines. Aluminum was more effective than zinc-phthalocyanine and sulfonation reduced viral killing, presumably because of reduced binding to the viral envelope. In whole blood, AIPcS4 was more effective than AIPc 54 probably because of a reduced binding of the sulfonated form to serum proteins. More dye is, therefore, available to bind with viral envelope.

New phthalocyanines containing an aluminum or silicon as the central ligand were also studied for their effects on RBC and virus inactivation. 58 These sensitizers have various siloxy groups as axial ligands (Fig 2). Some of these, especially Pc 5 and Pc 6, containing a silicon atom as their central metal and cationic axial ligand, have a higher virus-kill efficiency than AIPcS4; however, there is no clear relationship between the central ligand or charge with virus kill. RBC damage was more pronounced with Pc 5 and Pc 6, but was effectively reduced when cells were treated in plasma instead of buffer. 58

The mechanisms of virus inactivation appear to be different than those leading to RBC damage. From studies using different quenchers, which are specific for radicals generated by either electron transfer reactions (type I) or energy transfer reactions (type II), it was possible to conclude that virus killing is caused mainly by a mechanism involving singlet oxygen generation (type II). The influence of quenchers on RBC damage was studied by measuring hemolysis, circulatory survival in rab- bits, and binding of IgG to the RBC membrane. From these quencher studies, it was concluded that RBC damage was caused by a combination of type I and II reactions. Quenching of the radicals formed in a type I reaction with GSH (reduced glutathione) and mannitol is, therefore, an effective way of reducing RBC damage, without reducing virus kill. 59,6~ The potassium leakage, which occurs after treatment, could not be prevented with the quench-

20

ers mentioned earlier, but the water-soluble vitamin E analogue Trolox was able to reduce the leakage significantly following the 2-week storage of RBC. In addition, Trolox is able to prevent the phthalocyanine-induced reduction of negative charges on the RBC surface. Trolox did not interfere with the inactivation of VSV by the treatment with phthalocyanines and red light. 61

Another way to reduce RBC damage selectively without affecting virus inactivation is the use of a high light fluence rate (irradiance). A high fluence rate (80 mW/cm 2) is less damaging for RBC than a lower one (5 mW/cm 2) while virus kill is not affected by a change in fluence rate. The explana- tion for this phenomenon is not known, but the effect could be used to increase the selectivity of the treatment. 62

The silicon phthalocyanine Pc 4 (a form of the neutral Pc 5) proved to be capable of inactivating HIV in all its forms (cell free, cell associated, and latent) at doses that were even lower than those required for complete VSV kill (Table 1). In addition, more than 104 tissue culture infectious doses of T. cruzi trypomastigotes were inactivated in RBCC as well as the malaria parasite Plasmo- diumfalciparum, 63 at light doses much lower than that required for virus inactivation. Thus, RBCC treated for inactivation of human pathogenic viruses will also lose the ability to transmit malaria and Chagas disease.

In conclusion, the most promising method for the sterilization of RBCC appears to be the use of phthalocyanines in combination with different quenchers of type I reactions. Together with the proper choice of a light source with a high irradiance (eg, light-emitting diodes) damage to RBC as a result of the treatment can become insignificant.

Table 1. Virus Inactivation by Pc 4 in RBCC*

Log10 Virus Kill

HIV

Light Dose (J/cm 2) VSV Cell Free Cell Associated Latent

0 0 0 0 0 22.5 1.0 2.5 2.4 ~5.5 45 2.1 4.5 4.5 ~5.5

67.5 3.5 ~5.5 6.0 ~5.5

90 5.2 ~5.5 >6.5 ~5.5

Abbreviations: RBCC, red blood cell concentrates; VSV, vesicular stomatitis virus.

*RBCC spiked with the appropriate virus were irradiated with red light (25 mW/cm 2) in the presence of the quenchers

Trolox, glutathione and mannitol.

BEN-HUR ET AL

PHOTODYNAMIC PURGING OF BONE MARROW FOR AUTOLOGOUS

TRANSPLANTATION

The use of hematopoietic stem-cell transplantation in cancer treatment has increased dramatically in recent years. 64 To reduce the possible risk of relapse from the infused autologous stem cells, purging of these cells before infusion or storage is employed. Purging methods can be distinguished as positive or negative selection. In the former, stem cells are selected by biophysical or immuno- logical techniques or are preferentially expanded. Negative selection targets the cancer cells to eradi- cate them and leave the stem cells intact. This includes antibody methods as well as biochemical, biophysical, and biological methods. All of these methods usually achieve 3 to 5 logs of cancer cell depletion. However, 6 to 8 log reductions are needed to achieve complete elimination of tumor cells in the graft. This implies that more than two purging methods will likely be needed.

Photodynamic purging of cancer cells in bone marrow was pioneered by Sieber et a165'66 using merocyanine 540 as a photosensitizer. Despite a decade of research involving this dye, including a clinical trial, it has not been approved, presumably because it is not an effective sensitizer. More recently, other photosensitizers have been studied, such as aluminum phthalocyanine 6v and BPD. 68 The latter has been shown to lower tumor burden by 4 logs with virtually no loss of essential hemopoietic progenitors at extremely low concentration (25 ng/mL). Even more important is the observation that even multiple-drug-resistant (MDR) cells are responsive to photodynamic treatment. 69 This, however, was true only for Photofrin and not for BPD. Thus, the usefulness of the latter in bone marrow purging remains to be evaluated. The authors have found that the silicon phthalocyanine Pc 4 is highly effective in killing breast cancer cells and HL-60 leukemia cells. At a dose that eliminated 6 log10 of cancer cells (20 nM Pc 4 and 20 J/cm 2 red light) there was 100% recovery of bone marrow progenitors (CFU-GM).7~ If Pc 4-PDT will prove as effective against cancer cells express- ing the MDR phenotype this could be a highly efficient purging method. This approach could be further enhanced by including the tetrapeptide AcSDKP, which protects the CFU-GM progenitors but not leukemic cells (HL-60 and K-562) against PDT. 71

PHOTOCHEMICAL DECONTAMINATION OF BLOOD 21

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