U.S. patent application number 11/624487 was filed with the patent office on 2007-06-28 for removal of adenine during a pathogen reduction process in whole blood or red blood cells by dilution.
This patent application is currently assigned to GAMBRO, INC.. Invention is credited to Suzann K. Doane, Raymond P. Goodrich.
Application Number | 20070148630 11/624487 |
Document ID | / |
Family ID | 29270709 |
Filed Date | 2007-06-28 |
United States Patent
Application |
20070148630 |
Kind Code |
A1 |
Goodrich; Raymond P. ; et
al. |
June 28, 2007 |
Removal of Adenine During a Pathogen Reduction Process in Whole
Blood or Red Blood Cells by Dilution
Abstract
The methods of this invention involve preventing the formation
of a complex between adenine and riboflavin by reducing the amount
of adenine in a solution containing blood or blood components to be
pathogen reduced.
Inventors: |
Goodrich; Raymond P.;
(Lakewood, CO) ; Doane; Suzann K.; (Littleton,
CO) |
Correspondence
Address: |
GAMBRO, INC;PATENT DEPARTMENT
10810 W COLLINS AVE
LAKEWOOD
CO
80215
US
|
Assignee: |
GAMBRO, INC.
Lakewood
CO
|
Family ID: |
29270709 |
Appl. No.: |
11/624487 |
Filed: |
January 18, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
10423200 |
Apr 24, 2003 |
7183045 |
|
|
11624487 |
Jan 18, 2007 |
|
|
|
60375849 |
Apr 24, 2002 |
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Current U.S.
Class: |
435/2 ;
435/372 |
Current CPC
Class: |
A01N 1/0294 20130101;
A61L 2/0088 20130101; A61L 2/10 20130101; A61L 2/084 20130101; A61L
2202/22 20130101; A61L 2/16 20130101; A61L 2/08 20130101; A01N
43/90 20130101; A61K 41/17 20200101; A61P 7/08 20180101; A61L
2/0011 20130101; A61L 2/0082 20130101; A61L 2/0005 20130101; A61K
35/18 20130101; A01N 1/0205 20130101; A01N 1/0215 20130101 |
Class at
Publication: |
435/002 ;
435/372 |
International
Class: |
A01N 1/02 20060101
A01N001/02; C12N 5/08 20060101 C12N005/08 |
Claims
1. A method for treating a selected quantity of fluid to reduce
pathogens which may be present therein, the fluid containing one or
more components selected from the group consisting of blood and
blood components, the method comprising: (a) adding a sufficient
volume of a washing solution to the selected quantity of fluid; (b)
washing the selected quantity of fluid to create a washed fluid;
(c) mixing a pathogen reduction-effective, substantially non-toxic
amount of 7,8-dimethyl-10-ribityl isoalloxazine photosensitizer
with the washed fluid; (d) exposing the washed fluid containing the
photosensitizer to photoradiation of sufficient wavelength and
energy to activate the photosensitizer, whereby the pathogens are
reduced.
2. The method of claim 1 wherein the washing step further comprises
a plurality of washing steps.
3. The method of claim 1 wherein the method substantially prevents
damage to the blood or blood components contained in the fluid by
reducing the concentration of adenine in the fluid to prevent
formation of a photosensitizer-adenine complex.
4. The method of claim 1 wherein the method substantially prevents
an increase in oxidation reactions produced by photolysis of the
photosensitizer.
5. The method of claim 1 wherein the mixing step further comprises
placing the fluid in a container transparent to photoradiation,
adding the photosensitizer to the fluid in the container and
agitating the container.
6. The method of claim 1 wherein the washed fluid consists
essentially of platelets.
7. The method of claim 1 wherein the washed fluid consists
essentially of red blood cells.
8. A fluid for pathogen reduction that is substantially
adenine-free comprising: one or more components selected from the
group consisting of blood and blood components; an endogenous
photosensitizer or an endogenously-based derivative photosensitizer
and an adenine-free solution.
Description
PRIORITY CLAIM
[0001] This application is a divisional of U.S. Non-provisional
application Ser. No. 10/423,200 filed Apr. 24, 2003, which claims
priority from U.S. Provisional 60/375,849 filed Apr. 24, 2002.
BACKGROUND OF THE INVENTION
[0002] Contamination of blood supplies with infectious
microorganisms such as HIV, hepatitis and other viruses and
bacteria presents a serious health hazard for those who must
receive transfusions of whole blood or administration of various
blood components such as platelets, red cells, blood plasma, Factor
VIII, plasminogen, fibronectin, anti-thrombin III, cryoprecipitate,
human plasma protein fraction, albumin, immune serum globulin,
prothrombin, plasma growth hormones, and other components isolated
from blood. Blood screening procedures which are currently
available may miss contaminants. Thus, there is a need for
sterilization procedures that effectively neutralize all infectious
viruses and other microorganisms but do not damage cellular blood
components, do not degrade desired biological activities of
proteins, and preferably do not need to be removed prior to
administration of the blood product to the patient.
[0003] The use of photosensitizers, compounds which absorb light of
a defined wavelength and transfer the absorbed energy to an energy
acceptor, has been proposed as a solution to the contamination of
blood and blood components. Various photosensitizers have been
proposed for use as blood additives for pathogen inactivation of
blood or blood components. A review of commonly used
photosensitizers, and some of the issues of importance in choosing
photosensitizers for decontamination of blood products is provided
in Goodrich, R. P., et al. (1997), "The Design and Development of
Selective, Photoactivated Drugs for Sterilization of Blood
Products," Drugs of the Future 22:159-171.
[0004] Some photosensitizers that have been proposed for use for
blood component photoirradiation have undesirable properties. For
example, European Patent Application 196,515 published Oct. 8,
1986, suggests the use of non-endogenous photosensitizers such as
porphyrins, psoralens, acridine, toluidines, flavine (acriflavine
hydrochloride), phenothiazine derivatives, and dyes such as neutral
red and methylene blue, as blood additives. Another molecule,
chlorpromazine, has been used as a photosensitizer; however its
usefulness is limited by the fact that it should be removed from
any fluid administered to a patient after the decontamination
procedure because it has a sedative effect. Protoporphyrin, which
occurs naturally within the body, can be metabolized to form a
photosensitizer; however, its usefulness is limited in that it
degrades the desired biological activities of proteins.
[0005] Most preferred with respect to the reduction of pathogens in
blood or blood products are endogenous photosensitizers. 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. Examples of such endogenous
photosensitizers 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]), alloxazine mononucleotide (also known as flavine
mononucleotide [FMN] and riboflavine-5-phosphate), vitamin Ks,
vitamin L, their metabolites and precursors, and napththoquinones,
naphthalenes, naphthols and their derivatives having planar
molecular conformations. The term "alloxazine" includes
isoalloxazines.
[0006] The use of the endogenous alloxazine photosensitizers such
as those mentioned above to reduce pathogens which may be contained
in blood or blood products are disclosed in U.S. Pat. Nos.
6,258,577 and 6,277,337 issued to Goodrich et. al and are herein
incorporated by reference in their entirety to the amount not
inconsistent.
[0007] Endogenously-based derivative photosensitizers useful in
this invention 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. U.S. Pat. No. 6,268,120 to Platz et al.
discloses alloxazine derivatives which may also be used to
inactivate microorganisms contained in blood or blood components.
This patent is also incorporated by reference into the present
invention to the amount not inconsistent.
[0008] When certain endogenous photosynthesizers are used certain
components which are naturally occurring in blood plasma or in some
synthetic blood storage/collection solutions may interact with the
photosensitizer during the photoinactivation process and form
complexes. The presence of these complexes may increase the rate of
side reactions which occur during the photolysis of the
photosensitizer. One such complex which may form if
7,8-dimethyl-10-ribityl isoalloxazine (riboflavin) is used as the
photosensitizer, is a complex between riboflavin and adenine.
Adenine is found in blood plasma as well as being an additive
component of some synthetic blood collection/storage solutions.
[0009] It is toward this end of preventing damage to blood and
blood components to be pathogen reduced by preventing the formation
of a photosensitizer-plasma constituent complex (such as adenine)
that the present invention is directed.
[0010] Several U.S. Patents discuss the removal of plasma and
plasma proteins in a pathogen inactivation process using
photosensitizers. U.S. Pat. No. 5,360,734 issued Nov. 1, 1994 and
U.S. Pat. No. 5,597,722 issued Jan. 28, 1997 both to Chapman et al.
discuss treating a blood component containing red blood cells and
plasma proteins by removing a portion of the plasma proteins before
adding the photoactive agent benzoporphyrin. The treated blood
component is prevented from contacting plasma proteins for a period
of time (three to eighteen hours) after treatment to prevent
binding of the treated cells to IgG proteins in the plasma. These
patents do not disclose or suggest the removal of plasma to prevent
the formation of specific plasma constituent-photosensitizer
complexes which changes the efficiency of the photosensitizer.
BRIEF SUMMARY OF THE INVENTION
[0011] Adenine is found naturally occurring in small concentrations
in plasma and in some synthetic blood collection/storage solutions.
One method of this invention involves preventing the formation of a
complex between adenine and riboflavin by reducing the amount of
adenine in a solution containing blood or blood components to be
pathogen reduced by reducing the level of plasma.
[0012] Another aspect of this invention involves the collection of
blood or blood components to be pathogen reduced into pathogen
reduction/storage solutions which are adenine free.
[0013] If it is desired to pathogen reduce previously collected
blood or blood components which were initially collected and stored
in a collection/storage solution containing adenine, another aspect
of this invention involves washing the previously collected blood
components with saline or like solution, before the pathogen
reduction process.
[0014] Another method which may be used for reducing the
concentration of selected components of plasma such as adenine in a
fluid to be pathogen reduced may be by selective filtration.
[0015] After removal of adenine by any means known in the art, the
fluid containing the blood component to be pathogen reduced is
combined with a photosensitizer such as riboflavin and exposed to
photoradiation of the appropriate wavelength to activate the
photosensitizer. The amount of photoradiation used is sufficient to
activate the photosensitizer as described herein, but less than
that which would cause non-specific damage to the biological
components or substantially interfere with biological activity of
other proteins present in the fluid. Non-specific damage is damage
that damages all components.
BRIEF DESCRIPTION OF THE DRAWINGS
[0016] FIG. 1 is a Jablonski diagram showing chemical reactions of
7,8-dimethyl-10-ribityl isoalloxazine (riboflavin and other related
compounds) catalyzed by photoradiation, oxygen and other
components.
[0017] FIG. 2 is a top plan view of a bag set containing a filter
for removal of adenine for use in a pathogen reduction
procedure.
[0018] FIG. 3 shows an embodiment of this invention using a bag to
contain the fluid being treated with the photosensitizer and a
shaker table to agitate the fluid while exposing to photoradiation
from a light source.
[0019] FIG. 4 is a graph comparing the % hemolysis of pathogen
reduced red blood cells stored over time in pathogen
reduction/storage solutions with and without adenine.
DETAILED DESCRIPTION OF THE INVENTION
[0020] The pathogen reduction method of this invention using
endogenous photosensitizers and endogenously-based derivative
photosensitizers is exemplified herein using
7,8-dimethyl-10-ribityl isoalloxazine as the photosensitizer.
[0021] 7,8-dimethyl-10-ribityl isoalloxazine (riboflavin or vitamin
B2) absorbs light from about 200 to 500 nm. The ring system core of
7,8-dimethyl-10-ribityl isoalloxazine is resistant to
photodegradation but the ribityl side chain of riboflavin undergoes
photodegradation. ##STR1##
[0022] Photosensitizers of this invention include compounds which
preferentially adsorb to nucleic acids, thus focusing their
photodynamic effect upon the nucleic acids of microorganisms and
viruses with little or no effect upon accompanying cells or
proteins. Pathogen kill using riboflavin and related compounds also
occurs upon photoinactivation via singlet oxygen damage, thereby
disrupting the ability of the pathogen to function and reproduce or
both.
[0023] FIG. 1 is a Jablonski diagram showing the photochemical
reactions of 7,8-dimethyl-10-ribityl isoalloxazine (riboflavin and
other related compounds) which occur upon catalysis by
photoradiation, oxygen and other components. The photosensitizer in
its ground state is referred to as S.sub.0. Upon absorption of
light, riboflavin is converted to an electrically excited state
which in condensed phase immediately (<<10.sup.-11s) relaxes
to the lowest vibrational level of the lowest excited state
(S.sub.0). The lifetimes of S.sub.1 states in solution are usually
in the range of 1-10 ns and are controlled by internal conversion
(IC) and fluorescence (F) decay back to S.sub.0 by intersystem
crossing (ISC) to a paramagnetic triplet state (T.sub.1) and by
inter and intramolecular chemical reactions. As is known in the
art, internal conversion is the radiationless transition between
energy states of the same spin state. Intersystem crossing (ISC) is
a radiationless transition between different spin states. When the
riboflavin relaxes from the singlet state to the ground state, it
is called fluorescence. When the molecule relaxes from the triplet
state (S.sub.1) to the ground (unexcited) state (S.sub.0) this is
called phosphorescence.
[0024] The left arrow (first vertical, upward-pointing arrow) in
the diagram of FIG. 1 indicates that upon absorption of light
energy the riboflavin molecule can go from its ground state
(S.sub.0) to its excited sate (S.sub.1) and become involved in
chemical reactions including losing its ribityl moiety to become
lumichrome (7,8-dimethylalloxazine). Lumichrome is not photoactive
under visible light.
[0025] Alternatively, as shown by the second vertical, downward
pointing arrow the excited molecule may release its absorbed energy
and fluoresce to return to the ground state. The wavy arrows
indicate that energy is released.
[0026] The excited riboflavin molecule may also relax to its
triplet state (T.sub.1) through intersystem crossing (ISC) by
changing the spin of an electron (spin conversion). The wavy line
labeled ISC indicates intersystem crossing. If no oxygen is
present, the molecule in its triplet state can phosphoresce (second
wavy, downward pointing arrow) and return to its ground state. Or,
as indicated by the right arrow, the molecule in its triplet state
can react with other molecules in close proximity and return to its
ground state. IF oxygen is present, the molecule in its triplet
state can react with oxygen and return to its ground state
producing .sup.1O.sub.2 (singlet oxygen). Singlet oxygen can cause
DNA strand breaks, further contributing to pathogen kill.
[0027] One disadvantage of using the described photochemical
methods for pathogen reduction of blood products is that the
singlet oxygen species generated in the process of photolysis of
riboflavin may cause damage to blood products and compromise their
suitability for transfusions. If certain plasma proteins or other
components of plasma are present during the photolytic process, the
presence of such components may magnify this oxidative process.
[0028] One component found in blood plasma and in some commonly
used blood storage solutions which, if present, has been suggested
to have an effect on the oxidative process of riboflavin, is the
nucleoside adenine. Uchara et al. has shown that upon
photoactivation, a specific complex is formed between riboflavin
and adenine which increases the photodynamic efficiency of
riboflavin. The authors showed an accelerative effect of the
riboflavin-adenine complex on the photodynamic inactivation of
yeast alcohol dehydrogenase. (Kinachino Uehara, Tadashi Mizoguchi,
Morio Yonezawa, Saburo Hosomi and Ryogi Hayashi, Effect of Adenine
on the Riboflavin-sensitized Photoreaction 1. Effect of Adenine on
the Photodynamic Inactivation of Yeast Alcohol Dehydrogenase in the
Presence of Ribflavin, J. Vitaminology 17, 148-154 (1971.))
[0029] While the formation of a riboflavin-adenine complex may
appear to be a desirable side effect in that the presence of the
complex would help to decrease the time necessary to pathogen
reduce any pathogens contained in and/or around blood or blood
components, in fact, the presence of the complex speeds up the
oxidative chemistry of riboflavin. The increase in production of
reactive oxygen species produced during the oxidation of
riboflavin, increases the possibility of cell membrane damage.
Cells which are damaged during a pathogen reduction procedure are
unable to be reinfused into a patient.
[0030] Because adenine is naturally occurring in plasma, in one
embodiment of the present invention, the adenine content of fluid
to be pathogen reduced is reduced by reducing the plasma content.
One method suitable for the plasma reduction step is to dilute the
fluid containing plasma with an adenine-free diluting solution.
This will reduce the level of adenine in the fluid to be pathogen
reduced, thus reducing the amount of adenine available to form a
complex with riboflavin. The diluting solution used to reduce the
level of adenine to an amount which will not form a complex with
riboflavin may be one of many different solutions, including
saline; a physiologic buffer, which may comprise a variety of
different substances; a solution containing glucose, phosphate or
both, which may or may not act as a buffer; a solution containing
nutrients; a cryopreservative: an anticoagulant; a cell storage
solution known to the art or developed to provide cells with
suitable additives to enable them to be stored or infused; or other
suitable solution.
[0031] The diluting solution should not substantially interfere
with the inactivation of microorganisms or substantially destroy
the biological activity of the fluid. By "substantially interfere"
is meant interference which is sufficient to prevent pathogen
reduction from occurring at a desired level.
[0032] The diluting solution may also contain a substrate which
selectively binds to adenine, effectively removing it from the
fluid by rendering it unable to bind to riboflavin. Although in
this method adenine may still be present in the fluid to be
pathogen reduced, the adenine which is present is not available to
bind to riboflavin because it is bound to the adenine-binding
substrate. One such adenine-binding substrate which might be used
in this invention may be an antibody directed against adenine. The
antibody could be added directly to the adenine-containing solution
to be pathogen reduced, or could be coupled to a substrate such as
polymeric beads. Another substrate which may be used to remove
adenine from the fluid may be an ion exchange resin. Such a resin
would preferentially bind to adenine based upon the ionic charge of
adenine, thus effectively removing adenine from the fluid.
[0033] Another method which may be used for reducing the
concentration of selected components of plasma such as adenine in a
fluid to be pathogen reduced may be by selective filtration. Such
methods of filtering out unwanted substances such as adenine from
fluids are known in the art. One example of a filter which may be
used to selectively remove adenine is a hollow fiber filter. The
pore sizes of this filter would be small enough to allow adenine to
pass through the pores and be removed from the fluid, leaving the
blood component to be pathogen reduced behind.
[0034] Another method of selectively filtering out adenine which
may be useful with the present invention is to use a filter having
an absorption ligand on its surface which selectively binds to
adenine, thus effectively removing adenine from the fluid to be
pathogen reduced. This method would allows the plasma (minus
adenine) to be retained as part of the fluid to be pathogen
reduced.
[0035] FIG. 2 depicts one example of a bag set for use in a
pathogen reduction procedure containing a filter which may be used
to remove adenine from the fluid to be pathogen reduced. Fluid
containing blood and plasma, or a collected blood component which
has been previously collected in a collection storage solution
containing adenine is contained in bag 10. To substantially remove
all adenine which may be contained therein, the fluid to be
pathogen reduced flows out of bag 10 via exit port 2 through tubing
7 and into filter 5. Filter 5 may contain filter media having a
substrate thereon which selectively binds to adenine, thus removing
it from the fluid. After flowing through the filter, the now
substantially adenine-free fluid flows through tubing 9 and into
bag 12 via port 4. Bag 12 may be prepackaged to contain riboflavin,
or riboflavin may be added after the now adenine-free fluid to be
pathogen reduced is flowed into bag 12.
[0036] In another embodiment, the adenine removal filter may also
be contained within one of the bags 10 or 12. The adenine contained
in the fluid would bind directly to the filter contained within the
bag, and transfer of the now adenine-free fluid into another bag
would not be needed.
[0037] The adenine reducing step may also be carried out using
mechanical means such as centrifugation, to separate the fluid
containing adenine from the blood component to be pathogen reduced.
This centrifugation step may be done using an apheresis machine
such as the COBE Spectra.TM. or TRIMA.RTM. apheresis systems
available from Gambro BCT Inc. (Lakewood, Colo., USA) as well as
apheresis systems of other manufacturers. The separated blood
components may then be resuspended in a suitable solution which
does not contain adenine. The reduction step may also comprise
washing the separated blood component to be pathogen reduced one or
more times, as is known in the art. One machine suitable for
washing the blood or separated blood components is the COBE 2991
(also available from Gambro BCT Inc.) Washing is generally the
addition to the blood component to be pathogen reduced a solution
which does not contain adenine to dilute the percentage of plasma
(or of collection/storage solution) aid consequently the amount of
adenine. The wash solution is removed and a pathogen reduction
solution may be added to resuspend the washed components. The
process may be carried out one or more times depending on the
initial level of adenine contained in the fluid.
[0038] The fluid to be pathogen inactivated may also be initially
collected into a solution which does not contain adenine. If this
is the case, no step of removing adenine is needed.
[0039] In a batch-wise process, after substantially removing any
adenine initially present in the plasma or in the
collection/storage solution, the fluid to be pathogen reduced is
placed into bags which are photopermeable or at least sufficiently
photopermeable to allow sufficient radiation to reach their
contents to activate the photosensitizer. Photosensitizer is added
to each bag to substantially inactivate any pathogens which may be
contained therein, and the bag is preferably agitated while
irradiating, for a period of time to ensure exposure of
substantially all the fluid to radiation.
[0040] FIG. 3 depicts an embodiment of this invention in which
fluid to be decontaminated and which is substantially adenine-free
is placed in a bag 284 equipped with an inlet port 282, through
which photosensitizer 290 may be added from flask 286 via pour
spout 288. Shaker table 280 is activated to agitate the bag 284 to
mix the fluid to be decontaminated and the photosensitizer together
while photoradiation source 260 is activated to irradiate the fluid
and photosensitizer in bag 284. Alternatively, the bag can be
prepackaged to contain photosensitizer and the fluid to be pathogen
reduced is thereafter added to the bag.
[0041] It is also contemplated that the pathogen reduction process
can be done in a flow-through system. In a flow-through process,
after substantially removing any adenine initially present in the
plasma or in the collection/storage solution, a photosensitizer is
added to the fluid containing a blood component which is to be
pathogen reduced. The photosensitizer and blood component is flowed
past a photoradiation source, and the flow of the material
generally provides sufficient turbulance to distribute the
photosensitizer throughout the fluid. A mixing step may optionally
be added.
EXAMPLES
[0042] Blood to be pathogen reduced may be separated into
components by any mean is known in the art.
Example 1
[0043] The method of this example requires the removal of
substantially all adenine which may be contained in a solution used
to resuspend and/or collect platelets to be pathogen reduced.
Removal of adenine may be done using any of the methods set forth
above. If an adenine-free solution is used to resuspend or collect
the platelets to be pathogen reduced, no adenine removal step is
needed. After removal of any adenine which may be present, the
photosensitizer is mixed with the fluid containing platelets.
Mixing may be done by simply adding the photosensitizer or a
solution containing the photosensitizer to the platelets to be
pathogen reduced. In one embodiment, the material to be
decontaminated to which a 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 pathogen reduced. A
nixing step may optionally be added. In another embodiment, the
fluid and photosensitizer are placed in a photopermeable container
and irradiated in batch mode (see FIG. 2), preferably while
agitating the container to fully distribute the photosensitizer and
expose all the fluid to the radiation.
[0044] The amount of photosensitizer to be mixed with the fluid to
be pathogen reduced will be an amount sufficient to adequately
inactivate the reproductive ability of a pathogen. Preferably the
photosensitizer is used in a concentration of at least about 1
.mu.M up to the solubility of the photosensitizer in the fluid. For
7,8-dimethyl-10-ribityl isoalloxazine a concentration range between
about 1 .mu.M and about 160 .mu.M is preferred, preferably about 50
.mu.M.
[0045] The wavelength used will depend on the photosensitizer
selected, and the type of blood component to be pathogen reduced.
For platelets and plasma, a light source is used which provides
light in the range of about 200 nm to about 320 nm, and more
preferably about 308 nm may be used. For red blood cells, light in
the range of about 200 nm to about 600 nm is used, preferably about
447 nm.
[0046] The following storage solutions shown in Table 1a and 1b are
examples of commonly used platelet storage solutions which may be
used with this invention. These solutions may be used to resuspend
platelets to be pathogen reduced before the addition of the
photosensitizer, or may be used to resuspend platelets after a
pathogen reduction procedure. Other solutions not specifically
listed that do not contain adenine may also be used. It should be
noted that platelets may also be resuspended in buffer and/or
saline as long as no adenine is present. TABLE-US-00001 TABLE 1a
PAS II PSM1-pH PlasmaLyte A Molecular Conc. g/300 Conc. g300 Conc.
g/300 Weight (mMol/L) mLs (mMol/L) mLs (mMol/L) mLs Sodium Chloride
58.44 115.5 2.02 98 1.72 90 1.58 Potassium 74.55 0.00 5 0.11 5 0.11
Chloride Calcium Chloride 111 0.00 0.00 0.00 Magnesium 95.21 0.00
0.00 3 0.09 Chloride Magnesium 120.4 0.00 0.00 0.00 Sulfate
Tri-Sodium 294.1 10 0.88 23 2.03 23 2.03 Citrate Citric Acid 192.1
0.00 0.00 0.00 Sodium 84.01 0.00 0.00 0.00 Bicarbonate Sodium 142
0.00 25 1.07 0.00 Phosphate Sodium Acetate 82.03 30 0.74 0.00 27
0.66 Sodium 218.1 0.00 0.00 23 1.50 Gluconate Glucose 180.2 0.00
0.00 0.00 Maltose 360.3 0.00 0.00 0.00 Adenine 135.1 0.00 0.00 0.00
Note: Assumes that all salts are anhydrous
[0047] TABLE-US-00002 TABLE 1b SetoSol PAS III PAS Molecular Conc.
g/300 Conc. g/300 Conc. g/300 Weight (mMol/L) mLs (mMol/L) mLs
(mMol/L) mLs Sodium Chloride 58.44 90 1.58 77 1.35 110 1.93
Potassium 74.55 5 0.11 0.00 5.1 0.11 Chloride Calcium Chloride 111
0.00 0.00 1.7 0.06 Magnesium 95.21 3 0.09 0.00 0.00 Chloride
Magnesium 120.4 0.00 0.00 0.8 0.03 Sulfate Tri-Sodium 294.1 17 1.50
12.3 1.09 15.2 1.34 Citrate Citric Acid 192.1 0.00 0.00 2.7 0.16
Sodium 84.01 0.00 0.00 35 0.88 Bicarbonate Sodium 142 25 1.07 28
1.19 2.1 0.09 Phosphate Sodium Acetate 82.03 23 0.57 42 1.03 0.00
Sodium 218.1 0.00 0.00 0.00 Gluconate Glucose 180.2 23.5 1.27 0.00
38.5 2.08 Maltose 360.3 28.8 3.11 0.00 0.00 Adenine 135.1 0.00 0.00
0.00 Note: Assumes that all salts are anhydrous
Example 2
[0048] Example 2 is directed toward the removal of adenine in a
fluid containing red blood cells to be pathogen reduced. If a
riboflavin-adenine complex forms in a solution containing red blood
cells, the increased oxidation reactions caused by the presence of
the complex may damage the red blood cell membranes, causing
hemolysis and increased methemoglobin formation. Methemoglobin
formation is undesirable because methemoglobin does not allow the
red blood cells to efficiently bind and deliver oxygen.
[0049] This phenomenon is shown in FIG. 4, which shows the %
hemolysis of red blood cells over time in solutions with and
without adenine. Red blood cells were suspended in AS3 during a
pathogen reduction procedure using riboflavin and visible light.
AS3 is an AABB approved red blood cell preservative. AS3 contains
sodium chloride, dextrose, adenine, sodium phosphate, sodium
citrate and citric acid. As can be seen in FIG. 4, red blood cells
suspended in 5% AS3 show the highest percentage of red blood cell
hemolysis. Red blood cells subjected to a pathogen reduction
procedure in a solution containing no adenine (0% AS3) show less
than 2% hemolysis of red blood cells.
[0050] Red blood cells to be pathogen reduced should be collected
in an anticoagulant-preservation solution which does not contain
adenine. Other anticoagulant-preservation solutions not
specifically listed in Table 2a and 2b below that do not contain
adenine may also be used. As can be seen from Table 2a and 2b, none
of the anticoagulant-preservative solutions listed below contain
additional adenine. TABLE-US-00003 TABLE 2a ANTICOAGULANT
PRESERVATIVE SOLUTIONS CPD CP2D Molecular Conc. Conc. Weight
(mMol/L) mg/63 ml mg/100 ml (mMol/L) mg/63 ml mg/100 ml Sodium
Citrate 294.1 89.59 1660.00 2634.92 89.59 1660.00 2634.92 Citric
Acid 192.1 15.53 188.00 298.41 15.53 188.00 298.41 Dextrose 180.2
141.82 1610.00 2555.56 283.64 3220.00 5111.11 Monobasic 120 18.52
140.00 222.22 18.52 140.00 222.22 Sodium phosphate Adenine 135.1
0.00 0.00 0.00 0.00 0.00 0.00
[0051] TABLE-US-00004 TABLE 2b ANTICOAGULANT PRESERVATIVE SOLUTIONS
ACD-A ACD-B Molecular Conc. Conc. Weight (mMol/L) mg/100 ml
(mMol/L) mg/100 ml Dextrose 180.2 135.96 2450.00 81.58 1470.00
Adenine 135.1 0.00 0.00 0.00 0.00 Monobasic 120 0.00 0.00 0.00 0.00
sodium phosphate Mannitol 182.2 0.00 0.00 0.00 0.00 Sodium 58.45
0.00 0.00 0.00 0.00 Chloride Sodium 294.1 74.80 2200.00 44.88
1320.00 Citrate Citric Acid 192.1 41.64 800.00 24.99 480.00
[0052] Alternatively, if previously collected red blood cells are
to be pathogen reduced, the cells may be washed before undergoing a
pathogen reduction procedure to remove any adenine contained in the
solution used to collect and store the previously collected cells.
The washing procedure may be used to remove plasma (which contains
endogenous adenine), or to remove adenine from blood products which
were previously collected and stored in synthetic storage solutions
or anticoagulants containing exogenous adenine.
[0053] One red blood cell wash process which may be used with the
present invention is described below. However, any process for
washing cells known in the art may be used. Red cells can be washed
by manual centrifugation or with an automated cell washer such as
the COBE 2991 (available from Gambro BCT, Lakewood, Colo., USA).
The 2991 washes the red cells with 700 mL of 0.9% sodium chloride
and 300 mL of 500 .mu.M riboflavin in 0.9% sodium chloride.
[0054] The product of the wash step is a suspension of concentrated
red blood cells at a 60 to 70% hematocrit. The washed red cells are
mixed with a solution containing 550 .mu.M riboflavin in a 0.9%
sodium chloride to obtain a suspension with a hematocrit of 50% and
a volume of 276 mL. The solution may also be any of the
anticoagulant-preservative solutions set forth in the tables
above.
[0055] The washed red cells are transferred from the cell-washing
bag to a bag suitable for illumination and subsequent dilution to a
50% hematocrit. The washed red cells and riboflavin arc typically
illuminated with visible light at a wavelength of 447 nm and 120
J/cm2. After illumination, the extracellular fluid is expressed off
and a storage solution which may or may not contain adenine is
added in an amount necessary to increase the hematocrit of the red
cells to 55%. The pathogen reduced red blood cells may then be
stored or directly reinfused into a patient.
[0056] Removal of adenine may also be done using any of the other
methods set forth above.
[0057] The addition of "quenchers" or oxygen scavengers, may be
used to enhance the pathogen reduction process by further reducing
the extent of non-specific cell-damaging chemistry. Examples of
quenchers which may be used in this invention include electron rich
amino acids such as histidine, methionine, tyrosine and tryptophan.
Nucleotides such as cysteine, guanosine and adenoside
monophosphate. Sulfhidryl quenchers such as N-acetyl-L-cysteine and
glutathione. Antioxdants such as trolox, Vitamin E and
alpha-tocopherol acetate. Other quenchers such as propyl gallate,
ascorbate, mercaptopropionylglycine, dithiothreotol, nicotinamide,
BHT, BHA, lysine, serine, glucose, mannitol, glycerol, and mixtures
thereof may also be used. Quenchers may be added to the fluid to be
pathogen reduced either before or after the removal of adenine.
[0058] It will be appreciated that the instant specification and
claims are set forth by way of illustration and not of limitation,
and that various modifications and changes may be made without
departing from the spirit and scope of the present invention.
* * * * *