Photoinactivation of viral and bacterial blood contaminants using halogenated coumarins

A method for photoinactivating viral and/or bacterial contaminants in blood, blood products or cell cultures is presented. The blood, blood product or cell culture is mixed with an halogenated coumarin sensitizer and irradiated with UV light to inactivate the viral and/or bacterial contaminants.

FIELD OF THE INVENTION 
This invention relates to the general field of the inactivation of vital 
and bacterial contamination of blood and blood products including 
compositions comprising peripheral blood cells (red blood cells, 
platelets, leukocytes, stem cells, etc.), plasma protein fractions 
(albumin, clotting factors, etc.) from collected whole blood, the blood of 
vitally infected persons, ex vivo media used in the preparation of 
antiviral vaccines, and cell culture media such as fetal bovine serum, 
bovine serum or products derived from these sources. 
BACKGROUND OF THE INVENTION 
A major concern in the transfusion of donated, stored whole human blood or 
the various blood cells or protein fractions isolated from whole blood is 
the possibility of viral contamination. Of particular concern are the 
blood-borne viruses that cause hepatitis (especially hepatitis A, 
hepatitis B, and hepatitis C) and acquired immune deficiency syndrome 
(AIDS). While any number of cell washing protocols may reduce the viral 
contamination load for samples of blood cells, by physical elution of the 
much smaller virus particles, such washing alone is insufficient to reduce 
viral contamination to safe levels. In fact, some viruses are believed to 
be cell-associated, and unlikely to be removed by extensive washing and 
centrifugal pelleting of the cells. Current theory suggests that safe 
levels will ultimately require at least a 6 log (6 orders of magnitude) 
demonstrated reduction in infectious viral titer for cellular blood 
components. This 6 log threshold may be greater for plasma protein 
components, especially the clotting factors (Factor VIII, Factor IX) that 
are administered throughout the life of some hemophilia patients. 
All blood collected in the United States is now screened for six infectious 
agents: HIV-1, HIV-2, HTLV-1, hepatitis B virus, hepatitis C virus and 
syphilis. Additionally, donors are screened for risk factors, and 
potential donors are eliminated that are considered at risk for the HIV 
virus. Despite these measures, the risk of becoming infected by a 
potentially deadly virus or bacteria via the transfusion of blood or blood 
products remains serious. Screens for contaminants are by nature not 
foolproof. There is also the quite likely occurrence of new infectious 
agents that enter the blood supply before the significance of the event is 
known. For example, by the end of Jun. 1992, the Center for Disease 
Control reports that 4,959 AIDS cases could be traced to the receipt of 
blood transfusions, blood components or tissue. 
Viral inactivation by stringent sterilization is not acceptable since this 
could also destroy the functional components of the blood, particularly 
the erythrocytes (red blood cells) and thrombocytes (platelets) and the 
labile plasma proteins, such as clotting factor VIII. Viable RBC's can be 
characterized by one or more of the following: capability of synthesizing 
ATP; cell morphology; P.sub.50 values; filterability or deformability; 
oxyhemoglobin, methemoglobin and hemochrome values; MCV, MCH, and MCHC 
values; cell enzyme activity; and in vivo survival. Thus, if virally 
inactivated cells are damaged to the extent that the cells are not capable 
of metabolizing or synthesizing ATP, or the cell circulation is 
compromised, then their utility in transfusion medicine is compromised. 
Viral inactivation by stringent steam sterilization is not acceptable since 
this also destroys the functional components of the blood, particularly 
the blood cells and plasma proteins. Dry heat sterilization, like wet 
steam, is harmful to blood cells and blood proteins at the levels needed 
to reduce viral infectivity. Use of stabilizing agents such as 
carbohydrates does not provide sufficient protection to the delicate blood 
cells and proteins from the general effects of exposure to high 
temperature and pressure. 
Methods that are currently employed with purified plasma protein fractions, 
often followed by lyophilization of the protein preparation, include 
treatment with organic solvents and heat or extraction with detergents to 
disrupt the lipid coat of membrane enveloped viruses. Lyophilization 
(freeze-drying) alone has not proven sufficient to inactivate viruses, or 
to render blood proteins sufficiently stable to the effects of heat 
sterilization. The organic solvent or detergent method employed with 
purified blood proteins cannot be used with blood cells as these chemicals 
destroy the lipid membrane that surrounds the cells. 
Another viral inactivation approach for plasma proteins first demonstrated 
in 1958 has involved the use of a chemical compound, beta-propiolactone, 
with ultraviolet (UV) irradiation. This method has not found acceptance in 
the United States due to concern over the toxicity of beta-propiolactone 
in the amounts used to achieve some demonstrable viral inactivation and 
also due to unacceptable levels of damage to the proteins caused by the 
chemical agents. Concern has also been raised over the explosive potential 
for beta-propiolactone as well. 
There is significant interest in an effective viral inactivation treatment 
for human blood components, which will not damage the valuable blood cells 
or proteins. The treatment must be nontoxic and selective for viruses, 
while allowing the intermingled blood cells or proteins to survive 
unharmed. 
There is an immediate need to develop protocols for the inactivation of 
viruses that can be present in the human red blood cell supply. For 
example, only recently has a test been developed for Non A, Non B 
hepatitis, but such screening methods, while reducing the incidence of 
viral transmission, do not make the blood supply completely safe or virus 
free. Current statistics indicate that the transfusion risk per unit of 
transfused blood is as high as 1:3,000 for Non A, Non B hepatitis 
(hepatitis C), and ranges from 1:60,000 to 1:225,000 for HIV, depending on 
geographic location. Clearly, it is desirable to develop a method which 
inactivates or removes virus indiscriminately from the blood. 
Contamination problems also exist for blood plasma protein fractions, such 
as plasma fractions containing immune globulins and clotting factors. For 
example, new cases of non A, non B hepatitis and hepatitis A have occurred 
in hemophilia patients receiving protein fractions containing Factor VIII 
which have been treated for viral inactivation according to approved 
methods. Therefore, there is a need for improved viral inactivation 
treatment of blood protein fractions. 
The ability to inactivate bacterial contaminants from blood and blood 
products may be as critical as reducing viral contaminants. Between 1986 
and 1991, the Food and Drug Administration reported that 15.9% of all 
transfusion related fatalities were associated with the transfusion of 
bacterially contaminated blood components. Most of these fatalities were 
due to the transfusion of bacterially contaminated platelets. 
Psoralens are naturally occurring compounds which have been used 
therapeutically for millennia in Asia and Africa. The action of psoralens 
and light has been used to treat vitiligo and psoriasis (PUVA therapy; 
Psoralen Ultra Violet A) and more recently various forms of lymphoma. 
Psoralen will bind to nucleic acid double helices by intercalation between 
base pairs; adenine, guanine, cytosine and thymine (DNA) or uracil (RNA). 
Upon absorption of UVA photons the psoralen excited state has been shown 
to react with a thymine or uracil double bond and covalently attach to 
both strands of a nucleic acid helix. 
The crosslinking reaction is specific for a thymine (DNA) or uracil (RNA) 
base and will proceed only if the psoralen is intercalated in a site 
containing thymine or uracil. The initial photoadduct can absorb a second 
UVA photon and react with a second thymine or uracil on the opposing 
strand of the double helix to crosslink the two strands of the double 
helix. 
##STR1## 
Lethal damage to a cell or virus occurs when a psoralen intercalated into a 
nucleic acid duplex in sites containing two thymines (or uracils) on 
opposing strands sequentially absorb 2 UVA photons. This is an inefficient 
process because two low probability events are required, the localization 
of the psoralen into sites with two thymines (or uracils) present and its 
sequential absorption of 2 UVA photons. 
U.S. Pat. No. 4,748,120 of Wiesehan is an example of the use of certain 
substituted psoralens by a photochemical decontamination process for the 
treatment of blood or blood products. The psoralens described for use in 
the process do not include halogenated psoralens, or psoralens with 
non-hydrogen binding ionic substituents. Using traditional psoralens such 
as 8-MOP, AMT and HMT, it is imperative that additives be added into the 
blood product solution in conjunction with UV irradiation in order to 
scavenge singlet oxygen and other highly reactive oxygen species formed by 
irradiation of the psoralen. Without the addition of reactive oxygen 
species scavengers, cellular components and protein components in the 
blood product are seriously damaged upon irradiation. (See also, U.S. Pat. 
No. 5,176,921.) It is clear, therefore, that irradiation of psoralens such 
as 8-MOP and AMT in aqueous solution creates a competition between the 
inefficient photocrosslinking reaction and the generation of highly 
reactive oxygen species. It is also possible that much of the viral 
deactivation seen using these photosensitizers actually results from the 
action of the reactive oxygen species against the viral contaminants 
rather than the inefficient photocrosslinking mechanism. 
Attempts to inactivate viral decontaminants using photosensitizers and 
light have also been developed using some non-psoralen photosensitizers. 
The photosensitizers that have been employed are typically dyes. Examples 
include dihematoporphyrin ether (DHE), Merocyanine 540 (MC540) and 
methylene blue. 
In any event, an effective radiation photosensitizer must bind specifically 
to nucleic acids and must not accumulate in significant amounts in lipid 
bilayers, which are common to viruses, erythrocytes, and platelets. 
Although there is evidence that psoralens bind to nucleic acids by 
intercalation, neutral psoralens such as 8-MOP (8-methoxypsoralen) are 
uncharged and thus also have a high affinity for the interior of lipid 
bilayers and cell membranes. 
##STR2## 
The binding of 8-MOP to cell membranes, shown above, would be acceptable if 
the psoralen bound to the lipid was photochemically inert. However, Midden 
(W. R. Midden, Psoralen DNA photobiology, Vol II (ed. F. P. Gaspalloco) 
CRC press, pp. 1. (1988) has presented evidence that psoralens photoreact 
with unsaturated lipids and photoreact with molecular oxygen to produce 
active oxygen species such as superoxide and singlet oxygen that cause 
lethal damage to membranes. Thus, it is believed that 8-MOP is an 
unacceptable photosensitizer because it sensitizes indiscriminate damage 
to both cells and viruses. 
Positively charged psoralens such as AMT 
(4'-aminomethyl-4,5',8-trimethylpsoralen) will not bind to the interior of 
phospholipid bilayers (membranes) because of the presence of the charge. 
However, AMT contains an acidic hydrogen which can bind to the 
phospholipid head group by hydrogen bonding, shown below. 
##STR3## 
Thus AMT is believed to be an unacceptable photosensitizer because it will 
indiscriminately sensitize damage to viral membranes and to the membranes 
of erythrocytes and platelets. 
Studies of the affects of cationic sidechains on furocoumarins as 
photosensitizers are reviewed in Psoralen DNA Photobiology, Vol. I, ed. F. 
Gaspano, CRC Press, Inc., Boca Raton, Fla., Chapter 2. The following 
points can be gleaned from this review: 
1) The intent of this line of research was to improve the poor water 
solubility of the basic psoralen nucleus. 
2) None of the psoralens described were halogenated as are the 
photosensitizers of the present invention. 
4) Later conducted studies showed that a cationic group on a large linker, 
when added to the 5 or 8 position on the psoralen ring, gave the psoralen 
nucleus improved binding with native DNA relative to corresponding 5-MOP 
and 8-MOP analogues. 
5) Sidechain substitution at the 5 position was found to be less desirable 
than substitution at the 8 position. 
6) A study of 5-aminomethyl derivatives of 8-MOP showed that most of the 
amino compounds had a much lower ability to both photobind and form 
crosslinks to DNA compared to 8-MOP. These reports actually suggest that 
the primary amino functionality is the preferred ionic species for both 
photobinding and crosslinking. 
U.S. Pat. No. 5,216,176 of Heindel describes a large number of psoralens 
and coumarins that have some effectiveness as photoactivated inhibitors of 
epidermal growth factor. Included among the vast functionalities that 
could be included in the psoralen or coumarin backbone were halogens and 
amines. The inventors did not recognize the significance of either 
functionality or the benefits of a photosensitizer including both 
functionalities. 
U.S. patent applications Ser. Nos. 08/165,305 and 08/091,674 are commonly 
assigned with the present application, and are parent applications to this 
application. These applications disclose the use of a novel class of 
psoralen photosensitizers that are superior for use with irradiation to 
inactivate viral and bacterial contaminants in blood and blood products. 
The psoralens are characterized by the presence of a halogen substituent 
and a non-hydrogen binding ionic substituent to the basic psoralen side 
chain. See also, Goodrich et al. Proc. Natl. Acad. Sci. USA, 91:5552-56 
(1994). 
SUMMARY OF THE INVENTION 
The present invention provides a method for the inactivation of viral and 
bacterial contaminants present in blood and blood protein fractions. 
The present invention involves utilization of photosensitizers which bind 
selectively to a viral nucleic acid, coat protein or membrane envelope. 
The photosensitizer is also a moiety which can be activated upon exposure 
to radiation, which may be in the form of ultra-violet radiation or 
ionizing radiation, such as X-rays, which can penetrate the sample 
containing the contamination. 
The present invention is also applicable to inactivation of blood-borne 
bacterial contaminants, and to blood-borne parasitic contaminants, since 
such infectious organisms rely on nucleic acids for their growth and 
propagation. Since purified blood plasma protein fractions are 
substantially free of human nucleic acids, and mature human peripheral 
blood cells, particularly red blood cells and platelets lack their own 
genomic DNA/RNA, the use of nucleic acid-binding photosensitizers is 
especially useful for the problem of treating blood contaminants. 
The present invention may also be applied to viral inactivation of tissues 
and organs used for transplantation, and used in topical creams or 
ointments for treatment of skin disorders or for topical decontamination. 
The present invention may also be used in the manufacture of viral 
vaccines for human or veterinary use, particularly to produce live, 
nonviable or attenuated viral vaccines. The present invention may also be 
used in the treatment of certain proliferative cancers, especially solid 
localized tumors accessible via a fiber optic light device or superficial 
skin cancers. 
The present invention utilizes a class of compounds that have a selective 
affinity to nucleic acid. The class of compounds also contains a halogen 
substituent and a water solubilization moiety, such as, quaternary 
ammonium ion or phosphonium ion. These materials comprise a relatively low 
toxicity class of compounds, which can selectively bind to the nucleic 
acid (single-stranded DNA, double-stranded DNA, or RNA) that comprise the 
genetic material of viruses. The bound compound can be activated by 
exposure to radiation, such as ultraviolet radiation (UV light of a 
defined wavelength), or ionizing radiation such as x-rays, after which the 
activated compound damages the bound viral nucleic acid or viral membranes 
rendering the virus sterile and non-infectious. Activation of the 
selectively bound chemical photosensitizer focuses the photochemistry and 
radiation chemistry to the viral nucleic acid or viral membranes and 
limits exposure to nearby cellular components or plasma proteins. 
The preferred class of photosensitizers for use with the present invention 
are characterized generally as follows: a) they are intercalators, and 
they are comprised of either b) at least one halogen substituent or c) at 
least one non-hydrogen bonding ionic substituent. In preferred embodiments 
the photosensitizers comprise at least one halogen substituent and at 
least one non-hydrogen bonding ionic substituent. Particularly preferred 
photosensitizers are psoralens and coumarins comprising at least one 
halogen substituent and at least one non-hydrogen bonding ionic 
substituent. 
In one embodiment of the present invention, the preferred photosensitizers 
are intercalators that fluoresce and that are comprised of either a) at 
least one halogen substituent or b) at least one non-hydrogen bonding 
ionic substituent. The preferred photosensitizers according to this 
embodiment are the substituted coumarins having the structure as shown 
below. 
##STR4## 
The photosensitizers disclosed herein are suited for the inactivation of a 
variety of viral and bacterial contaminants associated with blood and 
blood products. The present invention specifically includes the 
photoinactivation of blood and blood products contaminated with Human 
Immunodeficiency Virus-1 (HIV-1), Sindbis virus, Cytomegalovirus, 
Vesicular Stomatitis Virus (VSV), and Herpes Simplex Virus Type 1 (HSV-1), 
using the photosensitizers of the present invention. 
The present invention also demonstrates the flexibility of adding one or 
more halogen atoms to any cyclic ring structure capable of intercalation 
between the stacked nucleotide bases in a nucleic acid (either DNA or RNA) 
to confer new photoactive properties to the intercalator. In the present 
invention essentially any intercalating molecule (psoralens, coumarins, or 
other polycyclic ring structures) can be selectively modified by 
halogenation or addition of non-hydrogen bonding ionic substituents to 
impart advantages in its reaction photochemistry and its competitive 
binding affinity for nucleic acids over cell membranes or charged 
proteins. 
In one embodiment, halogenation of psoralen enables the molecule, once 
intercalated within the nucleic acid, to undergo a strand cleavage 
reaction upon light activation that non-halogenated psoralens cannot 
initiate. The nucleic acid strand cleavage is due to a novel electron 
transfer pathway (see FIG. 1) created by the breaking of the 
carbon-halogen bond upon input of appropriate radiation energy. The 
mechanism for this alternate chemical reaction requires a single photon of 
light and is more efficient than the crosslinking reaction that normally 
occurs with non-halogenated psoralens. In addition, as shown in FIGS. 1 
and 2, the electron transfer reaction involves transfer from a donor 
(usually a guanine base when the intercalator is inserted in nucleic acid) 
and an acceptor (the carbon radical created by breakage of the 
carbon-halogen atom). Since the donor and acceptor species must be in 
close physical proximity for the transfer reaction to proceed, most damage 
is limited to the nucleic acid as desired for viral inactivation. 
In a second embodiment, halogenation of a coumarin imparts totally new 
photoactive properties useful for viral inactivation. Coumarins, unlike 
psoralens, do not have an inherent ability to crosslink nucleic acid 
strands upon exposure to radiation, and hence have not heretofore found 
application as photosensitizers. However, as shown in the present 
invention (FIG. 2), halogenation of this class of intercalating molecules 
confers the ability to undergo the electron transfer mechanism, thereby 
imparting new properties to the molecule. Without intending to limit the 
present invention, the inventors believe that the example of coumarin 
halogenation demonstrates that these principles can be extended to any 
intercalating molecule, to confer new photoactive properties. 
Due to the flexibility in adding halogen substituents or non-hydrogen 
bonding ionic substituents to virtually any cyclic or polycyclic ring 
structure, the inventors envision that new and useful molecules can be 
created by adapting the present invention to many known classes of ring 
compounds, whether those compounds comprise intercalating agents or not. 
For example, known classes of compounds that may be improved by the 
present invention include, porphyrins, phthalocyanines, quinones, 
hypericin, and many organic dye molecules (such as coumarins) including 
merocyanine dyes, methylene blue, eosin dyes, and others. 
Without intending to limit the present invention, the inventors anticipate 
that new classes of compounds prepared according to the principles of this 
invention will find application in numerous fields in addition to 
decontamination of blood and blood products. The new chemical reaction 
properties imparted by halogenation and the selective binding properties 
imparted by the use of non-hydrogen bonding ionic substituents, may be 
grafted onto known classes of molecules to impart advantageous chemical 
reaction and targeting properties to these molecules. Psoralens for 
example, such as 8-methyoxypsoralen (8-MOP) have been used in therapeutic 
photophoresis to treat cutaneous T-cell lymphoma, scleroderma, and other 
cancers or skin disorders. The modified psoralen derivatives of the 
present invention (or other classes of compounds modified according to the 
present invention) may prove more efficacious in therapeutic photophoresis 
applications. 
As a second example, organic dyes such as methylene blue (which is not 
considered a nucleic acid intercalating compound) have been used for viral 
inactivation treatments of blood plasma, with questionable success. It is 
contemplated that such organic dyes, modified according to the present 
invention, may prove more efficacious in such an application than the 
unmodified dye. 
Without intending to limit the present invention, the inventors further 
anticipate that the fluorescent coumarin photosensitizers described herein 
may also be used in combination with known photosensitizing molecules that 
absorb in the visible light wavelength region. FIG. 11 shows the 
fluorescence emission spectrum of one such coumarin molecule, 
Photosensitizer A, having an emission peak at 414 nm in the visible light 
spectrum. The emission spectrum of Photosensitizer A extends beyond 500 
nm, which can overlap the absorbance range of certain visible light 
activated molecules. It is therefore anticipated that a combination of a 
visible fluorescing photosensitizer with one or more photosensitizers that 
absorb in the visible light region may be utilized for enhanced virucidal 
or cytotoxic effect. Examples of photosensitizers that absorb in the 
visible light region include hypericin, pthalocyanines, porphyrins, and 
organic dyes such as methylene blue (see International Patent Application 
WO/94 14956 wherein hypericin is activated via a chemiluminescent reaction 
between luciferin and luciferase). 
Other fields of application wherein the present invention may find 
application include the preparation of non-infectious viral vaccines, 
therapeutic treatment of immune system disorders by photophoresis, 
elimination of viable nucleated cells such as leukocytes via the 
cytotoxicity of nucleic acid binding photosensitizers, and possible 
treatment for certain accessible cancers and tumors, again exploiting the 
cytotoxic effects of nucleic acid binding photosensitizers. 
The inventors further anticipate that the problem of singlet oxygen 
production by UV irradiation of traditional psoralen molecules can also be 
reduced by incorporating a quenching sidechain moiety onto the psoralen 
nucleus. An example of such a compound is shown below. 
##STR5## 
In this compound the non-hydrogen bonding ionic substituent of the present 
invention further comprises a quaternary ammonium pyridyl group. This 
quaternary ammonium pyridyl group will act as a quencher of the UV excited 
triplet state of the psoralen molecule (see FIG. 1). 
While not intending to be bound by theory, in principle the quenching 
pyridyl group or a comparable functional group will deactivate the triplet 
state of any psoralen or intercalator, thereby preventing formation of 
undesired singlet oxygen. The pyridyl group quenches the excited triplet 
state by promoting electron transfer. In the presence of the pyridium ion 
the halointercalator serves as the donor, and carbon centered radicals are 
not formed. The electron is transferred from the halointercalator to the 
pyridium ion and back. This reversible electron transfer shorts out the 
triplet state before it can react to make singlet oxygen. (Although in 
principle the pyridium ions could quench the excited singlet state of the 
halointercalator, the lifetime of the singlet state is so short that 
little quenching would occur.) 
Reduction of singlet oxygen production should minimize damage to lipid 
membranes or proteins. Attachment of a quenching group directly onto the 
psoralen nucleus provides proximity to the excited psoralen, and should 
obviate the need for addition of exogenous quenching agents (such as 
oxygen scavengers, reducing agents, or sugars) into the medium. Without 
limiting the scope of the present invention, the inventors anticipate that 
quenching sidechains that comprise both a non-hydrogen bonding ionic 
feature and a triplet quenching feature will be useful for selective viral 
inactivation of complex biological systems such as blood, blood plasma, or 
isolated blood cell fractions.

DETAILED DESCRIPTION OF THE INVENTION 
The present invention is directed to methods for reducing viral, bacterial 
and other parasitic contamination in blood, blood components, cell 
cultures or cell culture components by irradiation in the presence of a 
chemical photosensitizer. Photosensitizers are disclosed which are 
particularly useful to decontaminate liquid compositions, such as blood, 
blood components, reconstituted lyophilized cells, and the like, using UV 
irradiation. 
According to the present invention, a radiation sensitizing chemical 
compound is added to a liquid suspension of infectious viruses and/or 
bacteria and/or parasites, and the mixture is exposed to UV light or 
ionizing radiation. Assays of viral infectivity demonstrate the 
effectiveness of the compounds to inactivate the viruses, compared to 
radiation treatment alone. 
The present invention includes a method for reducing viral, bacterial and 
other parasitic contamination from a biological solution. Biological 
solutions include, but are not limited to, solutions comprising blood, a 
blood component, cell culture or a component of a cell culture. The method 
comprises mixing the composition in a liquid state with a photochemical 
photosensitizer capable of binding to the viral, bacterial or parasitic 
contamination. The photochemical photosensitizer is capable of being 
activated by irradiation under conditions of sufficient wavelength, 
intensity and period of exposure to inactivate the contaminant, while at 
the same time the conditions for irradiation are insufficient to produce 
reactive oxygen species in the composition at levels which substantially 
impair the physiological activity of the treated composition. The 
composition containing the photosensitizer is then irradiated under 
conditions where the concentration of biologically active contaminant is 
reduced and the physiological activity of the composition is substantially 
unimpaired. 
One of the most critical elements of the present invention is the use of a 
novel class of photosensitizer. A photosensitizer is defined for the 
purposes of this application as a chemical compound that has a 
light-absorbing chromophore that absorbs radiation in the ultraviolet or 
visible spectrum, and that is capable of inactivating viral or bacterial 
contaminants in blood or blood products. 
The photosensitizers of the present invention are characterized by their 
ability to bind to the nucleic acid components of the viral or bacterial 
contaminants that are to be inactivated. The blood and blood product 
compositions that are to be treated according to the method of this 
invention all contain at least some cellular components or complex 
proteins. 
In one embodiment of the invention, the photosensitizers of this invention 
are characterized as comprising a lipophilic moiety, a hydrophilic moiety 
and a photoreactive moiety. 
The photosensitizers of this invention are preferably nucleic acid 
intercalators that are comprised of either 1) at least one halogen atom; 
and b) at least one non-hydrogen bonding ionic moiety. Intercalators are 
defined broadly herein as any chemical compound that has a specific 
affinity to double or single stranded nucleic acid. More specifically, 
intercalators are chemicals -- not including nucleic acids, proteins or 
peptides -- that locate themselves between neighboring base pairs in 
nucleic acids. Intercalators are generally characterized by the presence 
of a relatively planar rigid, multi-cyclic pi-conjugated chemical 
backbone. Those skilled in the art are familiar with a relatively large 
number of intercalators, and are generally able to predict the types of 
chemical species that are able to function as intercalators based on the 
chemical structure of the backbone of the chemical species. Psoralens and 
coumarins, which are the preferred basic structure for the intercalators 
of the present invention, are just two examples of chemical backbone 
structures capable of nucleic acid intercalation. 
Preferred photosensitizers of the present invention comprise at least one 
halogen substituent. The halogens include F, Cl, Br and I. In the 
preferred embodiments of the present invention, the photosensitizer 
contains at least one bromine or chlorine atom. 
Preferred photosensitizers of the present invention comprise at least one 
non-hydrogen bonding ionic substituent. Chemical functionalities that are 
ionic and non-hydrogen bonding include quaternary ammonium functionalities 
and phosphonium functionalities. A variety of additional functionalities 
that are both ionic and non-hydrogen bonding are familiar to those skilled 
in the art, and equally applicable for use with this invention. 
In the preferred embodiments of the invention, the non-hydrogen bonding 
ionic substituent is linked to the backbone of the chemical intercalator 
via a spacer unit. The spacer can be selected from any of a number of 
chemical subunits known to those skilled in art, but in the preferred 
embodiments is composed of a saturated linear alkoxy group. In the most 
preferred embodiment the spacer element is --O(CH.sub.2).sub.3 --. 
The most preferred non-hydrogen bonding ionic functionalities are 
quaternary ammonium functionalities, more specifically trialkyl quaternary 
ammonium and even more specifically --O(CH.sub.2).sub.3 N.sup..noteq. 
(CH.sub.2 CH.sub.3).sub.3. 
Two preferred photosensitizers of the present invention are the following: 
##STR6## 
Compound A is a coumarin based photosensitizer, and compound B is a 
psoralen or furocoumarin based photosensitizer. 
Additional preferred embodiments of the present invention include the 
following coumarin based photosensitizers: 
##STR7## 
The synthesis of photosensitizer A is described in Example 9 below, 
according to the scheme shown in FIG. 7. The synthesis of photosensitizer 
D is described in Example 14 below, according to the scheme shown in FIG. 
13. 
Upon irradiation with UV light, compound A has been shown to be effective 
at viral inactivation while compound B has been shown to be effective at 
viral and bacterial inactivation. Compounds A, D and E also fluoresce upon 
UV irradiation. It is theorized by the present inventors that the 
fluorescence pathway for the dispersion of energy from the excited state 
of irradiated compounds A, D and E as depicted in FIG. 1, acts to reduce 
the production of highly reactive oxygen species in blood and blood 
components. The proposed reaction mechanism for the inactivation of viral 
contaminants using compound A and light is shown in FIG. 2. According to 
the proposed mechanism -- which is speculative and not intended to limit 
the scope of the invention -- the photoreaction is initiated by an 
electron transfer from a guanine residue to the photosensitizer in its 
executed singlet state. Electron transfer is followed by Br--C bond 
homolysis and the generation of a coumarin radical that can attack the 
nucleic acid backbone. 
Bromopsoralens, and photosensitizer B specifically, do not form free 
radicals upon irradiation in solution. A donor is required to activate 
photosensitizer B. Using fluorescence spectroscopy it has been shown that 
amino acids are not suitable donors to activate photosensitizer B. Thus 
any of these photosensitizers bound or associated with proteins should not 
generate radicals capable of damaging proteins. 
It is therefore one preferred embodiment of the method of the present 
invention to use a photosensitizer that is capable of fluorescence. 
Coumarins and furocoumarins that fluoresce are known to those skilled in 
the art, and the screening of photosensitizers to determine fluorescent 
properties is easily determined. 
Photosensitizers that are capable of fluorescence appear to be superior to 
non-fluorescent varieties. For a photosensitizer to be useful, there must 
be a mechanism for viral and bacterial inactivation. Non-halogenated 
psoralens may still function as useful photosensitizers if they are 
properly situated in the solution to be treated. Such compounds can 
inactivate viruses via the traditional photocrosslinking mechanism. Other 
photosensitizers, such as those having the coumarin backbone structure, 
must be halogenated in order to accomplish significant viral or bacterial 
inactivation. Thus, in this embodiment of the invention the preferred 
photosensitizers are intercalators, are capable of fluorescence; and 
either 1) are halogenated; or 2) have the psoralen backbone structure. 
According to an additional embodiment of the present invention, the 
photosensitizer of the invention may comprise a quenching sidechain moiety 
attached to the intercalating backbone. FIG. 1 provides a diagrammatic 
energy diagram for certain halogenated photosensitizers that are capable 
of fluorescence. According to the theory expressed herein, the ability to 
fluoresce provides a rapid means for the excited singlet state species to 
revert to ground energy state that competes with intersystem crossing to 
the triplet excited state. For photosensitizers that do not fluoresce in 
particular, the presence of a quenching moiety attached to the 
intercalator can also serve the same function. 
An example of a photosensitizer of this embodiment of the invention is as 
follows: 
##STR8## 
The non-hydrogen bonding ionic substituent comprises a quaternary ammonium 
pyridyl group. Such a compound can be easily prepared by one skilled in 
the art without undue experimentation. The quaternary ammonium pyridyl 
group can serve as a quencher of the UV excited triplet state of the 
psoralen compound. While not intending to be bound by theory, it is 
proposed that the quenching group will deactivate the triplet state of any 
intercalator, thereby preventing formation of undesired singlet oxygen. 
The reduction of singlet oxygen production as such minimizes damage to 
lipid membranes or proteins. The proximity of the quenching moiety to the 
intercalator should make quenching highly preferred to any reaction with 
oxygen in solution, and should also obviate the need for the addition of 
exogenous quenching agents (such as oxygen scavengers, reducing agents or 
sugars) into the medium. The quenching moiety may be attached to the 
backbone of the photosensitizer at any position, and can consist of any 
chemical functionality known to those skilled in the art to function as an 
PG,27 excited state quenching agent. 
The quaternary ammonium or phosphonium substituted halo-intercalators 
described herein do not accumulate in the interior of lipid bilayers 
(membranes) found in blood and blood products because of the presence of 
the charge, nor will they bind to the phospholipid head groups of the 
membrane because they lack acidic hydrogen for hydrogen bonding. 
Prior art psoralens (such as 8-MOP and AMT) must often be used in 
combination with a quencher (e.g. mannitol, dithiothreitol, vitamin E, 
etc.) to protect, repair or otherwise offset the deleterious effects of 
the photosensitizer and light on cell membranes, and to quench the 
production of free oxygen radicals in solution that cause indiscriminate 
damage. The photosensitizers described herein do not accumulate in viral 
membranes and as a consequence do not require the presence of a quencher 
additive to the blood product. In addition, the photosensitizers described 
herein containing halogen also should generate a minimal amount of free 
radicals in solution, thereby avoiding the need for quenchers. 
One preferred class of photosensitizers is selected from the group 
consisting of compounds of the formula (I): 
##STR9## 
wherein u is an integer from 1 to 6; X is an anionic counterion; Z is N or 
P; R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5, and R.sub.6 are 
independently halo; H; linear or branched alkyl of 1-10 carbon atoms; 
linear or branched alkoxy of 1-10 carbon atoms; (CH.sub.2)--.sub.m 
O(CH.sub.2).sub.p Z.sup..noteq. R', R", R'" or --O(CH.sub.2).sub.n 
Z.sup..noteq. R', R", R'" wherein n, m and p are independently integers 
from 1 to 10 and R', R", and R'" are independently H or linear or branched 
alkyl of 1 to 10 carbon atoms with the proviso that on each Z atom, not 
more than two of R', R", or R'" may be H; and at least on one of R.sub.1, 
R.sub.2, R.sub.3, R.sub.4, R.sub.5 or R.sub.6 is (CH.sub.2).sub.m 
O(CH.sub.2).sub.p Z.sup..noteq. R', R", R'" or --O(CH.sub.2).sub.n 
Z.sup..noteq. R', R", R'". Particularly preferred are compounds wherein 
R.sub.4 is --O(CH.sub.2).sub.n N.sup..noteq. R', R", R'", especially 
wherein R', R" and R'" are ethyl and n=3. Preferably, R.sub.6, R.sub.5, 
R.sub.2 and R.sub.1 are hydrogen and R.sub.3 is H or halo, preferably 
bromo. 
An additional preferred class of photosensitizers is selected from the 
group consisting of the formula (II). 
##STR10## 
wherein u is an integer from 1 to 6; X is an anionic counterion; Z is N or 
P; R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5, and R.sub.6 are 
independently halo; H; linear or branched alkyl of 1-10 carbon atoms; 
linear or branched alkoxy of 1-10 carbon atoms; (CH.sub.2).sub.m 
O(CH.sub.2).sub.p Z.sup..noteq. R', R", R'" or --O(CH.sub.2).sub.n 
Z.sup..noteq. R', R", R'" wherein n, m and p are independently integers 
from 1 to 10 and R', R", and R'" are independently H or linear or branched 
alkyl of 1 to 10 carbon atoms with the proviso that on each Z atom, not 
more than two of R', R", or R'" may be H; and at least on one of R.sub.1, 
R.sub.2, R.sub.3, R.sub.4, R.sub.5 or R.sub.6 is (CH.sub.2).sub. m 
O(CH.sub.2).sub.p Z.sup..noteq. R', R", R'" or --O(CH.sub.2).sub.n 
Z.sup..noteq. R', R", R'". Particularly preferred are compounds wherein 
R.sub.4 is --O(CH.sub.2).sub.n N.sup..noteq. R', R", R'", especially 
wherein R', R" and R'" are ethyl and n=3. Preferably, R.sub.3, R.sub.5, 
R.sub.2 and R.sub.1 are hydrogen and R.sub.3 is H or halo, preferably 
bromo. 
In general, the above compounds may be made by halogenating psoralens and 
isolating the appropriately substituted isomers. For compounds wherein the 
ring substituent is a quaternary ammonium alkoxy or phosphonium alkoxy 
group, that group may be made from the corresponding hydroxy-substituted 
psoralens, as exemplified by the following scheme. 
##STR11## 
As described above, the most preferred photosensitizers of the present 
invention are comprised of ionic functionalities that are non-hydrogen 
bonding. However, included within the scope of this invention are 
photosensitizers comprised of amine functionalities having one and in some 
cases two amine hydrogens. These compounds, of course, are capable of 
forming hydrogen bonds. It has been shown that there is a direct 
correlation between the number of hydrogens available on the amine and the 
cellular destruction-caused by a class of psoralen compounds. Goodrich, et 
al. Proc. Nat'l. Acad. Sci. USA, 91:5552-56 (1994). Thus, photosensitizers 
containing amine functionalities having two hydrogens are less preferred 
than those having one hydrogen, which are in turn less preferred than 
those having no hydrogen attached to the amine. 
Therefore, according to this invention, sensitizing compounds for viral 
inactivation preferably do not contain substituents which possess free 
hydrogen groups capable of exhibiting hydrogen bonding to the cell 
membrane. 
From the foregoing description, it will be realized that the invention can 
be used to selectively bind a chemical photosensitizer to 
blood-transmitted viruses, bacteria, or parasites. Also monoclonal or 
polyclonal antibodies directed against specific viral antigens (either 
coat proteins or envelope proteins) may be covalently coupled with a 
photosensitizer compound. 
Since cell compositions also comprise a variety of proteins, the method of 
decontamination of cells described herein is also applicable to protein 
fractions, particularly blood plasma protein fractions, including, but not 
limited to, fractions containing clotting factors (such as Factor VIII and 
Factor IX), serum albumin and/or immune globulins. The viral and bacterial 
inactivation may be accomplished by treating a protein fraction with a 
photosensitizer as described herein. 
Although described in connection with viruses, it will be understood that 
the methods of the present invention are generally also useful to 
inactivate any biological contaminant found in stored blood or blood 
products, including bacteria and blood-transmitted parasites. 
The halogenated psoralens and coumarins according to the present invention 
are improved and more efficient photosensitizers because they require only 
a single UVA photon for activation. The ability of the halogen 
photosensitizer to react with any base pair imposes no limitation for the 
site of intercalation. As shown in FIG. 2, absorption of a UVA photon by a 
bromocoumarin in the presence of guanine (or any nucleotide base) leads to 
electron transfer and the formation of bound radicals and ultimately 
nucleic acid cleavage and viral or cell death. This cleavage mechanism is 
more efficient than the conventional crosslinking reaction of 
non-halogenated psoralens. 
The coumarin radical 2 (FIG. 2) can inflict damage on the nucleic acid 
double helix to which it is bonded by abstraction of a ribose (RNA) or 
deoxyribose (DNA) sugar carbon hydrogen bond. This can lead to DNA 
cleavage by known mechanisms. The guanine radical cation shown as an 
example is also known to react with molecular oxygen, initiating a series 
of reactions which cleave DNA. The byproduct of the bound radical 
photochemistry is debrominated coumarin 4, which is incapable of forming 
crosslinks to DNA unlike psoralens. 
A preferred class of photosensitizers comprise nucleic acid intercalators 
which may be added to plasma or plasma fractions followed by UV radiation 
to reduce the viral contamination therein. According to the present 
invention, the reduction of viral contamination can be unexpectedly 
reduced by utilizing halogenated intercalators. For example, it was 
observed that the bromopsoralens are about 200,000 times more effective in 
reducing viral activity when compared to use of their non-brominated 
counterparts. 
The brominated intercalators are an improvement over the known psoralens 
and other substituted psoralens when used as photosensitizers because only 
one photon of light is required to activate the brominated photosensitizer 
whereas two photons are required to activate a non-brominated 
photosensitizer. Secondly, a brominated intercalator is effective in 
virtually every intercalative site, whereas a non-brominated 
photosensitizer is effective only in intercalation sites containing a 
uracil or thymine on different strands of the DNA or RNA. The brominated 
intercalators are also an improvement over the known coumarins, which 
unlike the known psoralens have no crosslinking ability and therefore have 
generally not been used previously as photosensitizers for viral 
inactivation, or as light activated drugs in therapeutic photophoresis 
procedures for certain cancer treatments and immune disorders. 
The use of the brominated or halogenated intercalators is particularly 
useful for activation in hydrated systems such as plasma, immune sera, 
tissue culture media containing animal serum or serum components (such as 
fetal calf serum), or recombinant products isolated from tissue culture 
media. 
The present invention may be applied to treatment of liquid blood in ex 
vivo irradiation, such as by methods and apparatus described in U.S. Pat. 
Nos. 4,889,129 and 4,878,891 and 4,613,322. 
The photosensitizers also may be utilized in vivo and delivered in 
liposomes (artificial cells) or drug-loaded natural cells. After 
introduction of the liposome or drug-loaded cell, the patient may be 
treated by radiation to activate the photosensitizer. 
The present invention is applicable to contaminants which comprise single 
or double-stranded nucleic acid chains, including RNA and DNA, and 
viruses, bacteria or other parasites comprising RNA and/or DNA. 
The present invention includes the inactivation of specific viral species 
that are found as contaminants in blood and blood products. Example 1 
below describes in great detail the experimental protocol for the 
inactivation of HIV-1 virus in platelet concentrate. The results obtained 
from this series of experiments validates the ability of the 
photosensitizers of the present invention to inactivate HIV-1 virus in a 
blood product. The results of this study are summarized in Table 1. 
Reductions in viral titer were obtained by subtracting the viral titer of 
treated samples from control samples. FIG. 3 and 4 show the results of the 
study graphically. FIG. 3 shows the viral reduction versus light intensity 
for a number of different concentrations of photosensitizer B, and FIG. 4 
shows viral reduction versus concentration of photosensitizer B. 
The procedure described in detail in Example 1 for the inactivation of the 
HIV-1 virus in platelets was typical of the type of experimental protocol 
utilized to examine the inactivation of a variety of viral species. 
Example 2 below describes the general protocol used to demonstrate the 
inactivation of Sindbis virus in human plasma. The results of the 
inactivation using photosensitizer A and photosensitizer B are depicted in 
FIG. 5. Example 3 below describes the general protocol used to demonstrate 
the inactivation of Cytomegalovirus in human platelet concentrates. The 
results of the inactivation using photosensitizer B are depicted in FIG. 
6. Example 4 below describes the general protocol used to demonstrate the 
inactivation of Vesicular Stomatitis Virus in human platelet concentrates. 
The results of the inactivation using photosensitizer B are depicted in 
FIG. 7. Example 5 below describes the general protocol used to demonstrate 
the inactivation of Herpes Simplex Virus Type I. The results of the 
inactivation using photosensitizer B are depicted in FIG. 8. 
Because the photosensitizers of the present invention are to be used to 
inactivate blood and blood products that will be used for transfusion into 
human patients, it is imperative that they be safe for transfusion 
following irradiation. Example 6 below describes the mutagenicity protocol 
used to verify the safeness of the photosensitizers of the present 
invention. The specific example provided in Example 6 is for 
photosensitizer B, before and after irradiation, under conditions suitable 
for the inactivation of viral and bacterial components in blood and blood 
products. The results of the mutagenicity tests for photosensitizer B 
demonstrate that a mixture of photosensitizer B photolysis products and a 
maximum residual photosensitizer B concentration of 4.36 .mu.g/mL per test 
plate did not cause any mutagenic effects in Salmonella strains TA98, 
TA100, TA1535, TA1537 and TA1538. The maximum residual concentrations of 
photosensitizer B under use conditions (25J/cm.sup.2 of UVA) corresponds 
to about 3.4 times the expected concentration of photosensitizer B per 
therapeutic dose of platelet concentrates of 1.28 .mu.g/mL. The results 
thus demonstrate that photosensitizer B is non-mutagenic when photolyzed 
in platelet concentrates such that the initial concentration is reduced by 
at least 60% under used conditions (&gt;25J/cm.sup.2 UVA and 12.8 .mu.g/mL 
photosensitizer B plate). 
The mutagenicity results for photosensitizer A showed that for both 
irradiated and non-irradiated solutions there was no significant increase 
in reversion rate with any of the five tester strains in the absence or 
presence of S-9 activation. 
Example 7 describes the mouse fibroblast protocol used to determine the 
cytotoxicity of the photosensitizers of the present invention. The results 
of these tests for photosensitizer B at 72hr are depicted in Table 2. 
Example 8 describes the Chinese Hamster Ovary, Hybridoma Cells and AE-L 
Cells protocol used to determine the cytoxicity of the photosensitizers of 
the present invention. The results of these tests for photosensitizer B 
are depicted in Tables 3 and 4. 
Compound A, 3-bromo-7-(.gamma.-triethylammonium propyloxy) coumarin 
bromide, is one of the most preferred photosensitizers of the present 
invention. The synthesis of Compound A is given in Example 9. The reaction 
scheme for the synthesis is shown in FIG. 9. 
One of the best measures of the effectiveness of potential photosensitizers 
is the extent to which the photosensitizer tends to associate with nucleic 
acids rather than to cellular membrane components or proteins in blood or 
blood products. Example 10 below describes the protocol that was employed 
for analyzing the specificity a variety of photosensitizers have for 
nucleic acids. 
Independent of the mechanism of photosensitized inactivation of viral and 
bacterial contaminants in blood or blood products, it is generally clear 
that the greater the preference the photosensitizer has to the nucleic 
acid components of the contaminants -- as opposed to cellular membranes or 
proteins in solution -- the better the performance of the photosensitizer. 
The quality of a photosensitizer being determined by the rate efficiency 
of contaminant inactivation, absolute contaminant inactivation, and as 
little impairment of the physiological activity of the treated composition 
as possible. Of course these factors are interrelated. The results of 
specificity experiments comparing the photosensitizers of the present 
invention with prior art photosensitizers reveals the superior properties 
of the novel photosensitizers disclosed herein. These results are shown in 
Table 5. 
The photosensitizers of the present invention have been examined to show 
the effects on the constituents of platelet concentrates under conditions 
that are sufficient for obtaining complete contaminant inactivation. The 
general procedure for conducting these experiments is disclosed in Example 
11 below. 
Table 6 presents a summary of the in vitro platelet properties after 
photoactivation in the presence of 300 .mu.g/mL of photosensitizer B, with 
and without bicarbonate. The bicarbonate was added to offset the effects 
on the pH of the solution that result from irradiation. Table 7 presents a 
summary of the pheresed platelet in vitro properties following 
photoinactivation in the presence 300 .mu.g/mL of photosensitizer B. Table 
8 summarizes the platelet in vitro properties following photoinactivation 
in the presence of photosensitizer A. The pH does not substantially change 
when photosensitizer A is employed. 
Additional experiments were conducted in order to compare the 
photosensitizers of the present invention with two prior art 
photosensitizers, 8-MOP and AMT. The protocol for this evaluation of 
photosensitizers irradiated in human platelet concentrates is described in 
Example 12 below. The results of this comparison can be summarized as 
follows: 
1. Complete inactivation of bacteriophage 6 (.gtoreq.6 logs of viral 
reduction) was obtained with photosensitizer B without alteration in 
platelet in vitro properties (HSR, morphology, aggregation response to 
collagen) under normal oxygen content at UVA fluence of 7.6 J/cm.sup.2. 
2. Equimolar concentrations of AMT and 8-MOP required 45 and 68 J/cm.sup.2 
of UVA energy respectively to obtain greater than 4 logs of viral 
inactivation. These conditions were associated with major alterations in 
platelet in vitro properties. 
3. Photoinactivated platelet concentrates using photosensitizer B (60 .mu.m 
photosensitizer concentration and 4.5 J/cm.sup.2) maintained normal 
properties following post-treatment storage for 5 days in a standard 
platelet incubator at 22 .+-. 2.degree. C. 
4. Virucidal efficacy of brominated psoralen was substantially higher than 
that for 8-MOP and AMT with respect to inactivation of non-enveloped 
bacteriophages such as lambda and R-17. 
Example 13 describes the results of a comparison study of the ability of a 
variety of photosensitizers of the present invention to inactivate Sindbis 
virus in human plasma. The compounds tested in this series of experiments 
were photosensitizers A, B, D and E, and non-halogenated forms of A, C and 
D. The results of these experiments are depicted graphically in FIG. 12. 
The results show that under the same conditions: 1) the coumarin-based 
photosensitizers A, C and D are superior to the psoralen-based 
photosensitizer B; 2) the non-halogenated coumarin-based photosensitizers 
are not suitable for photoactivated inactivation of virus; and 3) the 
methylated coumarins, photosensitizers D and E, appear to be the most 
efficient photosensitizers for viral inactivation. 
Example 14 describes the synthesis of photosensitizer D. The procedure 
follows the synthetic scheme depicted in FIG. 13. Following this general 
procedure, which is believed to be novel, one skilled in the art may also 
synthesize photosensitizer E and other photosensitizers of the present 
invention. (See, e.g., Sethna Chem. Rev., 36:10 (1945); Sethna et al. 
Organic Reactions, 7:1 (1953)). 
The following examples are presented in order to help define and enable the 
present invention. The examples are not to be considered as limiting the 
invention as described and claimed herein. 
EXAMPLES 
EXAMPLE 1: INACTIVATION OF HIV-1 VIRUS IN PLATELET CONCENTRATE. 
The experimental design for the viral validation studies involved the 
addition of photosensitizer B to platelet concentrates in standard 
platelet collection bags and subsequent activation of the photosensitizer 
upon exposure to ultraviolet light at 320-400 nm. The following studies 
were performed for the validation of elimination of HIV-1 from platelet 
concentrates. 
Photosensitizer Toxicity Test (PHASE 1): This study was performed to 
establish the degree of toxicity of the photosensitizer to the indicator 
cell lines used in the assay and to rule out any interference by the 
photosensitizer with the ability of the chosen viruses to infect the 
indicator cell lines. 
PHASE I: Photosensitizer Toxicity to Viral Indicator 
Cells 
Sample Set #1 
1. Platelet + Saline + Orbital Shaking + 30 min ambient light. 
2. Platelet + Saline + Orbital Shaking + 30 min UVA. 
3. Platelet + 100 .mu.g/mL Photosensitizer B + Orbital shaking + 30 min 
ambient. 
4. Platelet + 100 .mu.g/mL Photosensitizer B + Orbital Shaking + 30 min 
UVA. 
5. Platelet + 300 .mu.g/mL Photosensitizer B + Orbital Shaking + 30 min 
ambient. 
6. Platelet + 300 .mu.g/mL Photosensitizer B + Orbital Shaking + 30 min 
UVA. 
Sample Set #2 
7. Platelet + Saline + Orbital Shaking + 60 min ambient light. 
8. Platelet + Saline + Orbital Shaking + 60 min UVA. 
9. Platelet + 100 .mu.g/mL Photosensitizer B + Orbital Shaking + 60 min 
ambient light. 
10. Platelet + 100 .mu.g/mL Photosensitizer B + Orbital Shaking + 60 min 
UVA. 
11. Platelet + 300 .mu.g/mL Photosensitizer B + Orbital Shaking + 60 min 
ambient light. 
12. Platelet + 300 .mu.g/mL Photosensitizer B + Orbital Shaking + 60 min 
UVA. 
PHASE II 
Photosensitizer Dose Response: The main purpose of this study was to 
determine the optimum concentration of photosensitizer for complete 
inactivation of HIV-1. 
Kinetics of Inactivation: The main purpose of this study was to establish 
the optimal exposure time for effective inactivation of HIV-1. 
Variables Under Investigation 
1. Dose of Photosensitizer (Dose response studies). 
2. UVA Exposure time (Kinetics of inactivation). 
Fixed Parameters 
1. Light Source (UVA) 
2. Photosensitizer -- B 
3. Virus-HIV-1 
4. Suspending medium (Plasma) 
5. Light intensity (including distance of sample from the light source). 
6. Rotational speed for sample platform. 
7. Viral Titer (2 .times. 10.sup.7). 
8. Post-Photosensitizer incubation time (10 minutes). 
9. UVA Reactor (Orbital shaker) 
PHASE III: Elimination of HIV in Platelet Concentrates EXPERIMENTAL 
CONDITIONS 
Dose Response 
Kinetics of Inactivation 
Sample Set #1 
13. Platelet + Virus + Saline + Orbital Shaking + 5 min ambient light. 
14. Platelet + Virus + Saline + Orbital Shaking + 5 min UVA. 
15. Platelet + Virus + 50 .mu.g/mL Photosensitizer B + Orbital Shaking + 5 
min UVA. 
16. Platelet + Virus + 100 .mu.g/mL Photosensitizer B + Orbital Shaking + 5 
min UVA. 
17. Platelet + Virus + 200 .mu.g/mL Photosensitizer B + Orbital Shaking + 5 
min UVA. 
18. Platelet + Virus + 300 .mu.g/mL Photosensitizer B + Orbital Shaking + 
5 min UVA. 
18A. Platelet + Virus + 400 .mu.g/mL Photosensitizer B + Orbital Shaking + 
5 min UVA. 
Sample Set #2 
19. Platelet + Virus + Saline + Orbital Shaking + 15 min ambient light. 
20. Platelet + Virus + Saline + Orbital Shaking + 15 min UVA. 
21. Platelet + Virus + 50 .mu.g/mL Photosensitizer B + Orbital Shaking + 15 
min UVA. 
22. Platelet + Virus + 100 .mu.g/mL Photosensitizer B + Orbital Shaking + 
15 min UVA. 
23. Platelet + Virus + 200 .mu.g/mL Photosensitizer B + Orbital Shaking + 
15 min UVA. 
24. Platelet + Virus + 300 .mu.g/mL Photosensitizer B + Orbital Shaking + 
15 min I/VA. 
24A. Platelet + Virus + 400 .mu.g/mL Photosensitizer B + Orbital Shaking + 
15 min UVA. 
Sample Set #3 
25. Platelet + Virus + Saline + Orbital Shaking + 30 min ambient light. 
26. Platelet + Virus + Saline + Orbital Shaking + 30 min UVA. 
27. Platelet + Virus + 50 .mu.g/mL Photosensitizer B + Orbital Shaking + 30 
min ambient. 
28. Platelet + Virus + 50 .mu.g/mL Photosensitizer B + Orbital Shaking + 30 
min UVA. 
29. Platelet + Virus + 100 .mu.g/mL Photosensitizer B + Orbital Shaking + 
30 min ambient. 
30. Platelet + Virus + 100 .mu.g/mL Photosensitizer B + Orbital Shaking + 
30 min UVA. 
31. Platelet + Virus + 200 .mu.g/mL Photosensitizer B + Orbital Shaking + 
30 min ambient. 
32. Platelet + Virus + 200 .mu.g/mL Photosensitizer B + Orbital Shaking + 
30 min UVA. 
33. Platelet + Virus + 300 .mu.g/mL Photosensitizer B + Orbital Shaking + 
30 min ambient. 
34. Platelet + Virus + 300 .mu.g/mL Photosensitizer B + Orbital Shaking + 
30 min UVA. 
34A. Platelet + Virus + 300 .mu.g/mL Photosensitizer B + Orbital Shaking + 
30 min UVA. 
Sample Set #4 
35. Platelet + Virus + Saline + Orbital Shaking + 60 min ambient light. 
36. Platelet + Virus + Saline + Orbital Shaking + 60 min UVA. 
37. Platelet + Virus + 50 .mu.g/mL Photosensitizer B + Orbital Shaking + 60 
min ambient. 
38. Platelet + Virus + 50 .mu.g/mL Photosensitizer B + Orbital Shaking + 60 
min UVA. 
39. Platelet + Virus + 100 .mu.g/mL Photosensitizer B + Orbital Shaking + 
60 min ambient. 
40. Platelet + Virus + 100 .mu.g/mL Photosensitizer B + Orbital Shaking + 
60 min UVA. 
41. Platelet + Virus + 200 .mu.g/mL Photosensitizer B + Orbital Shaking + 
60 min ambient. 
42. Platelet + Virus + 200 .mu.g/mL Photosensitizer B + Orbital Shaking + 
60 min UVA. 
43. Platelet + Virus + 300 .mu.g/mL Photosensitizer B + Orbital Shaking + 
60 min ambient. 
44. Platelet + Virus + 300 .mu.g/mL Photosensitizer B + Orbital Shaking + 
60 min UVA. 
Note: Ambient means ambient laboratory light (Non-UVA light Source). 
METHODS -- PHASE I: 
I. Selection of uniform UVA exposure area: 
Step 1: Place a transparent sample platform at equidistant from the top and 
bottom UVA lamps. 
Step 2: Construct a square on the sample platform of the reactor. 
Step 3: Switch on the top-bank of UVA light and turn on the fan for 
maintenance of ambient temperature during photolysis. 
Step 4: Place the light intensity meter at both the four corners of the 
square and the center. Record the light intensity meter readings at these 
locations for the top bank of lights. 
Step 5: Repeat step 4 for the bottom bank of lights. 
Step 6: If the light intensity is different for the various locations, 
reconstruct the "square" such that light intensity is the same at all the 
different sections of the square. 
Step 7: Preparation of Stock Solution Photosensitizer B: Dissolve 
Photosensitizer B in 10mM of phosphate buffered saline (PBS) such that the 
final concentration is 40 mg/mL, solution A. Prepare the following working 
solutions from solution A: 
1. Solution B: 5 mg/mL 
2. Solution C: 10 mg/mL 
3. Solution D: 20 mg/mL 
4. Solution E: 30 mg/mL 
Preparation of Platelet Concentrates for UVA Irradiation Step 8: Pool four 
units of ABO compatible platelet concentrates together in a standard 
platelet collection bag to obtain a final volume of about 182 mL of 
platelet rich plasma (Platelet Suspension F). Place 50 mL of platelet 
concentrates into standard platelet collection bag for experiment in Phase 
1A (Platelet Suspension G). Save the remaining 132 mL of platelet 
concentrates for Phase II studies. 
Step 9: Samples 1-12: Place 7.0 mL aliquots of suspension G into 15 mL 
centrifuge tubes labeled for both control and test samples (100 and 300 
.mu.g/mL). 
Step 10: Pipette 71 .mu.l of working solutions C and E and add to platelet 
concentrates (from step 8) and allow samples to incubate with 
photosensitizer at 24.degree. C. for 10 minutes at ambient light. Add 71 
.mu.l of phosphate buffered saline (PBS) to control samples and incubate 
as described above. 
Step 11: Place 3.0 mL aliquots of treated and untreated samples from Step 
10 in 35 mm petri dishes. Place plastic covers on top of the petri dishes 
and irradiate samples according to the experimental conditions that are 
outlined for Phase IA studies. 
Step 12: At the end of the appropriate irradiation period, pour platelet 
samples into 5 mL test tubes. Test control and treated samples for (1) 
cellular toxicity for viral assay system; (2) viral interference for assay 
system. 
METHODS-PHASE II 
1. Selection of uniform UVA exposure area: Use the same area of uniform 
light distribution as in Phase I studies (i.e., Steps 1-6 are the same). 
Step 7: Preparation of Stock Solution B: (Same as in Phase I). 
Preparation of Platelet Concentrates with HIV-1 for OVA Irradiation 
Step 8: Add 8 mL of HIV-1 (2 .times. 10.sup.7 PFU of HIV-1/mL) to the 
remaining 132 mL of platelet concentrates (from step 8 of Phase I) such 
that the final HIV-1 titer is about 1.1 .times. 10.sup.6 (Platelet-HIV 
Suspension H). Divide platelet suspension H into the following aliquots 
for the different sample sets in the Phase II viral elimination studies: 
1. 27mL of Suspension H for Sample Set #1 
2. 27mL of Suspension H for Sample Set #2 
3. 39mL of Suspension H for Sample Set #3 
4. 39mL of Suspension H for Sample Set #4 
Step 9: Prepare samples for viral elimination studies. 
Step 10: Place 3.0 mL aliquots of treated and untreated samples from Step 4 
into 35 mm petri dishes. Place plastic covers on top of the petri dishes 
and irradiate samples according to the experimental conditions that are 
outlined above. 
Step 11: At the end of the appropriate irradiation period, pour platelet 
samples into 5 mL test tubes. Determine HIV-1 infectivity in control and 
treated samples. 
HIV Infectivity Assay: HIV is normally titrated in vitro by a MT-4 
syncytium assay. MT-4 is a cell line developed specifically to facilitate 
the recognition of HIV infection. These cells are adherent and abundantly 
express the CD4 receptor used by HIV during infection of a cell. Upon 
infection with HIV, these cells develop easily-detectable multinucleated 
cells or syncytium forming units. 
Buffer Toxicity/Viral Interference 
Twenty-four well cluster plates were seeded with MT-4 cells in a total 
volume of 1.0 ml/well. Each test dilution was inoculated into 3 wells at 
0.1 ml/well, then the cultures were incubated at 36.degree. C. 
.+-.1.degree. C. Observations for cytotoxicity and, if necessary, an 
estimation of the percentage of cells affected in each culture were 
performed on day 5 and day 7 post-inoculation. 
Viral Inactivation Assay 
The test article samples were spiked with human immunodeficiency virus type 
1. The spiked test article samples were carried through the inactivation 
process. All samples were tested undiluted or diluted in RPMI medium 
(negative control) at various dilutions. Retained samples were stored 
frozen at -60.degree. C. or below. 
Titration of Samples for the Presence of HIV-1 
Twenty-four well cluster plates were seeded with MT-4 cells in a total 
volume of 1.0 ml/well. From the spiked test article or positive control, 
ten fold serial dilutions were made in culture medium. At each dilution 
step quadruplicate 0.1 ml volume of the samples were tested. Cultures were 
fed twice a week by removal of 1.0 ml of medium and addition of 1.0 ml of 
fresh medium. On days 7, 14 and 28 the cultures were evaluated for the 
cytopathic effects to determine the TCID.sub.50. On days 7, 14 and 28, 1.0 
ml of each culture was removed for analysis by HIV-1 p24 antigen capture 
ELISA. 
The formula for the final titer calculation of TCID.sub.50 is based on the 
Karber method: negative logarithm of the endpoint titer = A - (S1/100-0.5) 
.times. B, where A =negative logarithm of the highest concentration 
inoculated, S.sub.1 = sum of the percentage positive at each dilution, and 
B = log.sub.10 (of the dilution factor). The values were converted to 
TCID.sub.50/ ml using a sample inoculum volume of 0.1 ml. 
The p24 assay is the Coulter HIV p24 Ag Assay which is an enzyme 
immunoassay for the detection of p24 antigen of HIV in plasma, serum or 
tissue culture media. It uses a murine monoclonal antibody (anti-HIV core 
antigen) coated onto microwell strips and if present the antigen binds to 
the antibody-coated microwells. The bound antigen is recognized by 
biotinylated antibodies to HIV which react with conjugated streptavidin 
horseradish peroxidase. Color develops from the reaction of the peroxidase 
with hydrogen peroxide in the presence of tetramethylbenzidine substrate. 
The intensity of the color developed is directly proportional to the 
amount of HIV antigen present in the sample. The p24 assay negative 
control was RPMI 1640 and the positive control was antigen reagent. 
Culture fluid from each well is analyzed by the HIV p24 assay and the 
absorbance value is compared to the cut off value for a positive result. 
The cut off value for a positive result was determined by adding the mean 
absorbance value of the ELISA negative control to a predetermined factor 
of 0.055. The expected range of the cut off value is 0.055 to 0.155. If 
the absorbance value for the well exceeds the cut off value, then the well 
is considered positive for HIV p24 antigen. The level of HIV p24 in each 
well is not quantitated. The TCID.sub.50 of the sample was determined from 
the sum of the percentage of wells positive for HIV p24 antigen at each 
dilution using the standard formula stated above. 
Materials 
______________________________________ 
Positive Control Article and 
Human immunodeficiency virus 
Spiking Virus: type 1 
Strain: IIIB 
Lot No.: VP012 H.1/8/93 
Titer = 10.sup.7.5 TCID.sub.50 /ml 
Source: Advanced Biotech- 
nologies, Inc. 
Columbia, Maryland 
Negative Control Article: 
RPMI 1640 Medium 
Source: Microbiological 
Associates, Inc. 
Rockville, Maryland 
Test System: MT-4 cells (L013-T) 
Source: National Institute 
of Health 
Bethesda, Maryland 
(Human T cells isolated from a 
patient with adult T cell 
leukemia; HTLV-I transformed) 
______________________________________ 
Results 
Cytotoxicity was observed with all the undiluted samples, however the 
cultures appeared to recover from the effects by day 7. Cytotoxicity was 
observed with all the samples diluted 1:10 on day 3, however the cultures 
recovered by day 7. These effects were most likely due to the excessive 
amount of cellular material in the samples. 
Results for samples taken at various points during the inactivation of 
HIV-1 study were obtained. The following samples showed no evidence of 
replication competent HIV-1: 34A, 42 and 44. One well of four inoculated 
with undiluted sample 34 and sample 32 was positive for CPE on day 28. Two 
wells of four inoculated with undiluted sample 40 were positive for CPE on 
day 28. The remaining samples had significant levels of replicating HIV-1. 
EXAMPLE 2: INACTIVATION OF SINDBIS IN PLASMA SOLUTION. 
Human plasma was spiked with Sindbis virus to a final concentration of &gt;7 
log.sub.10 plaque forming units (PFU)/mL. Photosensitizer was then added 
to a virus spiked plasma at 100 or 300 .mu.g/mL final concentration. After 
a 15 minute incubation at room temperature samples of photosensitizer 
treated virus spiked plasma was placed in a ultraviolet light (UV) 
irradiator and exposed to 24 J/cm.sup.2 of UVA energy. Treated samples 
were then assayed for residual infectious virus by plaque assay. Virus 
reduction (V.sub.R) was calculated by the equation V.sub.s -V.sub.f 
=V.sub.R where V.sub.s is the starting virus titer, and V.sub.f is the 
virus titer after treatment. 
EXAMPLE 3: INACTIVATION OF CYTOMEGALOVIRUS IN HUMAN PLATELET CONCENTRATES. 
Inactivation of Cytomegalovirus (CMV) in human platelet concentrates was 
conducted under normal ambient oxygen tension using a photosensitizer and 
long wavelength ultraviolet light (UVA) at 22.degree..+-.2.degree. C. Dose 
response and kinetics studies were conducted in order to determine the 
optimal conditions for inactivation of CMV in human platelet. 4-6 logs of 
CMV virus was added to standard units of human platelet concentrate. The 
contaminated platelet concentrates were incubated at ambient non-UVA 
laboratory light for 60.+-. 5 minutes with different concentrations of F 
(100-300 .mu.g/mL). Following incubation, the platelet concentrates were 
exposed to UVA at different fluences (14-43 J/cm.sup.2). Inactivation of 
CMV virus was evaluated by an infectivity assay using MRC-5 cells. 
Complete inactivation of CMV was obtained at 100 .mu.g/mL using 
Photosensitizer B and a UVA fluence of 21.6 J/cm.sup.2. 
EXAMPLE 4: INACTIVATION OF VESICULAR STOMATITIS VIRUS IN PLATELET 
CONCENTRATES. 
Inactivation of Vesicular Stomatitis (VSV) in human platelet concentrates 
was conducted under normal ambient oxygen tension using a photosensitizer 
and long wavelength ultraviolet light (UVA) at 22.degree..+-.2.degree. C. 
Dose response and kinetics studies were conducted in order to determine 
the optimal conditions for inactivation of VSV in human platelet 
concentrates, 6 logs of VSV virus was added to standard units of human 
platelet concentrate. The contaminated platelet concentrates were 
incubated at ambient non-UVA laboratory light for 10.+-. 5 minutes with 
different concentrations of photosensitizer B (30 and 150 .mu.g/mL). 
Following incubation, the platelet concentrates were exposed to UVA at 
different fluences (4.20-8.40 J/cm.sup.2). Inactivation of VSV virus was 
evaluated by an infectivity assay (plaque assay) using Vero cells. 
Inactivation of 6 logs of VSV using Photosensitizer B was obtained at a 
minimum UVA fluence of 4.20 J/cm.sup.2. 
EXAMPLE 5: INACTIVATION OF HERPES SIMPLEX VIRUS TYPE 1 IN CALF SERUM. 
Inactivation of Herpes Simplex Virus type 1 (HSV-1) in calf serum was 
conducted under normal ambient oxygen tension using a photosensitizer and 
long wavelength ultraviolet light (UVA) at 22.degree..+-.2.degree. C. 
Using a fixed concentration of Photosensitizer B (30 .mu.g/mL) kinetics 
studies were conducted in order to determine the optimal conditions for 
inactivation of HSV-1 in calf serum, 3 logs of HSV-1 virus were added to 
100 mL of calf serum. The contaminated sera were incubated at ambient 
non-UVA laboratory light for 10 .+-. 5 minutes. Following incubation, the 
sera were exposed to UVA at different fluences (4.20-8.40 J/cm.sup.2). 
Inactivation of HSV virus was evaluated by an infectivity assay. 
Inactivation of 3 logs of HSV-1 using Photosensitizer B was obtained at a 
UVA fluence of 12.6 J/cm.sup.2. 
EXAMPLE 6: MEASUREMENT OF PHOTOSENSITIZER MUTAGENICITY BY AMES MUTAGENICITY 
TEST. 
The Ames test is based upon the use of five specially constructed strains 
of Salmonella typhimurium containing a specific mutation in the histidine 
operon. These genetically altered strains, TA98, TA100, TA1535, TA1537, 
and TA1538 cannot grow in the absence of histidine. When they are placed 
in a histidine-free medium, only those cells which mutate spontaneously 
back to their wild type state (non-histidine-dependent by manufacturing 
their own histidine) are able to form colonies. The spontaneous mutation 
rate (or reversion rate) for any one strain is relatively constant, but if 
a mutagen is added to the test system, the mutation rate is significantly 
increased. Each tester strain contains, in addition to a mutation in the 
histidine operon, two additional mutations that enhance sensitivity to 
some mutagens. The rfa mutation results in a cell wall deficiency that 
increases the permeability of the cell to certain classes of chemicals 
such as those containing large ring systems that would otherwise be 
excluded. The second mutation is a deletion in the uvrB gene resulting in 
a deficient DNA excision-repair system. Tester strains TA98 and TA100 also 
contain the pKM 101 plasmid (carrying the R-factor). It has been suggested 
that the plasmid increases sensitivity to mutagens by modifying an 
existing bacterial DNA repair polymerase complex involved with the 
mismatch-repair process. TA98, TA1537 and TA1538 are reverted from 
histidine dependence (auxotrophy) to histidine independence (prototrophy) 
by frameshift mutagens. TA100 is reverted by both frameshift and base 
substitution mutagens and TA1535 is reverted only by mutagens that cause 
base substitutions. 
EXPERIMENTAL DESIGN FOR AMES MUTAGENICITY TEST 
The experiment was designed such that the concentrations of Photosensitizer 
B on the agar plate is equivalent to the expected final dose in a 
recipient given 5 units of platelet concentrates. Note that 5 units of 
platelet concentrates is equivalent to a standard single therapeutic dose 
(1TD). Calculation of the theoretical concentration of photosensitizer B 
is based on the following deductions assuming homogenous distribution of 
the drug in a 70 kg normal individual: 
1. Normal Blood Volume = 5600 mL 
2. 1 Unit of Platelet Concentrate = 50 mL 
3. 1 Therapeutic Dose (1TD) = 5 Units of Platelet Concentrates 
4. Volume of 1TD = 250 mL 
5. Starting Concentration of Photo-Sensitizer B 300 .mu.g/mL 
6. Irradiation for sufficient time to break down 90% of Photosensitizer B 
Based on the above assumptions, if a patient receives 5 Units of platelet 
concentrates the final concentration of Photosensitizer B in the body is 
derived as follows: 
##EQU1## 
A Salmonella/mammalian microsome mutagenicity test was conducted to 
determine whether a plasma test article solution of Photosensitizer B in 
platelet concentrates would cause mutagenic changes in histidine-dependent 
mutant strains of Salmonella typhimurium. The Ames mutagenicity test 
system has been widely used as a rapid screening procedure for the 
determination of mutagenic and potential carcinogenic hazards of pure 
compounds, complex compounds and commercial products. 
EXAMPLE 7: MEASUREMENT OF PHOTOSENSITIZER CYTOTOXICITY USING MOUSE 
FIBROBLASTS. 
In vitro mammalian cell culture studies have been used historically to 
evaluate the cytotoxicity of biomaterials and complex chemical compounds. 
Mouse fibroblasts (L-929) were grown to confluency in 25cm.sup.2 culture 
flasks using sterile minimum essential medium (MEM) supplemented with 5% 
fetal calf serum and nontoxic concentrations of penicillin, streptomycin 
and amphotericin B. Confluent monolayers of L-929 cells were exposed to 
extract dilutions of Photosensitizer B. A standard solution of 
Photosensitizer B was prepared by dissolving 12 mg in 20 mL of MEM 
supplemented with 5% bovine serum and then incubated at 37.degree. C. for 
24 hours. Following incubation, different dilutions (1:2 to 1:16) of 
standard stock of Photosensitizer B were prepared with fresh MEM. A 5 mL 
aliquot of the different dilutions of Photosensitizer B was added to 
confluent monolayers of L-929 cells and then incubated at 37.degree. C. 
for 72 hours. A 5 mL MEM aliquot was added as a negative control. After 
exposure to Photosensitizer B, the cells were examined microscopically at 
approximately 100.times. and scored for cytotoxic effects (CTE) at the end 
of the 24, 28 and 72 hours of incubation. Presence (+) or absence (-) of a 
confluent monolayer, vacuolization, cellular swelling and the percentage 
of cellular lysis were also recorded. CTE was scored as either Nontoxic 
(N), Intermediate (I) or Toxic (T). These data are shown in Table 2. The 
evaluation criteria are shown below: 
______________________________________ 
CTE SCORE MICROSCOPIC APPEARANCE OF CELLS 
______________________________________ 
Nontoxic (N) 
A uniform confluent monolayer with 
primarily elongated cells, with 
discrete intracytoplasmic granules 
present at the 24 hour observation. 
At the 48 and 72 hour observation 
periods, there should be an 
increasing number of rounded cells 
as cell population increases and 
crowding begins. Little or no 
vacuolization, crenation or swelling 
should be present. 
Intermediate (I) 
Cells may show marked vacuolization, 
crenation or swelling. Cytolysis 
(0-50%) of cells that results in 
"floating" cells and debris in the 
medium may be present. The 
remaining cells are still attached 
to the flask. 
Toxic (T) Greater than 50% of all cells have 
been lysed. Extensive 
vacuolization, swelling, or 
crenation are usually present in the 
cells remaining on the flask 
surface. 
______________________________________ 
EXAMPLE 8: MEASUREMENT OF PHOTOSENSITIZER CYTOTOXICITY USING CHINESE 
HAMSTER OVARY (CHO) HYBRIDOMA CELLS AND AE-L CELLS. 
Chinese hamster ovary, and AE-L cells were grown to confluency in 
25cm.sup.2 culture flasks using sterile Eagles Minimum Essential Medium 
(EMEM) supplemented with 2 mM L-glutamine, 1% Proline, 5% calf serum 
treated with different concentrations of Photosensitizer B (30-150 
.mu.g/mL) in the presence of UVA. Nontoxic concentrations of penicillin, 
streptomycin and amphoteric B were also added to the culture medium to 
prevent bacterial growth. Control samples contained nontreated calf serum. 
All samples were incubated at 37.degree. C. for 2 to 7 days. The number of 
viable cells were measured at the end of the incubation periods. Results 
show that the growth and viability of the two cell types were not affected 
by pretreatment of the sera with irradiated and non-irradiated 
Photosensitizer B. The viability of CHO cells as well as the expression of 
rhCg proteins on recombinant CHO cell lines were not affected. Note that 
upon UVA exposure of 30 minutes in the presence of 30 .mu.g/mL of 
Photosensitizer B, there were no adverse effects with the growth 
supporting functions of the treated sera or the expression of rhCg 
antigens. 
EXAMPLE 9: SYNTHESIS OF 3-BROMO-7-(.gamma.-TRIETHYLAMMONIUM PROPYLOXY) 
COUMARIN BROMIDE (PHOTOSENSITIZER A). 
Into a 1000 mL round bottom flask containing a 2.5 cm stirring bar was 
added 15 g of 7-hydroxycoumarin, 15 g of potassium carbonate, 500 mL of 
tetrahydrofuran (THF) and 70 mL of 1,3-dibromopropane. After stirring at 
reflux for 3-4 days (72-96 hours), the solution was filtered, the solids 
washed 4 times with 50 mL of dichloromethane and the combined filtrate 
concentrated by rotary evaporator. 50 mL of ethyl acetate was added to the 
concentrate followed by concentration under reduced pressure (25 in Hg.). 
Another 50 mL of ethyl acetate was added to the concentrate followed by 
filtration. The solids were washed 3 times with 10 mL of a 1:1 mixture of 
ethyl acetate and hexane. After drying, 15-20 g of the crude product was 
dissolved in 150-200 mL of dichloromethane and purified by flash 
chromatography (130-150 g SiO.sub.2 (70-230 mesh), 35 mm O.D. column, 
approximately 60 cm in length), using dichloromethane as the eluting 
solvent. The fractions were collected in 50-250 mL beakers and monitored 
by TLC (developing solvent: 4:6 mixture of ethyl acetate:hexane). The 
fractions containing product were combined, concentrated by rotary 
evaporator and dried. 
Synthesis of 3-bromo-7-(.gamma.-bromopropyloxy) coumarin. 
Into a 500 mL round bottom flask containing a 2.5 cm stirring bar was added 
13 g of 7-(.gamma.-bromopropyloxy) coumarin and 120-150 mL of THF. When 
the 7-(.gamma.-bromopropyloxy) coumarin was completely dissolved, 3 mL of 
bromine was added by syringe. After stirring for 2-5 hours at room 
temperature, the solution was concentrated by rotary evaporator. 50 mL of 
a 1:1 mixture of ethyl acetate and hexane was added to the concentrate and 
the mixture was stirred for 30 minutes at room temperature. The solution 
was then filtered and the solids washed 3 times with 1:1 ethyl acetate and 
hexane and then dried for 2 hours. To obtain additional product, the 
filtrate was concentrated by rotary evaporator and 30 mL of a 1:1 mixture 
of ethyl acetate and hexane was added to the concentrate. The resulting 
mixture was then filtered, the solids washed three times with 1:1 ethyl 
acetate and hexane and then dried for 2-5 hours. The product was checked 
by TLC (ethyl acetate:hexane (4:6)). 
The crude product (13-16 g) was dissolved in 100-170 mL of dichloromethane 
and purified by flash chromatography (100-150 g SiO.sub.2 (70-230 mesh), 
35 mm O.D. column, approximately 60 cm in length), using dichloromethane 
as the eluting solvent. Fractions were collected in a 50-250 mL beakers 
and monitored by TLC (developing solvent: ethyl acetate:hexane (4:6)). The 
fractions containing product were combined, concentrated by rotary 
evaporator and dried. 
Synthesis of 3-bromo-7-(.gamma.-triethylammoniumpropyloxy) coumarin 
bromide. 
Into a 500 mL round bottom flask containing a 2.5 cm stirring bar was added 
11 g of 3-bromo-7-(.gamma.-bromopropyloxy) coumarin, 150-200 mL of THF and 
60-70 mL of triethylamine. After stirring at reflux for 3-4 days (72-96 
hours), the solution was filtered and the solids washed 3 times with 10 mL 
of acetone, 3 times with 10 mL of hexane and then dried for 1 hour. The 
product was transferred to a 600 mL beaker and 80 mL of acetone was added. 
The mixture was stirred for 30 minutes, filtered, washed 3 times with 10 
mL of acetone and dried for 3-5 hours. The product was checked by TLC 
(ethyl acetate:hexane (4:6)). 
Photosensitizer A was found to fluoresce when irradiated with UV light. The 
absorption spectrum of photosensitizer A in water is shown in FIG. 10. The 
fluorescence spectrum of photosensitizer A is shown in FIG. 11. 
EXAMPLE 10: MEASUREMENT OF PHOTOSENSITIZER MIGRATION IN SOLUTION. 
Dialysis experiments were carried out using a custom-made polystyrene 
dialysis chamber. The unit consists of three chambers capable of holding a 
volume of 10 mL of solution. Each chamber was separated from the adjoining 
chamber by a dialysis membrane (MW cut off, 5000, Fisher). The center 
chamber was loaded with 100 .mu.M photosensitizer solution either in 
phosphate buffered saline (PBS) or plasma. The other two adjoining 
chambers were loaded with solutions containing the agents for which the 
binding was to be tested. Liposomes were prepared by vortexing dioleyl 
phosphatidylserine (4.0 mg/mL, Avanti polar lipids) solution in PBS. 
polyadynelic acid (Poly A; Sigma), Calf thymus DNA (DNA; Sigma) and bovine 
serum albumin (BSA) solutions were prepared in PBS (4.0 mg/mL). The 
dialysis cells containing solutions were allowed to equilibrate with 
constant agitation for a period of 24 hours at room temperature. At the 
end of 24 hours, the solutions were removed from individual chambers and 
absorbance was determined at 350 nm using a spectrophotometer. For 
experiments involving liposomes, 5% Titron X-100 (Sigma) was used to 
clarify the solutions prior to absorbance reading. Quantitative 
determination of photosensitizer in plasma and platelets was carried out 
by high performance liquid chromatography (HPLC) equipped with a C18 
reverse phase column. 
EXAMPLE 11: IRRADIATION OF PLATELET CONCENTRATES AND PHOTOSENSITIZER. 
Random donor platelet concentrates (24 hours old) were obtained from 
American Associate of Blood Banks accredited blood banks. Platelet units 
were aseptically pooled and subsequently split into controls and 
treatments. 10 mL of photosensitizer solution in 0.9% saline was added to 
50 mL platelet concentrates in CLX (Miles) containers to obtain the 
photosensitizer final preset concentration. After addition of the 
photosensitizer the platelet units were incubated at room temperature 
while mixing on a shaker for 10 minutes. Platelet concentrates containing 
photosensitizers were UVA irradiated from top and bottom in a prototype 
UVA reactor to deliver 25 J/cm.sup.2 fluence. During the irradiation, 
samples were placed on a linear shaker. After UVA exposure the samples 
were stored in a platelet incubator with shaking for an additional 4 days. 
During storage 3 mL aliquots from each unit were collected and 
subsequently analyzed for platelet in vitro properties. 
EXAMPLE 12: COMISON STUDY OF PHOTOSENSITIZER B, 8-MOP, AND AMT. 
Full units of one day old human platelet concentrates were collected in 
Cyrocyte bags (PL 269, Fenwal, Deerfield, IL) according to standard blood 
banking procedures (AABB Technical Manual, pg. 136, 13th Ed. 1989). 
Platelet units were spiked with 6 logs of bacteriophage 6. Equimolar 
concentrations (60 .mu.M) of 8-MOP, AMT and brominated psoralen were added 
to the platelet concentrations and then incubated at 22.degree..+-. 
2.degree. C. for 10 minutes. Treated samples were irradiated from top and 
bottom with a constant total ultraviolet-A light source intensity of 
7mW/cm.sup.2. During UVA exposure samples were continuously agitated to 
ensure adequate mixing. Virucidal properties were evaluated using a 
standard double agar plaque assay consisting of host bacteria Pseudomonas 
Syringe. In vitro platelet properties were evaluated using (1) aggregation 
response to collagen; (2) hypotonic shock response; (3) morphology 
according to method described by Kunicki, et al. (Transfusion 15:414-421, 
1975). Data represents the mean.+-.standard deviation of n=3. 
EXAMPLE 13: COMISON STUDY OF PHOTOINACTIVATION OF SINDBIS VIRUS IN HUMAN 
PLASMA. 
A comparison study was performed to evaluate the viral inactivation 
properties of photosensitizers A, B, D, and E of the present invention. 
Also examined were non-halogenated forms of photosensitizer A (hereinafter 
referred to as photosensitizer AX), photosensitizer C (hereinafter 
referred to as photosensitizer CX), and photosensitizer D and E 
(hereinafter referred to as photosensitizer DX). The TC1D50 assay was used 
to measure the affects of virus inactivation. 
The photosensitizer was added to virus spiked plasma. The virus employed 
was Sindbis and the plasma was spiked to a working titer of &gt;1 .times. 
10.sup.7. Each test unit was exposed to ultraviolet light at 320-400 nm 
(peak absorbance 365 nm) for 30 min. to achieve an irradiation of about 24 
J/cm.sup.2. Virus inactivation was quantitated by plaque assay. A 
monolayer of indicator cells were grown on a solid support and exposed to 
sample materials to allow for virus attachment. A foci of infection 
develops as virus replicate, lyse and released virus diffuses to, and 
infects, neighboring cells or virus infects neighboring cells via 
cell-cell fusion. CPE develops after several days of incubation. The virus 
titer in the sample is calculated from number of units exhibiting CPE. The 
results of this experiment are shown in FIG. 12. 
EXAMPLE 14: SYNTHESIS OF 3-BROMO-7-(.gamma.-TRIETHYLAMINO) 8-METHYL 
COUMARIN (PHOTOSENSITIZER D). 
Preparation of 7-hydroxy-8-methyl coumarin. 2-methyl resorcinol (0.161 
mmol, 20.024 g) and malic acid (0.165 mmol, 22.129 g) were dissolved in 
concentrated sulfuric acid. The reaction mixture was stirred at 80.degree. 
C. for 24h. The resulting solution was then poured over crushed ice, and 
the precipitate was collected by vacuum filtration. The precipitate was 
then washed with 5% NaHCO.sub.3 and again collected by vacuum filtration 
yielding an orange-yellow solid in a 20.64% yield. 
Preparation of 7-3'-bromopropyloxy-8-methyl coumarin. 7-hydroxy-8-methyl 
coumarin (0.286 mol, 5.045 g) and K.sub.2 CO.sub.3 (0.0317 mol, 4.379 g) 
were added to 50 ml of dibromopropane. The reaction was stirred at reflux 
for 24 h. The excess dibromopropane was removed by distillation. The 
remaining slurry was taken up in CHCl.sub.3 and gravity filtered to remove 
the K.sub.2 CO.sub.3. The CHCl.sub.3 was dried and removed in vacuo. The 
final solid was washed with hexanes to give a pale-yellow product in a 
76.0% yield. 
Preparation of 3 bromo-7-3'-bromopropyloxy-8-methyl coumarin. 
7-3'-bromopropyloxy-8-methyl coumarin (6.75 mmol, 2.01 g) was dissolved in 
THF and cooled to -76.degree. C. Approximately 3 ml of Br.sub.2 was slowly 
added to the solution. The mixture was stirred for 3h and then allowed to 
warm to room temperature. The resulting solution was dissolved in 
CHCl.sub.3 and washed with a 10% Na.sub.2 S.sub.2 O.sub.4 solution, a 10% 
NaHCO.sub.3 solution and finally water. The CHCl.sub.3 was dried and 
removed in vacuo. The resulting pale-yellow solid was washed with hexanes 
and collected by vacuum filtration to give a quantitative yield. 
Preparation of 3-bromo-7-(.gamma.-triethylaminopropyloxy)-8-methyl 
coumarin. 7-3'-bromopropyloxy-8-methyl coumarin (0.17 mmol, 0.064 g) was 
dissolved in 30 ml of CHCl.sub.3. Approximately 10 ml of triethylamine was 
added to the solution. By following TLC, the reaction required refluxing 
for 48h to ensure completion. The CHCl.sub.3 and Et.sub.3 N were removed 
in vacuo. The precipitate was washed with hexane, ethyl acetate and 
acetone several times. The resulting white solid was dried under high 
vacuum and obtained in a 65.4% yield. 
TABLE 1 
______________________________________ 
Reduction in Viral Titer 
at Different Concentrations of B 
(Log.sub.10 Reduction) 
UVA Fluence 100 200 300 400 
(J/cm.sup.2) 
50 .mu.g/mL 
.mu.g/mL 
.mu.g/mL 
.mu.g/mL 
.mu.g/mL 
______________________________________ 
4.32 0.50 0.75 1.50 1.0 0.75 
12.96 1.33 2.25 3.25 3.25 4.0 
25.92 1.50 2.50 5.25 5.25 5.25 
51.84 4.67 6.50 7.0 7.0 ND 
______________________________________ 
Abbreviation: ND, Not Determined 
TABLE 2 
__________________________________________________________________________ 
Evaluation of mouse fibroblast cells following 72 hour incubation after 
exposure to non-UVA irradiated solutions of B N = Indicates a negative 
or 
nontoxic response; I = Indicates an intermediate response, a subjective 
assessment of the extent of cellular response; T = Indicates a positive 
or toxic 
response consisting of greater than 50% cell death. 
Confluent 
Vacuoli- CTE 
Samples 
Monolayer 
zation 
Swelling 
Crenation 
% Lysis 
Score 
__________________________________________________________________________ 
600 .mu.g/mL 
+ + - - 0 N 
300 .mu.g/mL 
+ - - - 0 N 
150 .mu.g/mL 
+ - - - 0 N 
75 .mu.g/mL 
+ - - - 0 N 
37.5 .mu.g/mL 
+ - - - 0 N 
Control (-) 
+ - - - 0 N 
__________________________________________________________________________ 
CTE = Cytotoxic Effects Score 
N = Non toxic 
TABLE 3 
__________________________________________________________________________ 
Viability of AE-1 cells and CHO cells in calf sera that had been 
pretreated with 30 .mu.g/mL of B at different UVA fluences, 
Mean Viable CHO Cells 
CP38 Mean Viable AE-1 Cells (.times.10.sup.5 /mL) 
(.times.10.sup.5 /mL) 
Treatment 
.mu.g/mL 
Day 2 
Day 5 
Day 7 
Mean .+-. sd 
Day 4 
Day 7 
Average 
__________________________________________________________________________ 
Control 
0 1.83 
2.63 
3.09 
2.52 .+-. 0.64 
10.5 
0.87 
5.69 
OJ/cm.sup.2 
30 1.80 
2.24 
3.02 
2.35 .+-. 0.62 
12.9 
3.3 8.10 
4.2 J/cm.sup.2 
30 1.99 
2.06 
3.00 
2.35 .+-. 0.56 
12.4 
2.6 7.50 
8.4 J/cm.sup.2 
30 1.75 
2.00 
2.60 
2.12 .+-. 0.44 
10.7 
2.0 6.35 
16.8 J/cm.sup.2 
30 1.67 
2.32 
2.63 
2.21 .+-. 0.49 
11.3 
0.62 
5.96 
25.2 J/cm.sup.2 
30 1.78 
2.99 
4.10 
2.96 .+-. 1.16 
13.0 
2.2 7.6 
__________________________________________________________________________ 
TABLE 4 
__________________________________________________________________________ 
Viability of AE-1 cells and CHO cells in calf sera that had been 
pretreated with different concentrations of B 
Mean Viable AE-1 Cells (.times.10.sup.5 /ml) 
Mean Viable CHO Cells (.times.10.sup.5 /ml) 
Treatment 
Day 2 
Day 4 
Day 7 
Mean .+-. sd 
Day 4 
Day 7 
Day 11 
Mean .+-. sd 
__________________________________________________________________________ 
Control 
0.77 
1.6 6.1 2.82 .+-. 2.86 
27.2 
17.5 
19.9 
21.5 .+-. 5.1 
60 .mu.g/mL 
1.0 3.1 4.7 2.93 .+-. 1.86 
35.4 
16.6 
14.0 
22.0 .+-. 11.7 
90 .mu.g/mL 
1.1 3.3 4.6 3.00 .+-. 1.77 
32.4 
20.1 
15.1 
22.5 .+-. 8.9 
120 .mu.g/mL 
1.1 2.6 4.7 2.80 .+-. 1.81 
34.5 
19.5 
14.4 
22.8 .+-. 10.4 
150 .mu.g/mL 
1.1 2.3 4.7 2.70 .+-. 1.83 
33.3 
16.3 
11.0 
20.2 .+-. 11.7 
__________________________________________________________________________ 
TABLE 5 
______________________________________ 
Selective Binding Properties of Photosensitizers 
% Above Equilibrium 
Concentrate Ratio*: 
Compound % Lipid % DNA Lipid/DNA 
N 
______________________________________ 
B 7.2 .+-. 1.8 
11.2 .+-. 3.5 
0.7 5 
A 0 8.5 .+-. 1.1 
0 3 
C 0 20.6 .+-. 7.6 
0 3 
AMT 11.6 .+-. 0.7 
3.4 .+-. 2.9 
3.4 3 
Khellin 3.2 .+-. 0.3 
1.8 .+-. 0.2 
1.8 3 
Visnagin 2.7 .+-. 0.8 
5.0 .+-. 1.3 
0.5 3 
Methylene Blue 
0.0 .+-. 0 
19.7 .+-. 1.5 
0 3 
Ethidium Bromide 
1.0 .+-. 0.5 
25.8 .+-. 0.9 
0.04 3 
Proflavine 7.1 .+-. 2.4 
16.2 .+-. 1.0 
0.4 3 
______________________________________ 
*&gt;1, Lipid preference 
&lt;1, DNA preference 
1, No preference 
TABLE 6 
__________________________________________________________________________ 
Summary of platelet in vitro properties following photoinactivation 
using 
B (300 .mu.g/mL) and UVA light (25 J/cm.sup.2) 
__________________________________________________________________________ 
Day 0* Day 3* Day 4* 
Assay Control 
Treated 
Control 
Treated 
Control 
Treated 
__________________________________________________________________________ 
HSR 65 .+-. 10 
50 .+-. 7 
70 .+-. 8 
38 .+-. 22 
64 .+-. 12 
0 
% Control 78 .+-. 12 47 .+-. 22 0 
Morphology 
308 .+-. 27 
300 .+-. 27 
288 .+-. 27 
232 .+-. 24 
280 .+-. 40 
203 .+-. 7 
% Control 94 .+-. 5 81 .+-. 10 74 .+-. 10 
pH 7.41.+-. 0.10 
7.29 .+-. 0.08 
7.34 .+-. 0.18 
6.37 .+-. 0.25 
7.18 .+-. 0.33 
5.84 .+-. 0.19 
N 14 14 12 12 5 5 
__________________________________________________________________________ 
Post-UV treatment addition of 20 mM Bicarbonate (final concentration): 
Day 0* Day 3* Day 4* 
Assay Control 
Treated 
Control 
Treated 
Control 
Treated 
__________________________________________________________________________ 
HSR 56 .+-. 9 
46 .+-. 2 
64 .+-. 7 
41 .+-. 10 
57 + 9 
50 .+-. 18 
% Control 97 .+-. 3 64 .+-. 8 86 .+-. 19 
Morphology 
323 .+-. 4 
277 .+-. 22 
288 .+-. 20 
257 .+-. 14 
293 .+-. 41 
274 .+-. 31 
% Control 92 .+-. 6 90 .+-. 10 94 .+-. 7 
pH 7.32.+-. 0.04 
7.23 .+-. 0.05 
7.30 .+-. 0.07 
7.31 .+-. 0.04 
7.26 .+-. 0.11 
7.05 .+-. 0.14 
N 3 3 3 3 3 3 
__________________________________________________________________________ 
HSR, hypotonic shock response 
*Posttreatment storage (platelets were 24 hours old at Day 0) 
Random donor platelet concentrates were pooled and subsequently divided 
into control and treated units. 
Photoinactivation was carried out in CLX containers containing 50 mL 
platelet concentrates and 10 mL of sensitizer solution in 0.9% saline 
(CP38, 1.8 mg/mL). 2 mL of 5% sodium bicarbnoate solution (20 mM final 
concentration in platelet concentrates) was added following 
photoinactivation treatment. 
TABLE 7 
__________________________________________________________________________ 
Summary of pheresed platelet in vitro properties following 
photoinactivation using B (300 .mu.g/mL) and UVA light (25 J/cm.sup.2) 
Day 0* Day 3* Day 4* 
Assay Control 
Treated 
Control 
Treated 
Control 
Treated 
__________________________________________________________________________ 
Hypotonic shock 
77 .+-. 6 
69 .+-. 9 
76 .+-. 3 
71 .+-. 6 
75 .+-. 7 
42 .+-. 28 
response (HSR) 
% Control 88 .+-. 3 93 .+-. 8 55 .+-. 34 
Morphology score 
335 .+-. 15 
333 .+-. 12 
293 .+-. 31 
273 .+-. 20 
305 .+-. 15 
208 .+-. 40 
% Control 97 .+-. 4 92 .+-. 10 68 .+-. 13 
Collagen 79 .+-. 6 
67 .+-. 6 
71 .+-. 7 
61 .+-. 14 
71 .+-. 9 
41 .+-. 23 
aggregation 
% of control 86 .+-. 4 86 .+-. 18 58 .+-. 32 
GMP-140 11 .+-. 5 
10 .+-. 4 
14 .+-. 7 
30 .+-. 18 
22 .+-. 6 
68 .+-. 17 
expression 
pH 7.50 .+-. 0.05 
7.36 .+-. 0.06 
7.47 .+-. 0.05 
6.73 .+-. 0.13 
7.45 .+-. 0.03 
6.09 .+-. 0.20 
N 9 9 9 9 6 6 
__________________________________________________________________________ 
*Post-treatment storage (platelets were 24 hours old at Day 0) 
Pheresed platelet concentrates were divided into control and treated 
units. 
Photoinactivation was carried out in PL 732 containers containing 50 mL 
platelet concentrates and 10 mL of sensitizer solution in 0.9% saline (B 
1.8 mg/mL). 
HSR, hypotonic shock response. 
TABLE 8 
__________________________________________________________________________ 
Summary of platelet in vitro properties following photoinactivation 
using 
A (200 .mu.g/mL) and UVA light (29 J/cm.sup.2) 
Day 1 Day 4 Day 5 
Assay Control 
Treated 
Control 
Treated 
Control 
Treated 
__________________________________________________________________________ 
Cell Count 
0.73 .+-. 0.2 
0.63 .+-. 0.25 
0.71 .+-. 0.24 
0.74 .+-. 0.2 
0.71 .+-. 0.21 
0.78 .+-. 0.21 
(.times.10.sup.11) 
Morphology 
302 .+-. 25 
251 .+-. 16 
267 .+-. 26 
238 .+-. 15 
257 .+-. 36 
226 .+-. 21 
Score 
% Discs 57 .+-. 12 
36 .+-. 8 
46 .+-. 10 
30 .+-. 8 
41 .+-. 16 
23 .+-. 9 
HSR (%) 62 .+-. 9 
46 .+-. 7 
72 .+-. 4 
52 .+-. 7 
74 .+-. 6 
48 .+-. 7 
pH at 7.37 .+-. 0.09 
7.20 .+-. 0.05 
7.38 .+-. 0.16 
6.96 .+-. 0.16 
7.37 .+-. 0.18 
6.69 .+-. 0.22 
room temp. 
Lactate 5.6 .+-. 1.2 
5 .+-. 1 
16 .+-. 4 
16 .+-. 2 
15 .+-. 3 
18 .+-. 2 
(m mole/L 
pO2 87 .+-. 35 
59 .+-. 47 
113 .+-. 18 
135 .+-. 14 
53 .+-. 30 
33 .+-. 14 
(mm Hg) 
PCO2 30 .+-. 5 
37 .+-. 7 
16 .+-. 2 
13 .+-. 3 
17 .+-. 2 
20 .+-. 7 
(mm Hg) 
Aggreg. 39 .+-. 6 
21 .+-. 7 
36 .+-. 8 
21 .+-. 6 
34 .+-. 7 
18 .+-. 6 
Rate 
Aggreg. 48 .+-. 5 
35 .+-. 8 
45 .+-. 10 
34 .+-. 7 
43 .+-. 9 
31 .+-. 9 
Extent 
GMP-140 35 .+-. 16 
38 .+-. 14 
31 .+-. 10 
51 .+-. 11 
42 .+-. 11 
67 .+-. 9 
expression (%) 
N 6 6 6 6 6 6 
__________________________________________________________________________ 
One day old random donor platelet concentrates in CLX containers were 
subjected to photoinactivation under specified conditions in a prototype 
UVA reactor. Platelet in vitro properties were measured after treatment 
and subsequent storage in a platelet incubator with constant agitation. 
Aggregation response was measured with dual agonist ADP (80 .mu.M) and 
Collagen (8 .mu.g/mL) in an aggregometer.