U.S. patent application number 13/155983 was filed with the patent office on 2011-09-29 for induction of and maintenance of nucleic acid damage in pathogens using riboflavin and light.
This patent application is currently assigned to CARIDIANBCT BIOTECHNOLOGIES, LLC. Invention is credited to Raymond P. GOODRICH, Shawn D. KEIL, Vijay KUMAR, Robert Owen LOCKERBIE.
Application Number | 20110236876 13/155983 |
Document ID | / |
Family ID | 46329704 |
Filed Date | 2011-09-29 |
United States Patent
Application |
20110236876 |
Kind Code |
A1 |
LOCKERBIE; Robert Owen ; et
al. |
September 29, 2011 |
Induction of and Maintenance of Nucleic Acid Damage in Pathogens
Using Riboflavin and Light
Abstract
A process for damaging and maintaining damage to the nucleic
acids of pathogens such as white blood cells, bacteria and viruses
which may be contained in blood or blood components. This process
comprises adding to the blood or blood component containing
pathogens an effective amount of riboflavin, and exposing the fluid
to light of an appropriate wavelength to damage the nucleic acid of
the pathogen and to substantially maintain the damage to the
pathogenic nucleic acids to allow for subsequent transfusion into a
recipient.
Inventors: |
LOCKERBIE; Robert Owen;
(Littleton, CO) ; KUMAR; Vijay; (Gold River,
CA) ; KEIL; Shawn D.; (Wheatridge, CO) ;
GOODRICH; Raymond P.; (Lakewood, CO) |
Assignee: |
CARIDIANBCT BIOTECHNOLOGIES,
LLC
Lakewood
CO
|
Family ID: |
46329704 |
Appl. No.: |
13/155983 |
Filed: |
June 8, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11932629 |
Oct 31, 2007 |
7985588 |
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13155983 |
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10648536 |
Aug 25, 2003 |
7901673 |
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11932629 |
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10377524 |
Feb 28, 2003 |
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10648536 |
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09586147 |
Jun 2, 2000 |
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10377524 |
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60319488 |
Aug 23, 2002 |
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60319641 |
Oct 22, 2002 |
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Current U.S.
Class: |
435/2 |
Current CPC
Class: |
A61M 1/3683 20140204;
A61L 2202/22 20130101; A61L 2/084 20130101; A61M 1/3693 20130101;
A61L 2/0082 20130101; A61M 1/0213 20140204; A61M 2205/123 20130101;
A61M 1/3681 20130101; A61L 2/0011 20130101; A01N 43/90 20130101;
A61L 2/08 20130101; A61L 2/10 20130101; A61K 41/17 20200101; A61L
2/0088 20130101; A01N 43/90 20130101; A01N 25/00 20130101; A01N
43/90 20130101; A01N 2300/00 20130101 |
Class at
Publication: |
435/2 |
International
Class: |
A01N 1/02 20060101
A01N001/02 |
Claims
1. A process for preventing self-repair of nucleic acid of
pathogenic white blood cells, bacteria and/or viruses which may be
contained in blood components comprising the steps of: adding to
the blood components riboflavin acting as a photosensitizer at a
final concentration of between about 50-500 .mu.M; irradiating the
blood components and riboflavin acting as a photosensitizer with
light in a visible or UV range at an appropriate wavelength to
activate the riboflavin acting as a photosensitizer to fragment the
nucleic acid of the pathogenic white blood cells, bacteria and/or
viruses to cause permanent damage to the nucleic acid; preventing
self-repair of the nucleic acid; and wherein the permanent damage
to the nucleic acid caused by the photosensitizer and light is
maintained over time such that the pathogenic white blood cells,
bacteria and/or viruses will not reproduce in the blood
components.
2. The process of claim 1 further comprising adding to the blood
components and riboflavin acting as a photosensitizer a solution
containing additives to enhance blood component viability.
3. The process of claim 1 wherein the blood component further
comprises platelets.
4. The process of claim 1 wherein the blood component further
comprises red blood cells.
5. The process of claim 1 wherein the light used to irradiate the
blood components and riboflavin acting as a photosensitizer is in
the UVB range.
Description
PRIORITY CLAIM
[0001] This application is a continuation of U.S. application Ser.
No. 11/932,629 filed on Oct. 31, 2007, now allowed, which is a
continuation-in-part of U.S. application Ser. No. 10/648,536 filed
Aug. 25, 2003, now U.S. Pat. No. 7,901,673, which is a
non-provisional of U.S. Provisional Application No. 60/319,488
filed Aug. 23, 2002, now expired; a non-provisional of U.S.
Provisional Application No. 60/319,641 filed Oct. 22, 2002, now
expired; and a continuation of U.S. application Ser. No. 10/377,524
filed Feb. 28, 2003, now pending, which is a continuation of U.S.
application Ser. No. 09/586,147 filed Jun. 2, 2000, now
abandoned.
BACKGROUND
[0002] Whole blood collected from volunteer donors for transfusion
into recipients is typically separated into its components: red
blood cells, white blood cells, platelets, plasma and plasma
proteins, by apheresis or other known methods. Each of these blood
components are typically stored individually and are used to treat
a multiplicity of specific conditions and disease states. For
example, the red blood cell component is used to treat anemia, the
concentrated platelet component is used to control bleeding, and
the plasma component is used frequently as a source of Clotting
Factor III for the treatment of hemophilia.
[0003] After the components are separated, the white blood cell
component is typically discarded, unless the cells are needed for
specific applications such as photoimmune therapy or photophoresis.
Cell separation procedures are not 100% effective. There is
unusually some small percentage of other types of cells which are
carried over into a separated blood component. Undesirable cells
are typically cells of a different type which are carried over in
some percentage into a desirable component. Cells such as white
blood cells, which may transmit infections including HIV and CMV as
well as causing other transfusion-related complications such as
Transfusion-Associated Graft vs. Host Disease (TAGVHD),
graft-vs-tumor responses and cytokine production are considered
undesirable. The white blood cells present in the transfused blood
product can also induce recipient immune responses directly by
acting as antigen-presenting cells or indirectly by serving as a
source of antigen that can be processed and presented by
antigen-presenting cells. The recipient antidonor responses can
include responses that eliminate donor cells, production of
alloantiantibodies, and the induction of immunoregulatory responses
that can result in increased infections, increased risk of tumor
relapse, and enhanced survival of transplanted organs.
[0004] Ways to reduce the risks of these transfusion related
complications from white blood cells is either to reduce the number
of white blood cells transfused into a recipient, and/or to
effectively destroy the viability and capacity of any transfused
white blood cells to function post transfusion. White blood cells
include granulocytes, monocytes and lymphocytes.
[0005] Current methods used to deplete contaminating white blood
cells in blood products to be transfused include leukocyte
filtration, UV irradiation of platelet concentrates and gamma
irradiation of red blood cells and platelet concentrates. These
methods do not completely eliminate the white blood cells however,
and gamma and UV irradiation affect the cell quality of desired
blood components such as platelets and red blood cells.
[0006] The blood or blood component to be transfused may also be
contaminated with microorgansisms which may cause infections or
unwanted immune reactions in the transfusion recipient.
Microorganisms which may be present include, but are not limited
to, viruses, (both extracellular and intracellular), bacteria,
fungi, blood-transmitted parasites and protozoa.
[0007] Photosensitizers, or compounds which absorb light of a
defined wavelength and transfer the absorbed energy to an electron
acceptor may be a solution to the above problems, by inactivating
pathogens contaminating a blood product without damaging the
desirable components of blood. For the purposes of this invention,
the general term "pathogen" may encompass any undesirable organism
which may be found in blood or a blood product. Pathogens may be
undesirable cells such as white blood cells, or may include
microorganisms such as bacteria, parasites or viruses.
[0008] There are many pathogen reduction compounds known in the art
to be useful for inactivating microorganisms or other infectious
particles. Examples of such photosensitizers include porphyrins,
psoralens, dyes such as neutral red, methylene blue, acridine,
toluidines, flavine (acriflavine hydrochloride) and phenothiazine
derivatives, coumarins, quinolones, quinones, and
anthroquinones.
[0009] Dardare et al. showed in a study of the binding affinities
of several common photosensitizers that both psoralen and methylene
blue substantially bind to nucleic acids, phospholipid membranes
and proteins in typical pathogen eradication experiments.
[0010] Although many publications have shown that damage to nucleic
acids can be caused by photosensitizers and light, the issue of
whether induction of damage to the nucleic acids of pathogens and
undesirable cells is maintained over time, and after the pathogen
reduced cells have been infused into a recipient has not been
addressed.
[0011] One study done by R. Mababagloob et al. looked to see what
effect S-59 and S-303 (psoralens) had on the DNA repair mechanisms
of D. radiodurans, a bacteria which has multiple genomic copies and
redundant repair mechanisms. The authors found that the above
treatment in combination with UVA light or change in pH, resulted
in 1 S-303 adduct for every 114 genomic DNA base pairs and 1 S-59
adduct for every 58 genomic DNA base pairs. However, this study did
not examine whether the damage to the DNA was maintained after
treatment, or was repaired by the bacteria. (Mababangloob, R.,
Castro, G., Stassinopoulos, A.; Helinx Technology, Utilized in the
Intercept Blood System, Effectively Inactivates Deinoccus
radiodurans, a Bacterium with Highly Efficienty DNA Repair;
Abstract presented at the 44.sup.th Annual Meeting of the American
Society of Hematology, 2002).
[0012] It is towards the method of pathogen reducing blood and
blood components by inducing permanent damage to the nucleic acids
of pathogens that the present invention is directed. Permanent
damage means that the inactivated pathogens are unable to
re-activate upon storage or upon infusion into a patient.
SUMMARY
[0013] The present invention is directed towards a method of
substantially inactivating pathogens such as white blood cells, and
microorganisms such as bacteria and viruses which may be present in
a fluid to be transfused into a recipient. The invention is for a
process for substantially maintaining damage to pathogen nucleic
acid caused by a photosensitizer and light in a fluid containing
pathogens and blood components comprising the steps of adding to
the fluid a photosensitizer comprising riboflavin; irradiating the
fluid and photosensitizer with light at an appropriate wavelength
to activate the riboflavin to cause damage to the nucleic acid of
the pathogen; and substantially maintaining the damage to the
pathogen nucleic acid during storage and/or after transfusion of
the fluid into a recipient.
BRIEF DESCRIPTION OF THE DRAWINGS
[0014] FIG. 1 is a graph showing the production of IL-2 from Jurkat
cells after treatment with riboflavin and UV light.
[0015] FIG. 2 is a graph showing the production of IL-2 from Jurkat
cells after treatment with riboflavin and visible light.
[0016] FIG. 3a is a graph showing oxygen consumption of Jurkat
cells after treatment with riboflavin and UV light.
[0017] FIG. 3b is a graph showing oxygen consumption of Jurkat
cells after treatment with riboflavin and visible light.
[0018] FIG. 4 is a table showing fragmentation of Jurkat cell DNA
over a two-day period following treatment with riboflavin and
either UV or visible light.
[0019] FIG. 5 is a graph measuring the percent change in the
integrity of the genomic DNA of separated human leukocytes
following treatment with ultraviolet light and with and without
riboflavin.
[0020] FIG. 6a is an agarose gel showing laddering of DNA after
treatment with and without ultraviolet light and riboflavin.
[0021] FIG. 6b is a graph which quantifies the percentage of intact
(not fragmented) DNA from the gel shown in 6a.
[0022] FIG. 7 shows the log reactivation of lambda-phage virus
after treatment with riboflavin and ultraviolet light.
[0023] FIG. 8 is a graph showing the effect of riboflavin and light
treatment on the proliferative response of human MNCs to mitogenic
stimulation.
[0024] FIG. 9 is a graph showing the effect of riboflavin and light
treatment on the proliferative response of human MNCs to allogenic
stimulator cells.
[0025] FIG. 10 is a graph showing the effect of riboflavin and
light treatment on the proliferative response of human MNCs to
anti-CD3 or anti-CD3 and anti-CD28 stimulation.
[0026] FIG. 11 is a graph showing the effect of riboflavin and
light treatment on the ability of MNCs to stimulate the
proliferation of allogeneic responder cells.
[0027] FIG. 12 is a table showing the production of cytokines by
treated or untreated MNCs in response to LPS stimulation.
[0028] FIG. 13 is a table showing the production of cytokines by
treated or untreated MNCs in response to anti-CD3 or anti-CD3 and
anti-CD28 stimulation.
DETAILED DESCRIPTION OF THE INVENTION
[0029] A "photosensitizer" useful in this invention is defined as
any compound which absorbs radiation at one or more defined
wavelengths and subsequently utilizes the absorbed energy to carry
out a chemical process. In this case, the desired photosensitizer
will bind to any nucleic acids which are present in the fluid to be
decontaminated to which the photosensitizer has been added. The
chemical processes which occur damage any nucleic acids which may
be in the fluid. The fluid may contain blood or blood products, or
may be a solvent which requires sterility.
[0030] Endogenous photosensitizers are preferred for use in this
invention. The term "endogenous" means naturally found in a human
or mammalian body, either as a result of synthesis by the body or
because of ingestion as an essential foodstuff (e.g. vitamins) or
formation of metabolites and/or byproducts in vivo. When endogenous
photosensitizers are used, particularly when such photosensitizers
are not inherently toxic or do not yield toxic photoproducts after
photoradiation, no removal or purification step is required after
decontamination, and the decontaminated product can be directly
returned to a patient's body or administered to a patient in need
of its therapeutic effect.
[0031] Examples of such endogenous photosensitizers which may be
used in this invention are alloxazines such as
7,8-dimethyl-10-ribityl isoalloxazine (riboflavin),
7,8,10-trimethylisoalloxazine (lumiflavin), 7,8-dimethylalloxazine
(lumichrome), isoalloxazine-adenine dinucleotide (flavine adenine
dinucleotide [FAD]) and alloxazine mononucleotide (also known as
flavine mononucleotide [FMN] and riboflavine-5-phosphate). The term
"alloxazine" includes isoalloxazines.
[0032] Use of endogenous isoalloxazines as a photosensitizer to
pathogen reduce blood and blood components are described in U.S.
Pat. Nos. 6, 258,577 and 6,277,337 both issued to Goodrich et al.,
and are herein incorporated by reference to the amount not
inconsistent.
[0033] Endogenously-based derivative photosensitizers include
synthetically derived analogs and homologs of endogenous
photosensitizers which may have or lack lower (1-5) alkyl or
halogen substituents of the photosensitizers from which they are
derived, and which preserve the function and substantial
non-toxicity thereof. Such endogenously-based derivative
photosensitizers which may be used in this invention are disclosed
in U.S. Pat. No. 6,268,120 to Platz et al., which discloses
alloxazine derivatives which may also be used to inactivate
microorganisms contained in blood or blood components. This patent
is incorporated by reference into the present invention to the
amount not inconsistent herewith.
[0034] The photosensitizer compound riboflavin
(7,8-dimethyl-10-ribityl isoalloxazine) has been reported to attack
nucleic acids. Nucleic acids include both deoxyribonucleic acids
and ribonucleic acids. Photoalteration of nucleic acid in the
presence of riboflavin is discussed in Tsugita, A, et al. (1965),
"Photosensitized inactivation of ribonucleic acids in the presence
of riboflavin," Biochimica et Biophysica Acta 103:360-363; and
Speck, W. T. et al. (1976), "Further Observations on the
Photooxidation of DNA in the Presence of Riboflavin," Biochimica et
Biophysica Acta 435:39-44. Binding of lumiflavin
(7,8,10-trimethylisoalloxazine) to DNA is discussed in Kuratomi,
K., et al. (1977), "Studies on the Interactions between DNA and
Flavins," Biochimica et Biophysica Acta 476:207-217. Hoffmann, M.
E., et al. (1979), DNA strand breaks upon exposure to proflavine
and light are reported in Piette, J. et al. (1979), "Production of
Breaks in Single- and Double-Stranded Forms of Bacteriophage .PHI.X
174 DNA by Proflavine and Light Treatment," Photochemistry and
Photobiology 30:369-378, and alteration of guanine residues during
proflavine-mediated photosensitization of DNA is discussed in
Piette, J., et al. (1981), "Alteration of Guanine Residues during
Proflavine Mediated Photosensitization of DNA," Photochemistry and
Photobiology 33:325-333.
[0035] What has not been previously known or explored prior to the
instant invention, is whether the strand breaks in the nucleic
acids caused by the photolysis of riboflavin is permanent. That is,
whether the nucleic acid repair mechanisms of the pathogen are
unable to repair the damage caused by riboflavin and light and
therefore prevent the pathogen from replicating. In this context,
repair is defined as the molecular processes that are the basis for
pathogen reactivation. Reactivation, or the synonymous term
recovery, is defined as the regaining of, by a damaged pathogen,
the capability to propagate and to form a colony.
[0036] Substantially maintaining the damage means that any damage
sustained by the nucleic acids of pathogens is maintained over time
so that when the blood product, which has been treated with
riboflavin and light is transfused into a recipient, the
inactivated pathogen will not self-repair the damaged nucleic
acids, and reproduce in the transfusion recipient. Transfusion
related complications caused by viable pathogens which may be
contained in the pathogen reduced blood or blood product will
therefore be substantially reduced.
[0037] The method of this invention requires mixing the
photosensitizer with the whole blood or with the separated blood
component to be decontaminated. Mixing may be done by simply adding
the photosensitizer or a solution containing the photosensitizer to
a fluid to be decontaminated. In one embodiment, the material to be
decontaminated to which photosensitizer has been added is flowed
past a photoradiation source, and the flow of the material
generally provides sufficient turbulence to distribute the
photosensitizer throughout the fluid to be decontaminated. In
another embodiment, the fluid and photosensitizer are placed in a
photopermeable container and irradiated in batch mode, preferably
while agitating the container to fully distribute the
photosensitizer and expose all the fluid to the radiation.
[0038] The amount of photosensitizer to be mixed with the fluid
will be an amount sufficient to adequately inactivate any
pathogenic nucleic acids which may be present in the fluid, but
less than a toxic (to the desired components) or insoluble amount.
If riboflavin is used as the photosensitizer, it may be added to
the fluid at a final concentration of between about 50-500 .mu.M.
Pathogenic nucleic acid includes any undesirable nucleic acid such
as nucleic acid from white blood cells, bacteria or viruses.
Nucleic acids include either deoxyribonucleic acid (DNA),
ribonucleic acid (RNA) or both.
[0039] The fluid containing the photosensitizer is exposed to light
of the appropriate wavelength to activate the photosensitizer and
to cause substantially permanent damage to the pathogenic nucleic
acids. The pathogen reduced fluid may then be stored for a period
of time before being transfused into a recipient, or may be
transfused into a recipient directly after the pathogen reduction
procedure.
Example 1
[0040] To determine the effectiveness of isoalloxazine (riboflavin)
photosensitizer and light in damaging and maintaining the damage to
nucleic acids of white blood cells that may be contained in a
solution of red blood cells or platelets, Jurkat cells (a model
T-lymphocytic cell line) were spiked into solutions containing
either red blood cells or platelets. Jurkat cells were initially
grown in RPMI cell growth medium at 37.degree. C. in 5%
CO.sub.2.
[0041] Set forth below is one protocol which may be used in this
invention for determining the effectiveness of riboflavin and light
in causing damage to and subsequently maintaining the damage to the
nucleic acids of any contaminating white blood cells in a solution
containing red blood cells.
[0042] Fluid containing red blood cells and having 5% plasma
carryover may be placed into any container known in the art. A 1 L
bag is one such example, not meant to be limiting. A quencher may
also be optionally added to the fluid. Such quenchers may include
antioxidants or other agents to prevent damage to desired fluid
components or to improve the rate of inactivation of
microorganisms. Quenchers useful in this invention are exemplified
by glutathione, n-acetyl-cysteine, cysteine, adenine, histidine,
tyrosine, tryptophan, ascorbate, vitimin E, trolox, TPGS and
mixtures thereof. Riboflavin was added to the cells at a final
concentration of 500 .mu.M either with the quencher (if optionally
added) or separately. Any means known in the art for adding the
photosensitizer to the fluid to be decontaminated and for placing
the fluid in the container may be used. Such means typically
include flow conduits, ports, reservoirs, valves, and the like.
Additives which enhance blood component viability after treatment
with riboflavin and light may also be added either pre or post
illumination. Examples of additives which may be added to the fluid
may include methylxanthines and/or PGE1.
[0043] Air was added to the fluid containing red blood cells and
riboflavin at a volume of 133 mL for every 266 mL of fluid. Air may
be added to the fluid contained in the bag by any means known in
the art. The fluid containing red cells was then "spiked" with
Jurkat cells at a concentration of 10.sup.6/mL to simulate white
blood cell carryover in a collection procedure. The fluid
containing red blood cells and spiked Jurkat cells was illuminated
with light in the visible (from a source having a peak emission at
447 nm) spectrum at an intensity of 190 J/cm.sup.2. Treated cells
and untreated controls were incubated for 24 hours at 37.degree. C.
in growth medium to assess long term survival of cells which were
not killed during the illumination process. After treatments, the
Jurkat cells were recovered and assayed for cell viability markers,
such as production of IL-2, cell proliferation and fragmentation of
DNA.
[0044] A similar protocol was used to determine the effectiveness
of inactivating white blood cells and maintaining the inactivation
of the cells in a solution containing platelets. A final
concentration of 50 .mu.M isoalloxazine was added to platelets
collected by apheresis with a 90% plasma carryover. The fluid
containing platelets was "spiked" with Jurkat cells at a
concentration of 10.sup.6/mL to simulate white blood cell
contamination. The fluid containing platelets and spiked Jurkat
cells was illuminated with light in the ultraviolet (UV) spectrum
at 320 nm and an intensity of 7 J/cm.sup.2. After treatments, the
Jurkat cells were recovered and assayed for markers of cell
viability, such as production of IL-2, cell proliferation and
fragmentation of DNA.
[0045] Jurkat cell death was assessed with the Molecular Probes
Live/Dead Cytotoxicity/Viability assay. Cells were exposed to a
staining solution containing ethidium homodimer-1 which stains
non-viable (dying or dead) cells orange/red and calcein AM which
stains viable (live) cells green. Cells with holes in their
membrane appear red/orange when stain is excited with 495 nm light.
Intact cells stain green. Cells that appear orange and that contain
no green are counted as dead cells; green cells that contain no
orange stain are counted as live. Dead cells in a sample are
expressed as a percentage of the total number of cells present in a
sample.
[0046] FIGS. 1 and 2 show release of FL-2 over time by Jurkat cells
after stimulation with PMA and PHA. Jurkat cells were treated with
riboflavin (designated Rb in these Figs.) and either UV
(ultraviolet) (see FIG. 1) or visible light (see FIG. 2) and then
stimulated to produce IL-2. The addition of saline alone (no
riboflavin, no light) or riboflavin alone (no light) serves as the
experimental controls. Production of IL-2 by Jurkat cells upon
stimulation with PMA and PHA is a measure of T-cell viability.
After treating the cells with riboflavin and light, PMA and PHA are
added to induce production of IL-2 and the cells are incubated
overnight. IL-2 in the supernatent is detected using an ELISA
assay.
[0047] FIG. 1 shows IL-2 release from Jurkat cells after exposure
to riboflavin and UV light. Although difficult to see due to the
substantially similar results, UV alone, as well as riboflavin in
combination with UV light cause losses in cell viability and
subsequent decrease in IL-2 release.
[0048] FIG. 2 shows IL-2 release from Jurkat cells after exposure
to riboflavin and visible light. As can be seen from FIG. 2b, cells
treated with riboflavin and visible light cause substantial losses
in cell viability and subsequent decrease in IL-2 release. Visible
light alone also causes a decrease in the amount of cell viability
and subsequent release of IL-2, but not to the same extent as the
combination of riboflavin and visible light.
Example 2
[0049] FIGS. 3a and 3b are graphs showing oxygen consumption of
treated and control Jurkat cells. Oxygen consumption is a measure
of cell viability. Healthy cells respire and consume oxygen,
unhealthy and/or dead cells do not. A BD oxygen biosensor system
was used to monitor Jurkat cell metabolism for a period of 4 days
after treatment. Oxygen diffuses into the cell media from the
atmosphere. The presence of oxygen quenches a fluorescent marker
located at the bottom of the wells where the cells are grown. The
amount of oxygen consumed by the cells is measured by the amount of
fluorescence being produced. Where oxygen is used by healthy cells
for respiration, fluorescence is produced.
[0050] FIG. 3a is a graph measuring the amount of fluorescence
(NFRU) generated over time in Jurkat cells treated with riboflavin
(designated Rf in these Figs.) and UV light. Cells which have been
treated with riboflavin and UV light are damaged by the process.
Consequently, the cells are not metabolically active and therefore
consume no oxygen, thus producing little to no fluorescence.
[0051] FIG. 3b is a graph measuring fluoresce (NFRU) generated over
time in Jurkat cells treated with riboflavin and visible light.
Cells treated with riboflavin and visible light are damaged by the
process. As shown in FIG. 3b, treated cells are not metabolically
active and do not consume oxygen over a four day period. Little to
no fluorescence is produced.
Example 3
[0052] FIG. 4 shows fragmentation of DNA of Jurkat cells over a two
day period following treatment with riboflavin and either visible
or ultraviolet light. Strand breaks in the DNA of Jurkat cells were
measured by flow cytometry after treatment with riboflavin
(designated Rf in this Fig.) and light. % positive signifies
positive DNA damage. As seen in the table, on day one, UV light
alone causes DNA damage in 85.5% of cells. On day 2 however, only
0.4% of the cells exposed to UV light alone displayed DNA damage.
As shown in FIG. 4, exposure of the cells to UV light alone does
not maintain DNA damage over time. One hypothesis is that the cells
exposed to UV light alone may repair the damage. Another hypothesis
is that UV alone may not prevent the growth of new healthy
cells.
[0053] Upon treatment with riboflavin and UV light however, 36.1%
of the DNA in the treated cells were damaged, while on day 2, 87.5%
of the treated cells manifested DNA damage. The addition of
riboflavin appears to maintain the damage to DNA caused by exposure
to UV light.
[0054] Visible light alone did not cause DNA damage, however with
the addition of riboflavin, FIG. 4 illustrates that DNA was damaged
and the damage was substantially maintained over a two day
period.
[0055] From the data presented above, the addition of riboflavin or
other endogenous alloxazine derivatives appears to maintain the
damage to white blood cell DNA.
[0056] From FIG. 4 it can be seen that the addition of riboflavin
in combination with exposure to either visible or UV light not only
causes fragmentation of Jurkat DNA, but maintains the damage over
time.
[0057] FIG. 5 is a graph showing the percentage of DNA
fragmentation of human leukocyte genomic DNA on an agarose gel.
3.8.times.10.sup.5 autologous cells/mL in 90% plasma were added to
a 1 L ELP bag. Riboflavin at a final concentration of 400 .mu.M was
added and the cells were irradiated at 320 nm at a total intensity
of 5 J/cm.sup.2. As can be seen from this graph, the DNA of
autologous white blood cells showed around a 50% change in
integrity after being exposed to riboflavin and light as compared
to cells exposed to light alone. This result corresponds to the
results obtained with Jurkat cells.
Example 4
[0058] To measure the effect of riboflavin and light on bacterial
DNA kill, E. coli was spiked into 90% plasma. Riboflavin at a final
concentration of 50 .mu.M was added and the mixture was exposed to
light at an intensity range of 0 J/cm.sup.2, 10 J/cm.sup.2, 17
J/cm.sup.2 and 20 J/cm.sup.2. After light exposure, the bacterial
genomic DNA was purified using standard DNA purification
techniques, and the DNA fragmentation was analyzed by standard
agarose gel electrophoresis and fragmentation of the DNA was
quantified using standard imaging techniques.
[0059] FIG. 6b is a graph showing the percentage of fragmentation
of DNA (measured in terms of the percentage of DNA intact after
treatment) of E. coli after exposure to UV light either with or
without riboflavin. As can be seen in both the gel (see FIG. 6a)
and the graph, (see FIG. 6b) the combination of riboflavin and UV
light induced much greater bacterial DNA fragmentation than that
induced by UV light alone. More DNA degradation occurs at lower
energies with riboflavin than without.
Example 5
[0060] This study was done to determine if riboflavin and 320 nm
broadband UVB light causes irreparable damage to the DNA of
viruses.
[0061] It is known that UV light causes nucleic acid damage to
cells. However, exposure to UV light also causes up regulation of
cellular repair mechanisms. In the literature it has been reported
that viruses inactivated with UV light alone will reactivate a
small percentage of the time due to concurrent up regulation of the
host cell's nucleic acid repair mechanisms.
[0062] To test if riboflavin and light has the same inactivating
effect on the nucleic acid of viruses as it does on white blood
cells and bacteria, E. coli was chosen as the host cell for
lambda-phage virus. E. coli was irradiated with UV light to
initially up regulate the host cells nucleic acid repair
mechanisms. In this experiment, if the nucleic acid repair
mechanisms of the host cell are up regulated upon initial exposure
to UV light, any viral reactivation effect should be amplified. Any
prevention of viral reactivation should be easily quantified.
[0063] After initial exposure to UV light, host E. coli was placed
into Luria Broth growth media for 1-2 hours. Meanwhile, the lambda
phage virus was irradiated with 320 nm UVB light at an intensity of
0.08 J/cm.sup.2 in PBS and riboflavin at final concentrations of
between 0-300 .mu.M until a two log viral kill was achieved. The
irradiated virus was then incubated with either irradiated or
non-irradiated E. coli host cells for 20 minutes to allow for viral
absorption. The virus infected E. coli samples were plated and
allowed to grow overnight. At this time the plaques were counted to
determine the number of infectious particles.
[0064] FIG. 7 shows the amount of log virus reactivation at
increasing concentrations of riboflavin. The effect of riboflavin
on preventing virus reactivation was dose dependant. Approximately
0.8 log virus reactivation occurred over a concentration of 0-100
.mu.M riboflavin. At riboflavin concentrations greater than 100
.mu.M, reactivation was progressively prevented with no
reactivation occurring at 300 .mu.M.
[0065] These results suggest that irradiation of virus with both
riboflavin and light prevents reactivation of viral DNA.
Example 6
[0066] The high incidence of mortality associated with TAGVHD
eliminates the use of clinical trials to study the effectiveness of
various treatments in preventing TAGVHD. This has led to a search
for in vitro assays that would be representative of the in vivo
responses that contribute to the development of TAGVHD. Some of the
in vitro assays that may be used to measure this response include
proliferation of MNCs in response to a number of stimuli including
mitogens, allogeneic stimulator cells, or to T-cell antibodies; and
the ability to stimulate proliferation of normal responder cells.
These in vitro assays were used to test the effect of riboflavin
and light on the function of human mononuclear white blood cells
(MNCs).
Methods
[0067] The proliferative ability of MNCs in response to a number of
different stimuli was studied. MNCs were obtained by discontinuous
centrifugation with Ficol-Hypaque of WBCs obtained from the
leukoreduction portion of a standard apheresis platelet collection
on an automated blood collection machine. The MNCs were resuspended
in autologous platelelet poor plasma and split into two 250 mL
samples for a test sample and a control sample. The test samples
were treated with 35 mL of a 500 .mu.M riboflavin solution in 0.9%
saline. All samples were then exposed to 6.2 J UV light per mL
product for approximately 8 to 10 minutes. After treatment, the
cells from both test and control samples were pelleted by
centrifugation, washed and resuspended in RPMI 1640 containing 10%
fetal calf serum for the assays described below.
[0068] The proliferative response of MNCs to bind anti-CD3 and anti
CD28 antibodies was tested by adding 50 .mu.L of PBS containing 10
.mu.g per mL anti-CD3 or PBS containing 10 .mu.g per mL anti CD3
plus 4 .mu.g per mL anti CD28 or PBS alone to 96 well flat bottom
plates. The plates were incubated for 90 minutes at room
temperature, washed twice with PBS, and then 100 .mu.L RPMI 1640
containing 5% human AB serum and penicillin-streptomycin was added
to each well. After a 1 hour incubation at room temperature, 100
.mu.L it peripheral blood MNCs at 2.times.10.sup.6 cells per mL
were added to each well. The plates were incubated for 3 days and
pulsed with [.sup.3H] thymidine for 4 hours and harvested.
[0069] Cytokine levels were determined by measuring the
supernatants from the wells of MNCs (5.times.10.sup.6 MNCs/mL
RPMI1640 containing 10% FCS) that had been cultured for 24 hours
with or without 100 ng per mL lipoploysaccharide (LPS) or for 3
days with anti-CD3 or anti-CD3 and anti-CD28 coated plates
(prepared as described above) or with medium alone. The levels of
IL-10, IL-6, IL-8, IL-10, IL-12p70, TNF-.alpha., IL-2, IL-4, IL-5,
IL-10, TNF and IFN-.gamma., were measured with the CBA human
inflammatory cytokine kit (BD Biosciences, PharMingen).
Results
[0070] As seen in FIG. 8, the ability of MNCs treated with
riboflavin and light to proliferate in response to mitogenic
stimulation was tested by incubating riboflavin treated (see Graph
B) or control untreated (see Graph A) human MNCs with the mitogen
PHA. As seen in Graph B, treatment with riboflavin completely
inhibited the ability of the MNCs to proliferate in response to
mitogenic stimulation compared to the control treatment.
[0071] The effect of riboflavin and light on the proliferative
response of MNCs to allogeneic stimulator cells is shown in FIG. 9.
As in FIG. 8 above, treatment with riboflavin and light completely
inhibited the ability of MNCs to proliferate in response to
stimulation by allogenic stimulator cells compared to the control
treatment.
[0072] The effect of riboflavin and light on the proliferative
response to anti-CD3 or anti-CD3 plus anti-CD28 is shown in FIG.
10. The results again demonstrate that no detectable MNC
proliferation was induced by these stimuli in riboflavin-treated
cells as compared to the control treatment.
[0073] MNCs present in blood products are capable of presenting
antigens to recipient cells and inducing a response by the
recipient cells. As seen in FIG. 11, when riboflavin-treated MNCs
were tested for their ability to induce proliferation of allogeneic
responder cells, the riboflavin treated cells were unable to
stimulate any proliferation by the allogeneic responder cells. This
indicates that riboflavin and light are able to inhibit antigen
presentation capabilities of the riboflavin and light-treated
cells.
[0074] Another measure of the functionality of MNCs is to measure
the cytokines that are produced in response to stimulation.
Stimulation of MNCs with LPS produces cytokines as a result of
signaling via myeloid cells, while stimulation of MNCs with
anti-CD3 plus anti-CD28 produces cytokines as a result of signaling
via T-cells.
[0075] As shown in FIG. 12, incubation of MNCs with LPS resulted in
barely detectable levels of IL-2, IL-4, IL-5 and IL-12p70 in the
supernatants of either treated or control cells. However, LPS did
induce production of IL-1.beta., IL-10, IFN-.gamma., and
TNF-.alpha. in control cells but not in treated cells.
[0076] As seen in FIG. 13, increased production of IL-2, IL-10,
TNF-.alpha. and IFN-.gamma. was seen when control MNCs were
incubated with the T-cell mitogens anti-CD3 and anti-CD3 plus
anti-CD28, but not in the treated cells.
[0077] The results of these assays show that treatment of human
MNCs with riboflavin and light functionally inactivate MNCs, as
well as inducing irreparable damage to the nucleic acids of the
cells.
* * * * *