Note: Descriptions are shown in the official language in which they were submitted.
CA 02318508 2003-06-19
BATCH DEVICES FOR THE REDUCTION OF COMPOUNDS FROM
BIOLOGICAL COMPOSITIONS CONTAINING CELLS AND METHODS
OF USE
T'ECl-iNICAI. FIELD
The present invention relates to methods and devices for the reduction of
compounds in biological compositions. 'I"he compounds lave a molecular weight
ranging from about 100 g/rnol to about 30,000 g/mol.
BACKGROUND ART
An extensive body of research exists regarding the removal of substances
from blood products. The bulk of this research is directed at white cell
reduction.
See, e.g., M.N. Boomgaard et al., Transfusion 34:311 (1994); F. Bertalini et
al.,
Vox Sang 62:$2 {1992); and A.M. Joustra-Dijkhuis et al., Vox Sang 67:22
(1994).
Filtration of platelets is the most common method used in white cell reduction
of
platelet concentrates. See, e.g., M. Black et al., Trarrsjxrsion 31:333 {199X)
TM
(Sepacell PL-5A, Asahi, Tokyo, Japan); J.I~. Sweeney et al., Transfusion
35:131
TM
(1995) (Leukotrap PL, Miles Inc., Covina, CA)a and M. van Marwijk et al.,
Transfusion 30:34 (1990) (Cellselect, NPBi, Emmer-Compascuum, The
TM
Netherlands; Immugard Ig-500, 'I'erumo, Tokyo, Japan). These current
filtration
mechanisms, however are not amenable for the removal of relatively low
molecular weight compounds including for example psoralens, psoralen
photoproducts and other compounds commonly used in treating biological fluids.
The process of adsorption has been used to isolate selective blood
components onto phospholipid polymers. For example, several copolymers with
various electrical charges have been evaluated for their interactions with
blood
components, including platelet adhesion and protein adsorption. K. lshihara et
al.,
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J. Biomed. Mat. Res. 28:1347 (1994). Such polymers, however, are not designed
for the adsorption of low molecular weight compounds.
Various dialysis means are able to remove low molecular weight
compounds from plasma and whole blood. For example, dialysis can successfully
remove lowv molecular weight toxins and pharmaceutical compounds. Thus,
dialysis might be used to remove, for example, psoralens and psoralen
photoproducts from blood products. Unfortunately, current dialysis procedures
involve very complicated and expensive devices. As such, the use of dialysis
machines would not be practical for the decontamination of a large volume of
blood products.
The use of polystyrene divinylbenzene, silica gel, and acrylester polymers
for the adsorption of methylene blue has previously been described. For
example,
PCT Publication No. WO 91/03933 describes batch studies with free adsorbent
resin (e.g., Amberlites (Rohm and Haas (Frankfurt, Germany) and Bio Beads
(Bio-Rad Laboratories (Munich, Germany)). Without very careful removal of the
adsorbent resins after exposure to the blood product, however, these methods
create the risk of transfusion of the resin particles.
In addition, devices and processes for the removal of leukocytes and viral
inactivation agents (e.g., psoralens, hypericin, and dyes such as methylene
blue,
toluidine blue, and crystal violet) have also been disclosed. Specifically,
PCT
Publication No. WO 95/18665 describes a filter comprising a laid textile web
which includes a mechanically stable polymeric substrate. The web itself
comprises interlocked textile fibers forming a matrix with spaces and
fibrillated
particles disposed within the spaces. However, this device causes a
significant
decrease in the Factor XI activity, which may render the treated product
unsuitable for its intended use.
Simpler, safer and more economical means for reducing the concentration
of low molecular weight compounds in a biological composition containing cells
while substantially maintaining the biological activity of the treated
biological
composition containing cells are therefore needed.
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DISCLOSURE OF THE INVENTION
The present invention provides devices for reducing the concentration of
compounds in biological compositions containing cells. The devices include an
adsorption medium comprised of particles immobilized by an inert matrix and
are
of a batch configuration. Typically, the compounds reduced in biological
compositions using the device have molecular weights ranging from about 100
g/mol to about 30,000 g/mol. The biological composition containing cells
includes, for example, cells suspended in a biological medium, such as plasma
or
tissue culture media. The biological activity of the biological composition is
substantially maintained after contact with such devices.
Exemplary compounds include pathogen inactivating compounds, dyes,
thiols, plasticizers and activated complement. Devices are provided that
comprise
a three dimensional network of adsorbent particles immobilized by an inert
matrix. This immobilization reduces the risk of leakage of loose adsorbent
particles into the blood product. Furthermore, immobilization of the adsorbent
particles by an inert matrix simplifies manufacturing by reducing problems
associated with handling loose adsorbent particles. Immobilization of the
adsorbent particles may also enhance the ability of the adsorbent particles to
adsorb compounds in biological compositions containing cells without
mechanical
damage to the cells.
The present invention provides methods for reducing the concentration of
a biological response modifier in a biological composition containing cells,
wherein the method substantially maintains a desired biological activity of
the
biological composition. The method involves treating the biological
composition
with a device.
In one embodiment, the device comprises an inert matrix containing
highly adsorbent particles, wherein the diameter of the adsorbent particles
ranges
from about 100 ~m to about 1500 ~,m, and wherein the device is for use in a
batch
process.
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In another embodiment, the biological response modifier is activated
complement.
In another embodiment, the adsorbent particles of the device are
polyaromatic adsorbent particles that possess superior. wetting properties.
In another embodiment; the adsorbent particles of the device are activated
carbon particles derived from a synthetic source.
In another embodiment, the inert matrix of the device is a synthetic
polymer fiber, and the synthetic polymer fiber comprises a polymer core with a
high melting temperature surrounded by a sheath with a lower melting
temperature:
In another embodiment; the inert matrix of the device is a particulate
network, and the particulate network comprises polyethylene particles.
In another embodiment; the biological composition comprises platelets.
In another embodiment, the biological composition comprises red blood
cells.
In another embodiment, the method further reduces the concentration of a
psoralen derivative or an acridine derivative in the biological composition.
In another embodiment, the method further reduces the concentration of a
dye or a quencher in the biological composition.
In another embodiment; the present invention comprises a method for
reducing the concentration of a low molecular weight compound-in an aqueous
composition, wherein the method comprises contacting the aqueous composition
with an adsorption medium comprising porous adsorbent particles immobilized
by a matrix, wherein the diameter of the adsorbent particles ranges from about
-
100 ,gym to about 1500 ,um, wherein the adsorbent particles have an affinity
for
said low molecular-weight compound, wherein the low molecular-weight
compound is selected from the group consisting of biological response
modifiers, polyethylene glycols, plastic extractables, and polyamine
derivatives, and wherein said contacting occurs in a batch process.
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- 4A -
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 diagrammatically depicts a,perspective view of one embodiment of
a fiber; indicating its inner core and outer sheath, that forms the fiber
networks of
the immobilized adsorbent media.
FIG. 2 schematically represents a portion of one embodiment of the
immobilized adsorbent media of the present invention.
FIG. 3 diagrammatically represents a cross-sectional view of one
embodiment of immobilized adsorbent media in which the adsorbent beads are
secured to fibers that make up the fiberized resin. ,
FIG. 4 diagrammatically represents a cross-sectional view of one
embodiment of immobilized adsorbent media in which the adsorbent beads are
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immobilized within the fibers of the immobilized adsorbent media and the heat
seals that encompass samples of fiberized resin.
FIG. 5 is a graph showing a comparison of adsorption kinetics for removal
of aminopsoralens from platelets with Dowex~ XUS-43493 and Amberlite~
XAD-16 HP loose adsorbent beads and immobilized adsorbent media containing
Amberlite~ XAD-16.
FIG. 6 is a graph showing a comparison of adsorption kinetics for removal
of aminopsoralens from platelets with immobilized adsorbent media containing
Amberlite~ XAD-16 and immobilized adsorbent media with the two different
loadings of activated charcoal. Fiberized XAD-16 data is represented by
circles,
solid line; fiberized AQF-500-B as squares, short dashes; and, fiberized AQF-
375-
B as triangle, long dashes.
FIG. 7 is a graph showing a comparison of the adsorption kinetics for
removal of aminopsoralens from platelets with p(HEMA)-coated and uncoated
Dowex~ XUS-43493 beads.
FIG. 8 is a graph showing a comparison of the effect of pre-treatment with
solutions containing glycerol on the relative adsorption capacity of
Amberlite~
XAD-16 and Dowex~ XUS-43493 for aminopsoralens.
FIG. 9 is a graph showing a comparison of the effect of wetting solution
on 4'-(4-amino-2-oxa)butyl-4,5',8-trimethyl psoralen adsorption capacities for
dried adsorbent in 100% plasma for Amberlite~ XAD-16 (bottom) and Dowex~
XUS-43493 (top); the samples that were not wet in an ethanol solution are
labeled
"No Tx". Adsorption capacities are reported as percentages relative to the
capacity of optimally wet adsorbent.
FIG. 10 is a graph showing a comparison of adsorption of aminopsoralens
over a 3-hour period from plasma using Amberlite~ XAD-16 wet in several
different solutions.
FIG. 11 is a graph showing a comparison of the kinetics of adsorption of
methylene blue over a 2-hour period from plasma.
FIG. 12 depicts the chemical structures of acridine, acridine orange, 9-
amino acridine, and 5-[([3-carboxyethyl)amino]acridine.
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FIG. 13 is a graph showing plots the data for adenine capacity (y-axis)
and 5-(((3-carboxyethyl)amino]acridine capacity (x-axis) for various resins.
FIGS. 14A and 14B is a graph showing a comparison of the adsorption
kinetics for removal of 5-[((3-carboxyethyl)amino]acridine with Dowex~ XUS-
43493 and Purolite~ MN-200 and Amberlite~ XAD-16 HP.
FIG. 15 is a graph showing a comparison of the adsorption kinetics for
removal of 9-amino acridine and acridine orange with Dowex~ XUS-43493.
FIG. 16 is an illustration of a batch configuration for the immobilized
adsorption device (IAD).
FIG. 17 is a graph showing a comparison of the adsorption isotherms for
various Ambersorbs as compared to Purolite MN-200.
FIG. 18 is a graph showing a comparison of the levels of 5-[([3-
carboxyethyl)amino]acridine and GSH in the supernatant of 300 mL PRBC units
with continued or terminated exposure after 24 hours to a fiberized Pica 6277
IAD (S00 g/m2) over 4 weeks of storage at 4°C.
FIG. 19 is a graph showing the effect of enclosure material on adsorption
kinetics for 5-[((3-carboxyethyl)amino]acridine in PRBCs.
FIG. 20 is a graph showing a comparison of percent hemolysis for the
adsorbent devices containing non-immobilized and immobilized adsorbent
particle Purolite MN-200.
FIG. 21 is a graph showing a comparison of percent hemolysis for the non-
immobilized and immobilized adsorbent particle Pica G-277 activated carbon.
FIG. 22 is a graph showing kinetics for removal 4'-(4-amino-2-oxa) butyl-
4,5',8 trimethylpsoralen from platelet concentrates.
FIG. 23 is a graph showing psoralen adsorption kinetics for fiber matrix
IAD (AQF, squares) and particulate matrix IAD (Porex, triangles).
BEST MODE FOR CARRYING OUT THE INVENTION
The present invention provides devices for reducing the concentration of
compounds in biological compositions containing cells. The devices include an
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adsorption medium comprised of particles immobilized by an inert matrix and
are
of a batch configuration. Typically, the compounds reduced in biological
compositions using the device have molecular weights ranging from about 100
g/mol to about 30,000 g/mol. The biological composition containing cells
includes, for example, cells suspended in a biological medium, such as plasma
or
tissue culture media. The biological activity of the biological composition is
substantially maintained after contact with such devices.
Exemplary compounds include pathogen inactivating compounds, dyes,
thiols, plasticizers and activated complement. Devices are provided that
comprise
a three dimensional network of adsorbent particles immobilized by an inert
matrix. This immobilization reduces the risk of leakage of loose adsorbent
particles into the blood product. Furthermore, immobilization of the adsorbent
particles by an inert matrix simplifies manufacturing by reducing problems
associated with handling loose adsorbent particles. Immobilization of the
adsorbent particles may also enhance the ability of the adsorbent particles to
adsorb compounds in biological compositions containing cells without
mechanical
damage to the cells.
Definitions
The term "acridine derivatives" refer to a chemical compound containing
the tricyclic structure of acridine (dibenzo[b,e]pyridine; 10-azanthracene).
The
compounds have an affinity for (and can bind) to nucleic acids non-covalently
through intercalation. The term "aminoacridine" refers to those acridine
compounds with one or more nitrogen-containing functional groups. Examples of
aminoacridines include 9-amino acridine and acridine orange (depicted in
Figure
12).
The term "adsorbent particle" broadly refers to any natural or synthetic
particulate material which is capable of interacting with molecules in a
liquid thus
allowing the molecule to be removed from the liquid. Examples of naturally
occurring adsorbents include but are not limited to activated carbon, silica,
diatomaceous earth, and cellulose. Examples of synthetic adsorbents include
but
are not limited to polystyrene, polyacrylics, and carbonaceous adsorbents.
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Adsorbent particles are often porous, often possess high surface areas, and
may be
modified with a variety of functional groups (e.g. ionic, hydrophobic, acidic,
basic) which can affect how the adsorbent interacts with molecules.
The term "aromatic," "aromatic compounds," and the like refer broadly to
compounds with rings of atoms having delocalized electrons. The monocyclic
compound benzene (C6H6) is a common aromatic compound. However, electron
delocalization can occur over more than one adjacent ring (e.g., naphthalene
(two
rings) and anthracene (three rings)). Different classes of aromatic compounds
include, but are not limited to, aromatic halides (aryl halides), aromatic
heterocyclic compounds, aromatic hydrocarbons (arenes), and aromatic nitro
compounds (aryl nitro compounds).
The term "biocompatible coating" refers broadly to the covering of a
surface (e.g., the surface of a polystyrene bead) with a hydrophilic polymer
that
when in contact with a blood product does not result in an injurious, toxic,
or
immunological response and renders the surface more biocompatible by
decreasing cell adhesion, decreasing protein adsorption or improving cell
function. Suitable coatings are biocompatible if they have minimal, if any,
effect
on the biological material to be exposed to them. By "minimal" effect it is
meant
that no significant biological difference is seen compared to the control. In
preferred embodiments, biocompatible coatings improve the surface
hemocompatibility of polymeric structures. For example, poly(2-hydroxyethyl
methacrylate) (pHEMA) is frequently used for the coating of materials used in
medical devices (e.g., blood filters). .
The term "biocompatible housing" refers broadly to containers, bags,
vessels, receptacles, and the like that are suitable for containing a
biological
material, such as, for example, compositions containing platelets or red blood
cells. Suitable containers are biocompatible if they have minimal, if any,
effect on
the biological material to be contained therein. By "minimal" effect it is
meant
that no significant difference is seen in blood product function compared to
the
control as described herein, for red blood cells, platelets and plasma. Thus,
blood
products may be stored in biocompatible housings prior to transfusion to a
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recipient. In a preferred embodiment, biocompatible housings are blood bags,
including a platelet storage container or red blood cell storage container.
The term "container that is compatible with the biological composition"
refers to a container that is suitable for holding a biological composition
containing cells, such as, for example, cell culture compositions, as well as
compositions containing platelets or red blood cells. Such containers have a
minimal effect on a biological composition containing cells. Examples of such
containers include, without limitation, cell culture plates, cell culture
bottles and
blood bags.
The term " biological fluids" include media from cell cultures, synthetic
media for the storage of cells, human or non-human whole blood, plasma,
platelets, red blood cells, leukocytes, serum, lymph, saliva, milk, urine, or
products derived from or containing any of the above, alone or in mixture,
with or
without a chemical additive solution. Preferably, the fluid is blood or a
blood
product with or without a chemical additive solution, more preferably plasma,
platelets and red blood cells, most preferably apheresis plasma, red blood
cells
and platelets.
The term "blood bag" refers to a form of blood product container.
The term "blood product" refers to the fluid and/or associated cellular
elements and the like (such as erythrocytes, leukocytes, platelets, etc.) that
pass
through the body's circulatory system; blood products include, but are not
limited
to, blood cells, platelet mixtures, serum, and plasma. The term "platelet
mixture"
refers to one type of blood product wherein the cellular element is primarily
or
only platelets. A platelet concentrate (PC) is one type of platelet mixture
where
the platelets are associated with a smaller than normal portion of plasma. In
blood
products, synthetic media may make up that volume normally occupied by
plasma; for example, a platelet concentrate may entail platelets suspended in
35%
plasma/65% synthetic media. Frequently, the synthetic media comprises
phosphate.
The term "blood separation means" refers broadly to a device, machine, or
the like that is able to separate blood into blood products (e.g., platelets
and
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plasma). An apheresis system is one type of blood separation means. Apheresis
systems generally comprise a blood separation device, an intricate network of
tubing and filters, collection bags, an anticoagulant, and a computerized
means of
controlling all of the components.
The term "crosslinked" refers broadly to linear molecules that are attached
to each other to form a two- or three-dimensional network. For example,
divinylbenzene (DVB) serves as the crosslinking agent in the formation of
styrene-divinylbenzene copolymers. The term also encompasses
"hypercrosslinking" in which hypercrosslinked networks are produced by
crosslinking linear polystyrene chains either in solution or in a swollen
state with
bifunctional agents. A variety of bifunctional agents can be used for cross-
linking
(for example, see Davankov and Tsyurupa, Reactive Polymers 13:24-42 (1990);
Tsyurupa et al., Reactive Polymers 25:69-78 ( 1995).
The term "cyclic compounds" refers to compounds having one (i. e., a
monocyclic compounds) or more than one (i. e. , polycyclic compounds) ring of
atoms. The term is not limited to compounds with rings containing a particular
number of atoms. While most cyclic compounds contain rings with five or six
atoms, rings with other numbers of atoms (e.g., three or four atoms) are also
contemplated by the present invention. The identity of the atoms in the rings
is
not limited, though the atoms are usually predominantly carbon atoms.
Generally
speaking, the rings of polycyclic compounds are adjacent to one another;
however, the term "polycyclic" compound includes those compounds containing
multiple rings that are not adjacent to each other.
The term "dye" refers broadly to compounds that impart color. Dyes
generally comprise chromophore and auxochrome groups attached to one or more
cyclic compounds. The color is due to the chromophore, while the dying
affinities are due to the auxochrome. Dyes have been grouped into many
categories, including the azin dyes (e.g., neutral red, safranin, and
azocarmine B);
the azo dyes; the azocarmine dyes; the dephenymethane dyes; the fluorescein
dyes; the ketonimine dyes; the rosanilin dyes; the triphenylmethane dyes; the
phthalocyanines; and, hypericin. It is contemplated that the methods and
devices
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of the present invention may be practiced in conjunction with any dye that is
a
cyclic compound.
The term "fiberized resin" generally refers to immobilization of adsorbent
material, including for example, resins entrapped in or attached to a fiber
network.
In one embodiment, the fiber network is comprised of polymer fibers. In
another
embodiment, the fibers consist of a polymer core (e.g., polyethylene
terephthalate
[PET]) with a high melting point surrounded by a polymer sheath (e.g., nylon
or
modified PET) with a relatively low melting temperature. Fiberized resin may
be
produced by heating the fiber network, under conditions that do not adversely
affect the adsorbent capacity of the resin to a significant degree
(temperature
sufficient to melt the sheath but not the core). Where the resin comprises
beads,
heating is performed such that the adsorbent beads become attached to the
outer
polymer sheath to create "fiberized beads". By producing fiberized resin
containing a known amount of adsorbent beads per defined area, samples of
fiberized resin for use in the removal of cyclic compounds (e.g., psoralens,
and, in
particular, aminopsoralens) and other products can be obtained by cutting a
defined area of the fiberized resin, rather than weighing the adsorbent beads.
The term "filter" refers broadly to devices, materials, and the like that are
able to allow certain components of a mixture to pass through while retaining
other components. For example, a filter may comprise a mesh with pores sized
to
allow a blood product (e.g. red blood cell composition) to pass through, while
retaining other components such as resin particles. The term "filter" is not
limited
to the means by which certain components are retained.
The term "heterocyclic compounds" refers broadly to cyclic compounds
wherein one or more of the rings contains more than one type of atom. In
general,
carbon represents the predominant atom, while the other atoms include, for
example, nitrogen, sulfur, and oxygen. Examples of heterocyclic compounds
include furan, pyrrole, thiophene, and pyridine.
The phrase "high temperature activation process" refers to a high
temperature process that typically results in changes in surface area,
porosity and
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surface chemistry of the treated material due to pyrolysis and/or oxidation of
the
starting material.
'The term "Immobilized Adsorbent Device (IAD)" refers to immobilized
adsorbent material entrapped in or attached to an inert matrix. Where the
inert
matrix is a fiber network the term IAD can be used interchangeably with the
term
fiberized resin. For example, fiberized Ambersorb 572 and Ambersorb IAD
(AQF) refer to the same material.
The term "inert matrix" refers to any synthetic or naturally occurring fiber
or polymeric material which can be used to immobilize adsorbent particles
without substantially affecting the desired biological activity of the blood
product.
The matrix may contribute to the reduction in concentration of small organic
compounds although typically it does not contribute substantially to the
adsorption or removal process. In addition, the inert matrix may interact with
cellular or protein components resulting in cell removal (e.g. leukodepletion)
or
removal of protein or other molecules.
The term "isolating" refers to separating a substance out of a mixture
containing more than one component. For example, platelets may be separated
from whole blood. The product that is isolated does not necessarily refer to
the
complete separation of that product from other components.
The term "macropores" generally means that the diameter of the pores is
greater than about 500 ~. The term micropores refers to pores with diameters
less
than about 20 ~. The term mesopores refers to pores with diameters greater
than
about 20 ~. and less than about 500 ~.
The term "macroporous" is used to describe a porous structure having a
substantial number of pores with diameters greater than about 500 ~.
The term "macroreticular" is a relative term that means that the structure
has a high physical porosity (i.e., a large number of pores are present) a
porous
adsorbent structure possessing both macropores and micropores.
The term "mesh enclosure," "mesh pouch" and the like refer to an
enclosure, pouch, bag or the like manufactured to contain multiple openings.
For
example, the present invention contemplates a pouch, containing the
immobilized
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adsorbent particle, with openings of a size that allow a blood product to
contact
the immobilized adsorbent particle, but retain the immobilized adsorbent
particle
within the pouch.
The term "partition" refers to any type of device or element that can
separate or divide a whole into sections or parts. For example, the present
invention contemplates the use of a partition to divide a blood bag, adapted
to
contain a blood product, into two parts. The blood product occupies one part
of
the bag prior to and during treatment, while the adsorbent resin occupies the
other
part. In one embodiment, after treatment of the blood product, the partition
is
removed (e.g., the integrity of the partition is altered), thereby allowing
the treated
blood product to come in contact with the adsorbent resin. The partition may
either be positioned in the bag's interior or on its exterior. When used with
the
term "partition," the term "removed" means that the isolation of the two parts
of
the blood bag no longer exists; it does not necessarily mean that the
partition is no
longer associated with the bag in some way.
The term "photoproduct" refers to products that result from the
photochemical reaction that a psoralen or other dyes (e.g., methylene blue,
phthalocyanine) undergo upon exposure to ultraviolet radiation.
The term "polyaromatic compounds" refers to polymeric compounds
containing aromatic groups in the backbone, such as polyethylene terphalate,
or as
pendant groups, such as polystyrene, or both.
The term "polystyrene network" refers broadly to polymers containing
styrene (C6Fi5CH=CH2) monomers; the polymers may be linear, consisting of a
single covalent alkane chain with phenyl substituents, or cross-linked,
generally
with m- orp-phenylene residues or other bifunctional or hypercrosslinked
structure, to form a two-dimensional polymer backbone or 3D network.
The term "psoralen removal means" refers to a substance or device that is
able to remove greater than about $0% of the psoralen from, e.g., a blood
product;
preferably, greater than about 90%; most preferably greater than about 99%. A
psoralen removal means may also remove other components of the blood product,
such as psoralen photoproducts.
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The phrase "reducing the concentration" refers to the removal of some
portion of low molecular weight compounds from a biological composition.
While reduction in concentration is preferably on the order of greater than
about
70%, more preferably on the order of about 90%, and most preferably on the
order
S of about 99%.
The phrase "removing substantially all of said portion of a compound (e.g.
a psoralen, psoralen derivative, isopsoralen, acridine, acridine derivative,
dye,
plasticizer or activated complement) free in solution" refers preferably to
the
removal of more than about 80% of the compound free in solution, more
preferably to the removal of more than about 85%, even more preferably of more
than about 90%, and most preferably to the removal of more than about 99%.
The term "resin" refers to a solid support (such as particles or beads etc.)
capable of interacting and adsorbing to various small organic compounds,
including psoralens, in a solution or fluid (e.g., a blood product), thereby
decreasing the concentration of those elements in solution. 'The removal
process
is not limited to any particular mechanism. For example, a psoralen may be
removed by hydrophobic or ionic interaction. The term "adsorbent resin" refers
broadly to both natural organic substances and synthetic substances and to
mixtures thereof.
The term "agitation means" refers to any method by which a biological
composition can be mixed. Examples of agitation means include, without
limitation, the following mechanical agitators: reciprocating, orbital, 3-D
rotator
and rotator type agitators.
The term "shaker device" refers to any type of device capable of
thoroughly mixing a blood product like a platelet concentrate. The device may
have a timing mechanism to allow mixing to be restricted to a particular
duration.
The term "sintered medium" refers to a structure which is formed by
applying heat and pressure to a porous resin, including for example a
particulate
thermoplastic polymer. Porous resins can be prepared by mixing particulate of
relatively low melting polymers and heating them so the plastic particles
partially
fuse but still allow a path for fluids to penetrate the porous mass. Sintered
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CA 02318508 2002-04-12
adsorbent media can be prepared similarly by incorporating carbon or other
high
or non-melting adsorbent particle with that of the low melting powder and
heating. Methods of producing porous plastic materials are described in U.S.
Patent Nos. 3,975,481, 4,110,391, 4,460,530, 4,880;843 and 4,925;880.
The process causes fusing of the low melting
particles resulting in the formation of a porous solid structure. The sintered
medium can be formed into a variety of shapes by placing the polymer particles
in
a forming tool during the sintering process. Adsorbent particles can be
introduced
into the sintered medium by mixing adsorbent particles with the thermoplastic
polymer, particles before subjecting to the sintering process.
The term "stabilizing agent" refers to a compound or composition capable
of maintaining the adsorption capacity of certain adsorbents (e.g:,
Amberlites)
under drying conditions. Generally speaking, acceptable stabilizing agents
should
be soluble in water and ethanol (or other wetting agents), nonvolatile
relative to
I S water and ethanol, and safe for transfusion in small amounts. Examples of
stabilizing agents include, but are not limited to, glycerol and low molecular
weight PEGS. A "v~ietting agent" is distinguishable from a "stabilizing agent"
in
that the former is believed to reopen adsorbent pores ofthose resins that are
not
hypercrosslinked (e.g.; Amberlite XAD-4, Amberlite XAD-16). Wetting agents
generally will not prevent pores from collapsing under drying conditions,
whereas
stabilizing agents wih. A general discussion of wetting and wetting agents is
set
forth in U.S. Patent No. 5,501,795 to Pall et al.
The phrase "substantially maintaining a desired biological activity of the
biological composition" refers to substantially maintaining properties (e:g.,
cellular integrity) of the biological composition. In some embodiments, the
cellular integrity is reflective of the potential performance of the
composition in a
therapeutic setting. For example, where red blood cells are concerned, in vivo
activity is not destroyed or significantly lowered if ATP levels,
extracellular
potassium leakage, % hemolysis are substantially maintained in red blood cells
treated by the methods described herein. For example, the change in ATP level
of
the treated red blood cells should be less than about 10%. The hemolysis level
in
CA 02318508 2002-04-12
the treated red blood cells following storage should be less than about 1 %,
preferably less than about 0.8%. The change in extracellular potassium leakage
of
the treated red blood cells should be less than about 15%. Where platelets are
concerned, in vivo activity is not destroyed or significantly lowered if, for
.
example, platelet yield, pH, aggregation response, shape change, GMP-140,
morphology or hypotonic shock response are substantially maintained in
platelets
treated by the methods described herein. For example, platelet loss in a
biological
composition after storage is preferably less than 15%; more preferably 15%
after
5 days storage; even mare preferably 10% after S days storage. It is further
contemplated that the phrase substantially maintained for each of the
properties
associated with a described blood products may also include values acceptable
to
those of ordinary skill in the art as described in the literature, including
for
example' in Kiein H.G. ; ed. Standards for Blood Banks and Transfusion
Services,
17'~ Ed., Bethesda, MD: American Association of Blood Banks, 1996:
The term "equivalent thereto" when used in reference to a device of the
present invention refers to a device that functions equivalently with respect
to the
maintenance of biological activity of a biological composition. For example,
an
"equivalent" device or matrix containing adsorbent particles is one that
similarly
l 20 maintains cell viability or a suitable coagulation factor level.
The term "low molecular weight compound" refers to an organic or
biological molecule having a molecular weight ranging from about 100 g/mol to
about 30,000 g/mol. Low molecular weight compounds include, without
limitation, the following compounds: small organic compounds such as
psoralens; acridines or dyes; quenchers, such as glutathione; plastic
extractables,
such as plasticizers; biological modifiers, such as activated complement, that
possess a molecular weight between about 100 g/mol and about 30;000 g/mol;
and, polyamine derivatives.
The term "biological composition that is suitable for infusion" refers to a
biological composition that maintains its essential biological properties
(e.g.
platelet morphology) while having sufficiently low levels of any undesired
16
CA 02318508 2000-07-OS
WO 99/34914 PCT/US9$/14134
compounds (e.g. inactivation compounds, response modifiers) such that infusion
provides intended function without detrimental side effects.
The term "control," as used in phrases such as "relative to control," refers
to an experiment performed to study the relative effects of different
conditions.
For example, where a biological composition is treated with a device,
"untreated
control" would refer to the biological composition treated under the same
conditions except for the absence of treatment with the device, or treated
with an
alternative form of a device (e.g., immobilized particles vs. non-immobilized
particles).
The term "4'-(4-amino-2-oxa)butyl-4,5',8-trimethyl psoralen" is
alternatively referred to as "S-59."
The term "N-(9-acridinyl)-(3-alanine" is alternatively referred to as "5-[((3-
carboxyethyl)amino]acridine." It is further alternatively referred to as "S-
300."
The term "XUS-43493" is alternatively referred to as "Optipore 493."
Adsorbent Particles
Provided are adsorbent particles which are useful in a device for reducing
the concentration of compounds in a biological composition containing cells
while
substantially maintaining a desired biological activity of the biological
composition. Typically, the compounds that are reduced in the biological
composition have molecular weights ranging from about 100 g/mol to about
30,000 g/mol.
The adsorbent particles can be of any regular or irregular shape that lends
itself to incorporation into the inert matrix but are preferably roughly
spherical.
The particles are greater than about 100 ~m in diameter and less than about
1500
pm in diameter; preferably, the particles are between about 200 pm and about
1300 ~,m in diameter; more preferably, the particles are between about 300 p.m
and about 900 pm in diameter.
A high surface area is characteristic of the particles. Preferably, the
particles have a surface area between about 750 m2/g and about 3000 m2/g. More
preferably, the particles have a surface area between about 1000 m2/g and
about
3000 m2/g.
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WO 99/34914 PCT/US98/14134
Adsorbent particles suitable for use in the device of the present invention
can be any suitable material, with the limitation that the material does not
substantially adversely affect the biological activity of a biological
composition
upon contact. The adsorbent particle can be, for example, made of materials
such
as activated carbon, hydrophobic resins or ion exchange resins.
In one preferred embodiment the adsorbent particles are activated carbons
derived either from natural or synthetic sources. Preferably the activated
carbons
are derived from synthetic sources. Nonlimiting examples of activated carbons
include; Picatiff Medicinal°, which is available from PICA USA Inc.
(Columbus,
OH), Norit° ROX 0.8, which is available from Norit Americas, Inc.
(Atlanta,
GA), Ambersorb° 572, which is available from Rohm & Haas
(Philadelphia, PA),
and G-277°, which is available from PICA (Columbus, OH).
In another preferred embodiment, the particles can be hydrophobic resins.
Nonlimiting examples of hydrophobic resins include the following polyaromatic
adsorbents: Amberlite° adsorbents (e.g., Amberlite~ XAD-2, XAD-4, and
XAD-
16), available from Rohm and Haas (Philadelphia, PA); Amberchrom°
adsorbents
available from Toso Haas (TosoHass, Montgomeryville, PA);
Diaion~//Sepabeads° Adsorbents (e.g., Diaion° HP20),
available from
Mitsubishi Chemical America, Inc. (White Plains, NY); Hypersol-Macronet~
Sorbent Resins (e.g., Hypersol-Macronet~ Sorbent Resins MN-200, MN-150 and
MN-400) available from Purolite (Bala Cynwyd, PA); and Dowex°
Adsorbents
(e.g., Dowex~ XLTS-40323, XUS-43493, and XUS-40285), available from Dow
Chemical Company (Midland, MI).
Preferred particles are hydrophobic resins which are polyaromatic
adsorbents comprising a hypercrosslinked polystyrene network, such as Dowex~
XUS-43493 (known commercially as Optipore~ L493 or V493) and Purolite MN-
200.
Hypercrosslinked polystyrene networks, such as Dowex~' XUS-43493 and
Purolite MN-200 are non-ionic macroporous and macroreticular resins. The non-
ionic macroreticular and macroporous Dowex~ XUS-43493 has a high affinity for
18
CA 02318508 2000-07-OS
WO 99/34914 PCT/US98/14134
psoralens, including for example, 4'-(4-amino-2-oxa)butyl-4,5',8-trimethyl
psoralen, and it possesses superior wetting properties. The phrase "superior
wetting properties" means that dry (i. e. essentially anhydrous) adsorbent
does not
need to be wet with a wetting agent (e.g., ethanol) prior to being contacted
with
the blood product in order for the adsorbent to effectively reduce the
concentration of small organic compounds from the blood product.
Hypercrosslinked polystyrene networks, such as Dowex°~ XUS-43493
and
Purolite 1VIN-200 are preferably in the form of spherical particles with a
diameter
range of about 200 um to about 1300 um. Adsorbent particles, including for
example, Dowex~ XUS-43493, preferably have extremely high internal surface
areas and relatively small pores (e.g. average diameter 46 ~). The internal
surface
area of the particle can be from about 300 to about 1100 m2/g; preferably
about
900 to about 1100 m2/g; most preferably about 1100 m2/g. The majority of the
pores of the particle can be greater than 25~ and less than 800; preferably
from
about 25~ to about 150; most preferably from about 25~ to about 50~. While it
is not intended that the present invention be limited to the mechanism by
which
reduction of small organic compounds takes place, hydrophobic interaction is
believed to be the primary mechanism of adsorption. Its porous nature confers
selectively on the adsorption process by allowing small molecules to access a
greater proportion of the surface area relative to large molecules (i.e.,
large
proteins) and cells. Purolite~ has many similar characteristics to Dowex~ XUS-
43493, such as high affinity for psoralens and superior wetting properties,
and is
also a preferred adsorbent particle.
Polystyrene particles can be classified, based on their mechanism of
synthesis and physical and functional characteristics, as i) conventional
networks
and ii) hypercrosslinked networks. Preferred adsorbents have a high surface
area,
have pores that do not collapse upon drying, do not require wetting for
biological
compositions comprising red blood cells or platelets, and have extremely low
levels of small particles and foreign particles (e.g. dust, fibers, non-
adsorbent
particles, and unidentified particles). In addition, preferred adsorbents have
low
19
CA 02318508 2000-07-OS
WO 99/34914 PCT/US98/14134
levels of extractable residual monomer, crosslinkers and other organic
extractables.
The conventional networks are primarily styrene-divinylbenzene
copolymers in which divinylbenzene (DVB) serves as the crosslinking agent
(i.e.,
the agent that links linear polystyrene chains together). These polymeric
networks
include the "gel-type" polymers. The gel-type polymers are homogeneous, non-
porous styrene-DVB copolymers obtained by copolymerization of monomers.
The macroporous adsorbents represent a second class of conventional networks.
They are obtained by copolyrnerization of monomers in the presence of diluents
that precipitate the growing polystyrene chains. The polystyrene network
formed
by this procedure possess a relatively large internal surface area (up to
hundreds
of square meters per gram of polymer); Amberlite~ XAD-4 is produced by such a
procedure.
In contrast to the conventional networks described above, the preferred
adsorbents of the present invention (e.g., Dowex~ XUS-43493) are
hypercrosslinked networks. These networks are produced by crosslinking linear
polystyrene chains either in solution or in a swollen state with bifunctional
agents;
the preferred bifunctional agents produce conformationally-restricted
crosslinking
bridges, that are believed to prevent the pores from collapsing when the
adsorbent
is in an essentially anhydrous (i. e., "dry") state.
The hypercrosslinked networks are believed to possess three primary
characteristics that distinguish them from the conventional networks. First,
there
is a low density of polymer chains because of the bridges that hold the
polystyrene
chains apart. As a result, the adsorbents generally have a relatively large
porous
surface area and pore diameter. Second, the networks are able to swell; that
is, the
volume of the polymer phase increases when it contacts organic molecules.
Finally, the hypercrosslinked polymers are "strained" when in the dry state;
that
is, the rigidity of the network in the dry state prevents chain-to-chain
attractions.
However, the strains relax when the adsorbent is wetted, which increases the
network's ability to swell in liquid media. Davankov and Tsyurupa, Reactive
CA 02318508 2002-04-12
Polymers 13 :27-42 ( l 990); Tsyurupa et al., Reactive Polymers 25:69-78 (
1995).
Several cross-linking agents have been successfully employed to produce
the bridges between polystyrene chains, including p-xylene dichloride (XDC);
S monochlorodimethyl ether (MCDE),1,4-bis-chloromethyldiphenyl (CMDP), 4,4'-
bis-(chloromethyI)biphenyl (CMB), dimethylformal (DMF),pp'-bis-
chloromethyl-1,4-diphenyibutane (DPB), and tris-(chloromethyl)-mesitylene
(CMM). The bridges are formed between polystyrene chains by reacting one of
these cross-linking agents with the styrene phenyl rings by means of a Friedel-
Crafts reaction. Thus, the resulting bridges link styrene phenol rings present
on
two different polystyrene chains. See, e:g.; U.S. Patent No. 3,729,457.
The bridges are especially important because they generally eliminate the
need for a "wetting" agent. That is, the bridges prevent the pores from
collapsing
when the adsorbent is in an essentially anhydrous (d. e. , "dry") state, and
thus they
do not have to be "reopened" with a wetting agent prior to the adsorbent being
contacted with a blood product. In order to prevent the pores from collapsing,
conformationally-restricted bridges should be formed. Some bifunctional agents
like DPB do not result in generally limited conformation; for example, DPB
contains four successive methylene units that are susceptible to conformation
rearrangements. Thus, DPB is not a preferred bifunctional agent for use with
the
present invention.
Some of the structurally-related characteristics of the above-described
adsorbent particles are summarized in Table A.
TABLE A
Cttet~ical ?mature Weaeiyrf~ae Mead ~~ ~rlesh .
'' A~~'~~'~ ;, . ... iDiarre~ E~~: . . ~~ (t~~~!:
Ambertite~ Adsorbents -Rohm and Haas
XAD-2 polyaromatic 300 90 20-60
XAD-4 polyaromatic 725 40 20-60
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CA 02318508 2000-07-OS
WO 99/34914 PCT/US98/14134
TABLE A
XAD-7 polymethacrylate450 90 20-60
XAD-16 polyaromatic 800 100 20-60
XAD-1180 polyaromatic 600 300 20-60
XAD-2000 polyaromatic 580 42 20-60
XAD-2010 polyaromatic 660 280 20-60
Amberchrom~
Adsorbents
- Toso Haas
CG-71m polymethacrylate450-550 200-300 50-100
CG-71c polymethacrylate450-550 200-300 80-160
CG-161m polyaromatic 800-950 110-175 50-100
CG-161c polyaromatic 800-950 110-175 80-160
Diaion~//Sepabeads~
Adsorbents
- Mitsubishi
Chemical
Hl'20 polyaromatic 500 300-600 20-60
SP206 brominated 550 200-800 20-60
styrenic
SP207 brominated 650 100-300 20-60
styrenic
SP850 polyaromatic 1000 50-100 20-60
HP2MG polymethacrylate500 200-800 25-50
HP20SS polyaromatic 500 300-600 75-150
SP20MS polyaromatic 500 300-600 50-100
Dowex~ Adsorbents
- Dow Chemical
Company
XUS-40285 functionalized800 25 20-50
XUS-40323 polyaromatic 650 100 16-50
XUS-43493 polyaromatic 1100 46 20-50
Processing the Adsorbent Particles
The adsorbent particles may be further processed to remove fine particles,
salts, potential extractables, and endotoxin. The removal of these extractable
components is typically performed by treatment with either organic solvents,
steam, or supercritical fluids. Preferably the particles are sterilized.
Several companies currently sell "cleaned" (i.e., processed) versions of
commercially available adsorbent particles. In addition to processing the
adsorbent particles (e.g. resins), these companies test the adsorbents, and
the final
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CA 02318508 2000-07-OS
WO 99/34914 PCTNS98/14134
adsorbent is certified sterile (USP XXI), pyrogen-free (LAL), and free of
detectable extractables (DVB and total organics).
Thermal processing (e.g., steam) is an effective method for processing
adsorbent particles. F. Rodriguez, Principles Of Polymer Systems, (Hemisphere
Publishing Corp.), pp. 449-53 (3rd. Ed., 1989). Supelco, Inc. (Bellefonte, PA)
uses a non-solvent, thermal proprietary process to clean the Dowex~ XUS-43493
and Amberlite adsorbents. The main advantage of using steam is that it does
not
add any potential extractables to the adsorbent. One big disadvantage,
however,
is that this process can strip water from the pores of the resin beads;
effective
performance of some adsorbents requires that the beads be re-wet prior to
contacting the blood product.
One advantage of the cleaned/processed adsorbent is an extremely low
level of particles with diameters less than 30 um. Preliminary testing on
adsorbents (Dowex~ XUS-43493 and Amberlite~ XAD-16) processed by Supelco
was performed to determine particle counts. The results of these tests
indicated
that foreign particles (e.g., dust, fibers, non-adsorbent particles, and
unidentified
particles) were absent and that fine particles (< 30 um) were essentially
absent.
The Use of Wetting Agents and Stabilizing Agents with Adsorbent Resins
Methods may be used for preventing drying and loss of adsorption
capacity of particles, such as Amberlite~ which lose some of their adsorption
capacity under certain conditions (e.g., drying).
In one method, particles, materials or devices may be manufactured in a
wet state which is sealed and not capable of drying. This method is associated
with several important drawbacks. The shelf life of the products could be
reduced
since levels of extractables from the materials could increase over time.
Sterilization may be limited to a steam process because Y-irradiation of wet
polymers is typically not performed. Manufacturing a device that requires that
a
component be maintained in a wet state is, in general, more difficult than
manufacturing a dry device; for example, bioburden and endotoxin may become
of concern if there is a long lag time between device assembly and terminal
sterilization.
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CA 02318508 2000-07-OS
WO 99/34914 PCT/US98/14134
A second method for preventing loss of adsorption capacity involves the
use of an adsorbent which is not adversely affected by drying. As previously
set
forth, macroreticular adsorbents possessing highly crosslinked porous
structures
(e.g., Dowex~ XLJS-43493 and Purolite~ MN-200) generally do not require a
wetting agent because the crosslinks prevent the pores from collapsing. Unlike
Amberlite~ XAD-16, these ma,croreticular adsorbents retain a very high
proportion of their initial activity when they are dried.
In a third method, loss of adsorption capacity upon drying may be
prevented by hydrating Amberlite~ XAD-16 and related adsorbents (e.g.,
Amberlite~ XAD-4) in the presence of a non-volatile wetting agent. For
example,
when using Amberlite~ XAD-16 as the adsorbent, the adsorbent beads may
partially dry prior to use during handling, sterilization, and storage. When
the
water content of these adsorbents drops below a critical level, a rapid loss
in
adsorption capacity occurs (probably due to "collapse" of the pores); thus,
for
optimum effectiveness, the pores have to be "reopened" with a wetting agent
prior
to use.
Stabilizing agents are effective in maintaining adsorption capacity near its
maximum when certain adsorbent resins are subjected to drying conditions. It
is
believed that the use of stabilizing agents serves to prevent the adsorbent
pores
from collapsing.
An acceptable stabilizing agent should be soluble in water and ethanol,
nonvolatile relative to ethanol and water, and safe for transfusion in small
amounts. Glycerol and low molecular weight polyethylene glycol (e.g., PEG-200
and PEG-400) are examples of stabilizing agents possessing these
characteristics.
Glycerol has a positive hemocompatibility history. It is frequently added to
blood
as a cryo-preservative agent in the frozen storage of red blood cell
preparations.
See, e.g., Chaplin et al., Transfusion 26:341-45 (1986); Valeri et al., Am. J.
Vet.
Res. 42(9)1590-94 (1981). Solutions containing up to 1% glycerol are routinely
transfused, and glycerol solutions are commercially available (e.g.,
Glyerolite 57
Solution, Fenwal Laboratories, Deerfield, IL). Adsorbent beads like Amberlite~
XAD-16 may be stabilized in ethanol and glycerol.
24
CA 02318508 2002-04-12
Low molecular weight polyethylene glycols, commonly used as
pharmaceutical bases; may also be used as stabilizing agents. PEGs are liquid
and
solid polymers of the general chemical formula H(OCH2CH2)"OH, where n is
greater than or equal to 4. PEG formulations are usually followed by a number
that corresponds to its average molecular weight; for example, PEG-200 has a
molecular weight of 200 and a molecular weight range of 190-210. PEGs are
commercially available in a number of formulations (e.g., Carbowax; Poly-G;
and
Solbase).
Inert Matrices for Particle Immobilization
The adsorbent particles are immobilized by an inert matrix. The inert'
matrix can be made of a synthetic or natural polymer. For example, the inert
matrix can be a synthetic or natural polymer fiber, for example, a fiber
network.
The inert matrix can be sintered polymers. The inert matrix, as with the other
components of the device, preferably is biocompatible and does not
substantially
adversely affect the biological activity of a material upon contact.
Most preferably, the synthetic fibers are polyester fibers (Air Quality
Filtration (AQF), a division of Hoechst Celanese (Charlotte; N.C.)). Other
preferred examples of synthetic fibers are polyethylene or polyamide fibers.
Other exemplary synthetic fibers include polyolefin, polyvinyl alcohol and
polysulfone fibers.
In a preferred embodiment, the synthetic polymer fiber includes a first
polymer core with a high melting point surrounded by a sheath with a lower
melting temperature. The polymer core can be a polyester(polyethylene
terephthalate). The sheath can be a nylon, or a modified polyester. Fibers are
commercially available from Unitika (Osaka, Japan) and Hoechst Trevira GmbH
& Co. (Augsberg, Germany).
Exemplary natural polymer fibers include cellulose fibers derived from a
variety of sources, such as jute, kozu, kraft and manila hemp. Networks of
synthetic or natural polymer fibers have been used to make filters as
described in
U.S. Patent Nos. 4,559,145 and 5,639,376.
CA 02318508 2002-04-12
..,
Synthetic polymers suitable for the construction of sintered particles are
high density polyethylene, ultra high molecular weight polyethylene,
polypropylene, polyvinyl fluoride, polytetrafluoroethylene, nylon 6. More
preferably the sintered particles are polyoIefins, such as polyethylene.
Polymeric fibers such as those described above maybe adsorbent resins
without the attachment of adsorbent particles. Such fibers may be formed into
a
fiber network or may be immobilized on a fiber network of a less adsorbent
fiber.
Such fibers are contemplated by the present invention; such fibers preferably
contain a large, porous, adsorptive surface area or other adsorptive means to
facilitate reduction in the concentration of low molecular weight compounds.
Immobilization of Partictes
In one embodiment, the adsorbent particles are immobilized by an inert
matrix to produce an adsorption medium for reducing the concentration of small
organic compounds in a material. The inert matrix can be a three dimensional
network including a synthetic or natural polymer faber network with adsorbent
particles immobilized therein.
Preferably, the adsorption medium comprises small porous adsorbent
particles with highly porous structures and very high internal surface areas,
as
described above, immobilized by the inert matrix. Preferably, when a
biological
material is brought into contact with the adsorption medium, the adsorption
medium does not substantially adversely affect the biological activity or
other
properties of the material.
Technology for immobilization of adsorbent beads on a fiber network to
construct air filters has been described in U.S. Patent No. 5,486,410 and U.S.
Patent No. 5,605,746. . As depicted in Figure l,
the polymerfibers 600 of the fiber network consist of a polymer core 602
(e.g.,
polyethylene terephthalates (PET)) with a high melting point surrounded by a
polymer sheath 604 (e.g., nylon) with a relatively low melting temperature.
See.
U.S. Patent No. 5,190,65? to Heagle et al. The
fiberized resin is prepared by first evenly distributing the adsorbent beads
in the
fiber network. Next, the network is rapidly heated (e.g.; 180°C x 1
min.) causing
26
CA 02318508 2002-04-12
the polymer sheath of the fibers 600 to melt and bond to the adsorbent beads
606
and other fibers, forming a cross-linked fiber network, represented in Figure
2.
As depicted in Figure 3 and Figure 4 (not to scale), generally speaking, the
fiber
networks contain three layers; two outer layers 647 that are densely packed
with
fibers 600 and a less dense inner layer 609 that contains the adsorbent beads
606
and fewer fibers 600. In a preferred embodiment, the edges of the fiberized
resin
may be sealed with polyurethane or other polymers. Alternatively, as depicted
in
Figure3 and Figure 4, heat seals 608 may be made in the resulting fiberized
resin
at predetermined intervals; for example, heat seals can be made in the
fberized
resin in a pattern of squares. Thereafter, the fiberized resin can be cut
through the
heat seals to form samples of resin containing a desired mass (e.g:,
preferably less
than 5.0 g and more preferably less than 3.0 g) of adsorbent beads and of a
size
suitable for placement within a t~lood product container. The heat seals serve
to
prevent the cut fiberized resin from fraying and help to immobilize the
adsorbent
beads. However, the use of such heat seals is not required in order to
practice'the
present invention. In an alternative embodiment, depicted in Figure 4, the
adsorbent beads 606 are not secured to the fibers themselves, hut rather are
immobilized between the denser outer layers 607 of fibers and with the heat
seals
608; this embodiment may also result in samples of fiberized media
containing.a
defined amount of adsorbent after being cut through the heat seals.
The present invention also contemplates the use of an adhesive (e.g., a
bonding agent) to secure the adsorbent resin to the fibers. Moreover, while it
is
preferable that the adsorbent beads be chemically attached to the fiber
network,
the beads may also be physically trapped within the fiber network; this might
be
accomplished, for example, by surrounding the beads with enough fibers so as
to
hold the beads in position.
Other ways that the adsorbent particles may be immobilized in a fiber
network are also contemplated. The particles can be immobilized using a dry-
laid
process, as described in U:S. Pat. Nos. 5;605;746 and 5,486,410 (AQF patents),
The particles can be immobilized
using a wet-laid process, as described in U.S. Pat: Nos. 4,559,145 and
4,309,247.
27
CA 02318508 2002-04-12
The particles can be immobilized
using amelt-blown process; as described in U.S. Pat. No. 5,616,254.
Where a wet-laid process is used o construct a
matrix from natural polymer fibers, the inert matrix preferably includes a
binding
S agent to bond the adsorbent particles to the fibers. NonIimiting examples of
binding, agents include melamine, polyamines and polyamides. The matrix
typically contains 1 % or less of such binding agents.
Where the inert matrix is constructed from particles of synthetic polymers
which are sintered with adsorbent particles, it is important that the
adsorbent
particle have a higher melting temperature than. the matrix.
In a preferred embodiment, the adsorbent particles are immobilized in a
fiber matrix that is formed by thermal bonding of a biocomponent fiber
network.
An alternative embodiment involves immobilizing adsorbent particles in non-
biocomponent fibers and using a wet strength resin system, adhesives or
additional fusible f bees to form bonds between the fibers and adsorbent
particles.
Nonlimiting examples of useful fibers include polyester, nylon and polyolefin.
(Suppliers of fibers for the nonwovens industry have been listed in "A Guide
to
Fibers for Nonwovens," Nonwovens Industry, June 1998, 66-87.) Examples of
wet strength resin systems include melamine/formaldehyde, epichlorohydrin-
based resins, polyamines and polyamides. The use of heat fusible fibers for
immobilizing particles in fiber matrices has been disclosed. See, e.g. ~ U.S.
Pat.
No. 4,160,059.
Preferably, the resulting adsorption medium comprises known amounts of
adsorbent per area. The adsorbent per area is from about 100 glm2 to about 500
g/m2, preferably from about 250 g/m2 to about 350 glm2. Thus, the appropriate
amount of adsorbent contemplated for a specific purpose can be measured simply
by cutting a predetermined area of the fiberized resin (i.e., there is no
weighing: of
the fiberized resin).
The adsorption medium preferably is biocompatible (i.e., not producing: a
toxic, injurious, or immunologic response); has a minimal impact on the
properties of the material such as blood product (e.g., platelets and clotting
28
CA 02318508 2002-04-12
factors); and is not associated with toxic extractables. The immobilized
adsorbent
particles of the adsorption medium preferably have high mechanical stability
(i.e.,
no fine particle generation). The adsorption medium for a batch device
contains
about 25-85% adsorbent by weight, preferably about 50-80% adsorbent at a
loading of about 100-500 gJm2, more preferably about 50-80% adsorbent at a
loading of about 250-350 glm2.
Coating the Adsorbent Particles
The surface hernocompatibility of the particles, matrices or adsorption
medium can be improved by coating their surfaces with a hydrophilic polymer.
Exemplary hydrophilic polymers include poly(2-hydroxyethyl methacrylate)
(pHEMA), which may be obtained from, e.g., Scientific Polymer Products, Inc.
(Ontario; NY) and cellulose-based polymers, e.g., ethyl cellulose, which may
be
obtained from Dow Chemical {Midland, MI). See, e.g., Andrade et al., Trans.
Amer. Soc. Artif. Int. Organs XViI:222-28 (1971). Other examples of coatings
include polyethylene glycol and polyethylene oxide, also available from
Scientific
Polymer Products, Inc.. The polymer coating can increase hemocompadbility and
reduce the risk of small particle generation due to mechanical breakdown.
T'he adsorbent surface may also be modified with immobilized heparin. In
addition,: strong anion exchange polystyrene divinylbenzene adsorbents may be
modified via heparin adsorption. Heparin, a polyanion, will adsorb very
strongly
to the surfaces of adsorbents which have strong anion exchange
characteristics. A
variety of quaternary amine-modified polystyrene divinyl benzene adsorbents
are
commercially available.
The coating can be applied in a number of different methods, including
radio frequency glow discharge polymerization; as described in U:S. Patent
number 5,455,040, and the Wurster
coating process (performed by International Processing Corp. (Winchester, KY).
In one embodiment, the Wurster coating process can be applied by
suspending the adsorbent particles (generally via air pressure) in a chamber
such
that the hydrophilic polymer, such as pHEMA, can be sprayed evenly onto all
surfaces of the adsorbent particle. As illustrated in Example 3, Dowex~ XUS-
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WO 99/34914 PCT/US98/14134
43493 sprayed evenly with pHEMA demonstrated an increase in platelet yield as
well as a dramatic effect on platelet shape change with increasing amounts of
coating. It was found that the Wurster coating process selectively coated the
outside surface of the adsorbent surface, leaving the inside porous surface
nearly
unaffected.
In a preferred embodiment, the coating can be applied by soaking the
immobilized adsorption medium in the hydrophilic polymer (see Example 3).
This process is simpler and less expensive than spraying the adsorbent
particles
with the hydrophilic polymer.
The process is not limited to a process that applies the coating of the
adsorption medium at any particular time. For example, in one embodiment, the
pHEMA coating is applied after production of the adsorption medium, but prior
to
heat sealing the adsorption medium. In another embodiment, the adsorption
medium is first heat sealed, and then the pHEMA coating is applied. In
addition
to coating the adsorption medium, the rinsing process associated with pHEMA
application serves to remove loose particles and fibers.
As the amount of coating is increased, it becomes more difficult for some
small organic compounds to cross the coating to reach the particle surface,
resulting in a decrease in adsorption kinetics. Thus, as the amount of coating
is
increased, an increased mass of adsorbent must generally be used to achieve
the
same removal kinetics as coated adsorbent. In one embodiment, the optimum
level of pHEMA coating is the minimum coating at which a protective effect on
platelet yield and in vitro platelet function is observed (0.1-0.5%).
The coatings may be sensitive to sterilization. For example, gamma
sterilization may result in cross-linking and/or scission of the coating.
Therefore,
the type (E-beam vs. gamma irradiation) and dose of sterilization may
influence
the properties of the coated adsorbent. Generally, E-beam sterilization is
preferred.
Devices
Devices are provided for reduction of compounds from biological
compositions containing cells. The device is a batch device. An example of a
CA 02318508 2002-04-12
batch device is shown in Figure 16: Batch devices are known in the literature
and
are described, for example, in PCT publication WO 96140857.
Batch devices of the invention may comprise a container, such as a blood
bag; including the adsorbent medium containing immobilized particles. In one
embodiment a blood product is added to a blood bag containing he adsorbent
medium and the bag is agitated for a specified period of time.
For example, in one embodiment, an adsorption medium, e.g:,
immobilized Dower' XUS-43493, is placed inside a blood product container
(e.g., a PL 146 Plastic container (BaxterHealthcare Corp. (Deerfield, IL))
kept on
either a platelet shaker (Helmer Laboratories (Novesvill, IN)) or rotator
{Helmer
Laboratories (Novesvill, IN)) for about 24 hours at room temperature and
stored
under various temperature conditions. The size of the blood product container
can
be from about 600 to about 1200 mL. The storage temperature can be from about
4°C to about 22°C.
Methods can be used to reduce the presence of adsorbent particles that
may come loose from the adsorbent medium.
The present invention contemplates a batch device including the
immobilized adsorbent medium retained in a container such as a mesh bag/pouch.
The mesh/pouch can be constructed of a woven, non-woven or membranous
enclosure. In one embodiment, the woven mesh pouch can be constructed of
medical-grade polyester or nylon. The preferred embodiment is polyester.
Commercially-available membranes include, but are not limited to, Supor~ 200,
800,.1200 hydrophilic polyethylene sulfonate (PES) membranes (German
Sciences (Ann Arbor, MI)); Durapore~ hydrophilic modified polyvinylidene
difluoride (PVDF} (Mantee America Corp. (San Diego, CA)) and hydrophilic
modified polysuIfone membranes with integrated hydrophobic vents, e.g. Gemini
membranes, (Millipore (Marlborough, MA)); and membranes comprising
polycarbonate with a polyvinylidene coating {Poretics (Livermore, CA)). The
containers can be sterilized after addition of the adsorption medium.
Preferred Embodiment for Biological Compositions Containing Cells
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WO 99/34914 PCT/US98/14134
The present invention provides devices for reducing the concentration of
low molecular weight compounds in biological compositions containing cells.
The devices comprise an adsorption medium, which is comprised of particles
immobilized by an inert matrix.
Biological response modifiers like the anaphalatoxin C3a and the terminal
membrane attack complex SCSb-9 have been shown to be produced by the
processing, (e.g., leukofiltration, pheresis, recovery of shed blood, etc.)
and
storage of whole blood and its components. These biological response modifiers
have been implicated in adverse events in surgery and transfusion.
In some embodiments, the device of the present invention reduces or
controls the concentration of activated complement in biological compositions
containing cells. The concentration of activated complement in the composition
is
reduced or controlled when it is treated with the device, as opposed to a
composition that has not been treated with the device. In one embodiment, the
adsorption device comprises fiberized Ambersorb IAD, for example, as produced
by AQF. In this embodiment, exposure of biological compositions containing
cells to the device results in a reduction in the C3a complement fragment and
SCSb-9 terminal component complex over control. In one embodiment, exposure
to the device for 5 days results in at least about a 10% reduction of C3a
complement fragment over control. In another embodiment, exposure to the
device for 5 days results in at least about a 30% reduction of C3a complement
fragment over control. In another embodiment, exposure to the device for 5
days
results in at least about a 50% reduction of C3a complement fragment over
control.
In one embodiment, the invention provides a device for reducing the
concentration of compounds in a biological composition comprising platelets.
The biological activity of the platelets is substantially maintained after
treatment
with the device. The adsorption medium of this embodiment comprises adsorbent
particles immobilized by an inert matrix. Preferred particles are highly
porous
and have a surface area greater than about 750 m2/g.
Particularly preferred particles for this embodiment are polyaromatic
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WO 99/34914 PC'T/US98/14134
adsorbents comprising a hypercrosslinked polystyrene network, such as Dowex~
XUS-43493 or Purolite IvIN-200. The preferred inert matrix includes a
synthetic
or natural polymer fiber. In a preferred embodiment the inert matrix includes
a
synthetic polymer fiber which includes a first polymer core with a high
melting
point surrounded by a sheath with a lower melting temperature. The polymer
core
can be a polyethylene terphthalate core. The sheath can be a nylon sheath or a
modified polyester sheath. Staple fibers are commercially available from
Unitika
(Osaka, Japan) and Hoechst Trevira.
Exemplary compounds that are reduced or controlled by the devices,
materials and methods of this embodiment are psoralens, psoraten derivatives,
isopsoralens, psoralen photoproducts, acridines, acridine derivatives,
methylene
blue, plastic extractables, biological response modifiers, quenchers and
polyamine
derivatives.
Biological compositions comprising platelets are typically used within 3
days of donation but may be stored for up to 7 days at room temperature,
therefore, it would be advantageous to allow the platelet compositions to
remain
in contact with the adsorption medium for the entire storage period.
Preferably,
the procedure would result in an acceptable platelet yield (e.g., iess than
10%
loss). One method contemplated by the present invention allows extended
storage
by improving the hemocompatibility of the adsorbent surface.
The use of an adsorption medium comprising adsorbent particles
immobilized by an inert matrix permits the concentration of low molecular
weight
compounds to be reduced without a substantial loss in platelet count. The
phrase
"without a substantial loss" refers to a platelet preparation that is suitable
for its
intended purpose, for example, is suitable for infusion into humans, and may
refer
to, for example, a loss of platelet count or function of less than about 10%,
preferably less than about 5% over a period of time, more preferably at least
S
days. Furthermore, the time that the platelets may be contacted with the
adsorption medium without substantial loss in platelet count is greater than
the
amount of time that the platelets can be contacted with the adsorbent
particles
alone. The immobilization of the particles unexpectedly permits both a longer
33
CA 02318508 2000-07-OS
WO 99/34914 PCT/US98/14134
contact time and a reduction in loss of platelet count. The platelets
typically
cannot be contacted with non-immobilized adsorbent particles for more than
about
20 hours without a significant loss of platelet count e.g. about 20% loss. In
contrast, platelets may be contacted with the adsorption medium comprising
adsorbent particles immobilized by an inert matrix for more than 20 hours,
e.g.
about 1 to 7 days without a substantial loss in platelet count.
Additionally in vitro platelet function (e.g., shape change, GMP-140, pH)
is improved for platelets stored in the presence of the adsorption medium in
comparison to the storage of the platelets without the adsorption medium over
time. Platelets stored in the presence of the adsorption medium can have a pH
from greater than about 6 to less than about 7.5.
In another embodiment, the invention provides a device for reducing the
concentration of low molecular weight compounds (e.g., small organic
compounds) in a biological composition comprising red blood cells while
substantially maintaining the biological activity of the red blood cells.
Typically,
the compounds removed by the device have a molecular weight ranging from
about 100 g/mol to about 30,000 g/mol. The adsorption medium comprises
adsorbent particles immobilized by an inert matrix. Preferred particles for
this
embodiment are highly porous and have a surface area greater than about 750
m2/g.
Preferably, a device used for red blood cell compositions is a device that
substantially maintains the biological activity of the red blood cells after
reduction
of the concentration of low molecular weight compounds. In one embodiment,
the red blood cell device does not substantially adversely affect the
biological
activity of a fluid upon contact. The device embodiment comprises an
adsorption
medium containing particles immobilized by an inert matrix and optionally a
particle retention device.
In one embodiment the particles used in devices fox red blood cell
compositions are activated charcoal. Preferably the activated carbons are
derived
from synthetic sources. Nonlimiting examples of activated carbons include
Picactif Medicinal, which is available from Pica U.S.A. (Columbus, Ohio);
Norit
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WO 99/34914 PCT/US98/14134
ROX 0.8, which is available from Norit Americas Inc. (Atlanta, GA); and G-277,
which is available from Pica U.S.A. (Columbus, OH).
In one embodiment, the adsorbent is preferably an activated carbon
derived from a synthetic source, such as Ambersorb 572. The Ambersorbs are
synthetic activated carbonaceous (i.e. rich in carbon) adsorbents that are
manufactured by Rohm & Haas (Philadelphia, PA). Ambersorbs are generally
large spherical (300-900 p,m) particles that are more durable than typical
activated
carbons. The Ambersorbs are synthetically manufactured by treating highly
sulfonated porous polystyrene beads with a proprietary high temperature
activation process. These adsorbents do not require pre-swelling to achieve
optimal adsorption activity.
In another embodiment, the particles used in devices for use with
compositions containing red blood cells ("red blood cell devices") can be
hydrophobic resins. Nonlimiting examples of hydrophobic resins include the
following polyaromatic adsorbents: Amberlite~ adsorbents (e.g., Amberlite~
XAD-2, XAD-4, and XAD-16), available from Rohm and Haas (Philadelphia,
PA); Amberchrom~ adsorbents available from Toso Haas (Toso Haas,
Montgomeryville, PA); and Diaion~//Sepabeads~ Adsorbents (e.g., Diaion~
HP20), available from Mitsubishi Chemical America, Inc. (White Plains, NY). In
a particularly preferred embodiment the particles are Hypersol-Macronet~
Sorbent
Resins (e.g., Hypersol-Macronet~ Sorbent Resins MN-200, MN-150 and MN-
400) available from Purolite (Bala Cynwyd, PA) or Dowex~ Adsorbents (e.g.,
Dowex~ XUS-43493, and XUS-40285), available from Dow Chemical Company
(Midland, MI).
The preferred inert matrix of a red blood cell device includes a synthetic or
natural polymer fiber. In a preferred embodiment the inert matrix includes a
synthetic polymer fiber which includes a first polymer core with a high
melting
point surrounded by a sheath with a lower melting temperature. The polymer
core
can be a polyethylene terephthalate or polyester core. The sheath can be a
nylon
sheath or a modified polyester sheath. Fibers are commercially available from
CA 02318508 2000-07-OS
WO 99/34914 PCTNS98/14134
Unitika (Osaka, Japan) and Hoechst Trevira (Augsberg, Germany).
In some embodiments, the adsorption medium of a red blood cell device is
in an enclosure. In one embodiment, the device comprises an adsorption medium,
and a housing. In another embodiment, the device comprising an adsorption
medium and a housing may also include a particle retention medium. In one
embodiment, the housing comprises a blood bag of a volume between about 600
ml and about 1 L. In another embodiment, the housing comprises a blood bag of
a
volume between about 800 ml and about 1 L. The particle retention medium may
comprise a polyester woven, polyester non-woven, or synthetic membranous
enclosure.
It is preferable that the device contact the red blood composition at about 4
° C or about 22 °C (room temperature), in the presence of
agitation, over a time
period of 1 to 35 days. In one embodiment, the red blood composition is
contacted with the device at about 22 °C for no more than about 36
hours: In
another embodiment, the red blood cell composition is contacted with the
device
at about 22 °C for no more than about 24 hours. In another embodiment,
the red
blood composition is contacted with the device at about 22 °C for no
more than
about 12 hours. In another embodiment, the red blood composition is contacted
with the device at about 22 °C for no more than 6 hours.
In some embodiments, the temperature is changed after the red blood
composition is brought into contact with the device. In one embodiment, the
device is contacted with the red blood composition at about 22 °C for a
time
ranging from about 0.5 to about 24 hours and then stored at about 4 °C
for up to
about 5 weeks. In another embodiment, the device is contacted with the red
blood
composition at about 22 °C for a time ranging from about 0.5 to about
12 hours
and then stored at about 4 °C for up to about 5 weeks. In another
embodiment,
the device is contacted with the red blood composition at about 22 °C
for a time
ranging from about 0.5 to about 6 hours and then stored at about 4 °C
for up to
about 5 weeks.
The use of an adsorption medium comprising adsorbent particles
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CA 02318508 2000-07-OS
WO 99/34914 PCT/US98/14134
immobilized by an inert matrix permits the treatment of the red blood cell
composition without a substantial loss in red blood cell function. The phrase
"without a substantial loss" refers to a product that would be allowable for
transfusion, or its intended purpose, and in some instances may refer to less
than
about 1 % hemolysis; preferably less than about 0.8% hemolysis, greater than
about 80% recovery of red blood cells; preferably greater than 90% recovery of
red blood cells, less than about 10% difference from no-device control red
blood
cells in change in ATP concentration, and less than about 15% difference from
no-device control red blood cells in change in extracellular potassium
concentration. At 35 days, the change in hemolysis is at least 10% lower for
the
IAD compared to the non-immobilized particles, preferably, 20% lower and more
preferably 50%. Most preferably, the change is hemolysis is at least 90% lower
for the IAD compared to the non-immobilized particles.
Red blood cell function can be assayed using standard kits. In
particular, hemolysis may be determined by measuring the absorbence at 540 nm
of a red blood cell supernatant sample in Drabkin's reagent ((Sigma Chemical
Company), St. Louis, MO). Potassium leakage can be assayed using a Na+/K+
analyzer. (Ciba-Corning Diagnostics, Medfield, MA). Quantitative enzymatic
determination of ATP in total Red Blood Cell samples is possible using a
standard
kit (Sigma Diagnostics, St. Louis, MO) and measuring absorbance at 340 nm
compared to a water background.
Where the biological composition to be treated is a red blood cell
containing composition, the device can reduce the concentration of low
molecular
weight compounds in a red blood cell sample. Preferably the device can reduce
the concentration of both acridine derivatives and thiols in a red blood cell
sample. More preferably, the device can reduce the concentration of both S-
[((3-
carbethyoxyethyl)amino]-acridine and glutathione in a red blood cell sample.
Standard HPLC assays can be used to determine concentrations of 5-[((3-
carboxyethyl)amino]acridine and glutathione in red blood cells contacted with
the
device. Assay mobile phases are 10 mM H3P04 in HPLC water and 10 mM
H3P04 in acetonitrile. Zorbax SB-CN and YMC ODSAM-303 columns are
37
CA 02318508 2002-04-12
available from MacMod Analytical; Inc. (Chadds Ford, PA) and YMC; Inc.
(Wilmingtion; N.C.).
Where a device is brought into contact with a biological composition
containing cells in the presence of agitation, the agitation can be constant
or
intermittent. The agitation is provided through any suitable means which
maintains the functionality of the cells, including mechanical agitators of
the
following types: reciprocating; orbital, 3-D rotator and rotator type
agitators. In
one embodiment, the agitation is provided by an orbital agitator and is
constant.
In another embodiment, the agitation is provided by an orbital agitator and is
intermittent. In another embodiment, the agitation is provided by a
reciprocating
agitator:
Applications
The present invention contemplates reducing the concentration of low
molecular weight compounds from biological compositions containing cells.
Such compounds include, for example, pathogen-inactivating agents such as
photoactivatibn products, aminoacridines, organic dyes and phenothiazines.
Exemplary pathogen inactivating agents include furocoumarins, such as
psoralens
and acridines. Following treatment of a blood product with a pathogen
inactivating compound as described for example in U.S. Patent Numbers
5,459,030 and 5,559,250, the concentration of
pathogen inactivating compounds in the blood product can be reduced by
contacting the treated blood product with a device of the invention.
In one embodiment the present invention contemplates a method of
inactivating pathogens in solution, wherein the method comprises: a)
providing, in
any order: r) a cyclic compound; ii) a solution suspected of being
contaminated
with said pathogens, and iii) fiberized resin; b) treating said solution with
said
cyclic compound so as to create a treated solution product wherein said
pathogens
are inactivated; and c) contacting said treated solution product with said
fiberized
resin, and further comprising a device far reducing the concentration of small
organic compounds in a blood product while substantially maintaining a desired
biological activity of the blood product, the device comprising highly porous
38
CA 02318508 2002-04-12
adsorbent particles, wherein the adsorbent particles are immobilized by an
inert
matrix.
In addition to the pathogen inactivating compound, reactive degradation
products thereof can be reduced from the material such as a blood product, for
S example prior to transfusion.
The materials and devices disclosed herein can be used in apheresis
methods: Whole blood can be separated into two or more specific components
(e.g., red blood cells, plasma and platelets). The term "apheresis" refers
broadly
to procedures in which blood is removed from a donor and separated into
various
components, the components) of interest being collected and retained and the
other components being returned to the donor. The donor receives replacement
fluids during the reinfusian process to help compensate for the volume and
pressure loss caused by component removal. Apherersis systems are described in
PCT publication W096/4085'7,
Low Molecular Weight Compounds
A device of the present invention reduces the concentration of a low
molecular weight compound in a composition containing cells. The term "low
molecular weight compound" refers to an organic or biological molecule having
a
molecular weight ranging from about 100 g/mol to about 30,000 g/mol. Low
molecular weight compounds include; without limitation, the following
compounds: small organic compounds such as psoralens, acridines or dyes;
quenchers, such as giutathione; plastic extractables, such as plasticizers;
biological modifiers; such as activated complement, that possess a molecular
weight between about 100 g/mol and about 30;000 g/mol; and, polyamine
derivatives.
Small Organic Compounds
A diverse set of small organic compounds can be adsorbed by the device
of the present invention. The molecules can be cyclic or acyclic. Inane
embodiment the compounds are preferably, cyclic compounds such as psoralens,
acridines or dyes. In another embodiment the compounds are thiols.
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WO 99/34914 PCT/US98/14134
Nonlimiting examples of cyclic compounds include actinomycins,
anthracyclinones, mitomyacin, anthramycin, and organic dyes and photoreactive
compounds such as benzodipyrones, fluorenes, fluorenones, furocoumarins,
porphyrins, protoporphyrins, purpurins, phthalocyanines, hypericin, Monostral
Fast Blue, Norphillin A, phenanthridines, phenazathionium salts, phenazines,
phenothiazines, phenylazides, quinolines and thiaxanthenones. Preferably the
compounds are furocoumarins or organic dyes. More preferably the compounds
are furocoumarins.
Nonlimiting examples of furocoumarins, include psoralens and psoralen
derivatives. Specifically contemplated are 4'-aminomethyl-4,5',8-
trimethylpsoralen, 8-methoxypsoralen, halogenated psoralens, isopsoralens and
psoralens linked to quaternary amines, sugars, or other nucleic acid binding
groups. Also contemplated are the following psoralens: S'-bromomethyl-4,4',8-
trimethylpsoralen, 4'-bromomethyl-4,5',8-trimethylpsoralen, 4'-(4-amino-2-
I S aza)butyl-4,5',8-trimethylpsoralen, 4'-(4-amino-2-oxa)butyl-4,5',8-
trimethylpsoralen, 4'-(2-aminoethyl)-4,5',8-trimethylpsoralen, 4'-(5-amino-2-
oxa)pentyl-4,5',8-trimethylpsoralen, 4'-(S-amino-2-aza)pentyl-4,5',8-
trimethylpsoralen, 4'-(6-amino-2-aza)hexyl-4,5',8-trimethylpsoralen, 4'-(7-
amino-2,S-oxa)heptyl-4,5',8-trimethylpsoralen, 4'-(12-amino-8-aza-2,5-
dioxa)dodecyl-4,5',8-trimethylpsoralen, 4'-{13-amino-2-aza-6,11-dioxa)tridecyl-
4,5',8-trimethylpsoralen, 4'-(7-amino-2-aza)heptyl-4,5',8-trimethylpsoralen,
4'-
(7-amino-2-aza-5-oxa)heptyl-4,5',8-trimethylpsoralen, 4'-(9-amino-2,6-
diaza)nonyl-4,5',8-trimethylpsoralen, 4'-(8-amino-5-aza-2-oxa)octyl-4,5',8-
trimethylpsoralen, 4'-(9-amino-5-aza-2-oxa)nonyl-4,5',8-trimethylpsoralen, 4'-
(14-amino-2,6,11-triaza)tetradecyl-4,5',8-trimethylpsoralen, 5'-(4-amino-2-
aza)butyl-4,4',8-trimethylpsoralen, 5'-(6-amino-2-aza)hexyl-4,4',8-
trimethylpsoralen and 5'-(4-amino-2-oxa)butyl-4,4',8-trimethylpsoralen.
Preferably, the psoralen is 4'-(4-amino-2-oxa)butyl-4,5',8-trimethylpsoralen.
Acridines
Nonlimiting examples of acridines include acridine orange, acriflavine,
quinacrine, N1, NI-bis (2-hydroxyethyl)-N4-(6-chloro-2-methoxy-9-acridinyl)-
CA 02318508 2002-04-12
1,4-pentanediamine, 9-(3-hydroxypropyl~mmoacridine, N-(9-acridinyl)glycine,
S-(9-acridinyl)-glutathione. In a preferred embodiment the acridine is N-(9-
acridinylrø-alanine, alternatively, named 5-[(~-carboxyethyl)amino)acridine.
Dyes
Nonlimiting examples of dyes include phenothia2ines such as methylene
blue, neutral red, toluidine blue, crystal violet and azure A, phenothiazones
such
as methylene violet Bernthsen, phthalocyanines such as aluminum 18,15,22-
tetraphenoxy-29H,3I H-phthalocyanine chloride and silica analogues, and
hypericin. Preferably, the dye is methylene blue or toluidine blue. More.
preferably, the dye is methylene blue.
The term "thiazine dyes" includes dyes that contain a sulfur atom in one or
more rings. The most common thiazine dye is methylene blue [3,7-
Bis(dimethylamino)-phenothiazin-S-ium chloride). Other thiazine dyes include,
but are not limited to; azure A, azure C and thionine, as described e.g. in
U.S.
Patent No. 5,571,666 to Schinazi.
The term "xanthene dyes" refers to dyes that are derivatives of the
compound xanthene. The xanthene dyes may be placed into one of three major
categories: i) fluorenes or amino xanthenes, ii) the rhodols or
aminohydroxyxanthenes, and iii) the fluorones or hydroxyxantheses. Examples of
xanthene dyes contemplated for use with the present invention include rose
Bengal
and eosin Y; these dyes may be commercially obtained from a number of sources
(e.g., Sigma Chemical Co., St. Louis, MI), and as described e.g. in U.S.
Patent
No. 5,571,666 to Schinazi.
Quenchers
The concentration of a variety of compounds may be reduced. Other
exemplary compounds include quenching compounds. Methods for quenching
undesired side reactions of pathogen inactivating compounds that include a
functional group which is, or which is capable of forming, an electrophilic
group,
are described in the co-owned U.S. Patent Application, "Methods for Quenching
Pathogen Inactivators in Biological Systems", Docket Number282173000600;
filed January 6, 1998. = In this
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WO 99/34914 PCT1US98/14134
method, a material, such as a blood product, is treated with the pathogen
inactivating compound and a quencher, wherein the quencher comprises a
nucleophilic functional group that is capable of covalently reacting with the
electrophilic group. In one embodiment, the pathogen inactivating compound
includes a nucleic acid binding ligand and a functional group, such as a
mustard
group, which is capable of reacting in situ to form the electrophilic group.
Examples of quenchers include, but are not limited to, compounds including
nucleophilic groups. Exemplary nucleophilic groups include thiol, thioacid,
dithoic acid, thiocarbamate, dithiocarbamate, amine, phosphate, and
thiophosphate groups. The quencher may be, or contain, a nitrogen heterocycle
such as pyridine. The quencher can be a phosphate containing compound such as
glucose-6-phosphate. The quencher also can be a thiol containing compound,
including, but not limited to, glutathione, cysteine, N-acetylcysteine,
mercaptoethanol, dimercaprol, mercaptan, mercaptoethanesulfonic acid and salts
thereof, e.g., MESNA, homocysteine, aminoethane thiol, dimethylaminoethane
thiol, dithiothreitol, and other thiol containing compounds. Exemplary
aromatic
thiol compounds include 2-mercaptobenzimidazolesulfonic acid, 2-mercapto-
nicotinic acid, napthalenethiol, quinoline thiol, 4-nitro-thiophenol, and
thiophenol.
Other quenchers include nitrobenzylpyridine and inorganic nucleophiles such as
selenide salts or organoselenides, thiosulfate, sulfite, sulfide,
thiophosphate,
pyrophosphate, hydrosulfide, and dithionitrite. The quencher can be a peptide
compound containing a nucleophilic group. For example, the quencher may be a
cysteine containing compound, for example, a dipeptide, such as GlyCys, or a
tripeptide, such as glutathione.
Compounds that may be removed by the device of the present invention
may include thiols such as methyl thioglycolate, thiolactic acid, thiophenol,
2-
mercaptopyridine, 3-mercapto-2-butanol, 2-mercaptobenzothiazole, thiosalicylic
acid and thioctic acid.
Plastic Extractables
The concentration of a group of low molecular weight compounds that are
extractables from plastic storage containers and tubing used to handle
biological
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CA 02318508 2000-07-OS
WO 99/34914 PCT/US98/14134
compositions may also be reduced in a biological composition using a device of
the present invention. Examples of extractables include, but are not limited
to,
plasticizers, residual monomer, low molecular weight oligomers, antioxidants
and
lubricants. See, e.g., R. Carmen, Transfusion Medicine Reviews 7(1):1-10
(1993).
S The sterilization of plastic components by-steam, gamma irradiation or
electron
beam can produce oxidative reactions and/or polymer scission, resulting in the
formation of additional extractable species.
Plasticizers are commonly used to enhance properties of plastics such as
processability and gas permeability. The most common plasticizer found in
blood
storage containers is di(2-ethylhexyl) phthalate (DEHP), which is used in PVC
formulations. DEHP has been identified as a potential carcinogen. Alternative
plasticizers have been developed, including, without limitation, the following
compounds: tri (2-ethylhexyl) trimellitate (TEHTM), acetyl-tri-n-hexyl citrate
(ATHC), butyryl-tri-n-hexyl-citrate (BTHC), and di-n-decyl phthalate.
A device of the present invention may be used to reduce or control the
concentration of plastic extractables in a biological composition in a variety
of
settings. Such settings include, but are not limited to, the following: blood
treatment; blood storage; and, extracorporeal applications such as
hemodialysis
and extracorporeal membrane oxygenation.
Biological Response Modifiers (BRMs)
The concentration of a group of low molecular weight compounds broadly
referred to as biological response modifiers (BRMs) may also be reduced or
controlled in a biological composition using a device of the present
invention.
BRMs are defined as "a wide spectrum of molecules that alter the immune
response." Illustrated Dictionary oflmmunology, J.M. Cruse and R.E. Lewis.
General groups of BRMs include, without limitation, the following types of
compounds: small molecules such as histamine and serotonin; lipids such as
thromboxanes, prostaglandins, leukotrienes and arachidonic acid; small
peptides
such as bradykinin; larger polypeptides that contain further groups, including
activated complement fragments (C3a, CSa); cytokines such as IL-l, IL-6 and IL-
8; and chemokines such as RANTES and MIP.
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The accumulation of BRMs in a blood product during storage can
adversely ai~ect the desired biological activity of a biological composition.
Complement activation, for example, has been demonstrated to occur during
storage of platelets under standard blood bank conditions. Complement
activation
has been associated with a loss of platelet function and viability termed
"platelet
storage lesion." See, e.g., V.D. Mietic and O. Popovic, Transfusion 33(2):150-
154 (1993). The accumulation of BRMs in a stored blood products can also, for
example, adversely affect a patient that receives the blood product: the
accumulation of BRMs in platelet concentrates during storage has been
associated
with non-hemolytic febrile transfusion reactions in patients receiving
platelets.
See, e.g., N.M. Heddle, Current Opinions in Hematology 2(6):478-483 (1995).
Polyamine Derivatives
The concentration of a group of low molecular weight compounds known
as polyamine derivatives may also, for example, be reduced in a biological
composition using a device of the present invention. Polyamine derivatives are
compounds that contain multiple nitrogen atoms in a carbon backbone.
Polyethylene Glycols
Other exemplary compounds include activated polyethylene glycols
(aPEG), which may be used for the modification of the surface of cells or
materials in order to provide immunomasking properties or pacification toward
protein binding, respectively. The device may be used for the reduction of
either
the excess activated polyethylene glycol or the unreactive derivative of the
PEG
resulting from the reaction of the activated PEG with water or small
nucleophiles
such as phosphate, phosphate esters or thiols, such as glutathione. Other
compounds that may be removed include impurities in the activated PEG
preparation, which may affect the function of the blood products or make them
unsuitable for transfusion (eg. toxic compounds). Finally, small molecules
(leaving groups) such as N-Hydroxy succinimide which are released during the
reaction of the aPEG with cell surface nucleophiles may also be reduced.
Examples of compounds that may be removed by the device of the
present invention include linear or branched polyethylene glycols attached to
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activating moeities which may include cyanuryl chloride, succinimidyl esters,
oxycarbonyl imidazole derivatives, nitrophenyl carbonate derivatives, glycidyl
ether derivatives, and aldehydes.
It is to be understood that the invention is not to be limited to the exact
details of operation or exact compounds, composition, methods, or procedures
shown and described, as modifications and equivalents will be apparent to one
skilled in the art. From the above, it should be clear that the methods and
devices
can be incorporated with apheresis systems and other devices and procedures
currently used to process blood products for transfusion.
EXAMPLES
The following examples serve to illustrate certain preferred embodiments
and aspects of the present invention and are not to be construed as limiting
the
scope thereof.
In the experimental disclosure which follows, the following abbreviations
apply: eq (equivalents); M (Molar); uM (micromolar); N (Normal); mol (moles);
mmol (millimoles); umol (micromoles); nmol (nanomoles); g (grams); mg
(milligrams); ug (micrograms}; Kg (kilograms); L (liters); mL (milliliters);
uL(microliters); cm (centimeters); mm (millimeters); um (micrometers); nm
(nanometers); min. (minutes); s and sec. (seconds); J (Joules, also watt
second);
°C {degrees Centigrade); TLC (Thin Layer Chromatography); HPLC (high
pressure liquid chromatography); pHEMA and p(HEMA) (poly[2-hydroxyethyl
methacrylate]); PC(s) (platelet concentrate(s)}; PT (prothrombin time); aPTT
(activated partial thromboplastin time); TT (thrombin time); HSR (hypotonic
shock response); FDA (United States Food and Drug Administration); GMP
(good manufacturing practices); DMF (Drug Masterfiles); SPE (Solid Phase
Extraction); Aldrich (Milwaukee, WI); Asahi (Asahi Medical Co., Ltd., Tokyo,
Japan); Baker (J.T. Baker, Inc., Phillipsburg, NJ); Barnstead
(Barnstead/Thermolyne Corp., Dubuque, IA); Becton Dickinson (Becton
Dickinson Microbiology Systems; Cockeysville, MD); Bio-Rad (Bio-Rad
CA 02318508 2000-07-OS
WO 99/34914 PCT/US98/14134
Laboratories, Hercules, CA); Cerus (Cerus Corporation; Concord, CA); Chrono-
Log (Chrono-Log Corp.; Havertown, PA); Ciba-Corning (Ciba-Corning
Diagnostics Corp.; Oberlin, OH); Consolidated Plastics (Consolidated Plastics
Co., Twinsburg, OH); Dow (Dow Chemical Co.; Midland, MI); Eppendorf
(Eppendorf North America Inc., Madison, WI); Gelman (Gelman Sciences, Ann
Arbor, MI); Grace Davison (W.R. Grace & Co., Baltimore, MD); Helmer (Helmer
Labs, Noblesville, IN); Hoechst Celanese (Hoechst Celanese Corp., Charlotte,
NC); International Processing Corp. (Winchester, KY); Millipore (Milford, MA);
NIS (Nicolet, a Thermo Spectra Co., San Diego, CA); Poretics (Livermore, CA);
Purolite (Bata Cynwyd, PA); Rohm and Haas (Chauny, France); Quidel (San
Diego, CA); Saati (Stamford, CT); Scientific Polymer Products (Ontario, NY);
Sigma (Sigma Chemical Company, St. Louis, MO); Spectrum (Spectrum
Chemical Mfg. Corp., Gardenia, CA); Sterigenics (Corona, CA); Tetko, Inc.
(Depew, NY); TosoHaas (TosoHass, Montgomeryville, PA); Wallac (Wallac Inc.,
Gaithersburg, MD); West Vaco (Luke, W.Va.); YMC (YMC Inc., Wilmington,
NC); DVB (divinyl benzene); LAL (Limulus Amoebocyte Lystate); USP (United
States Pharmacopeia); EAA (ethyl-acetoacetate); EtOH (ethanol); HOAc (acetic
acid); W (watts); mW (milliwatts); NMR (Nuclear Magnetic Resonance; spectra
obtained at room temperature on a Varian Gemini 200 MHz Fourier Transform
Spectrometer); ft3/min (cubic feet per minute); m.p. (melting point); g/min
and
gpm (gallons per minute); UV (ultraviolet light); THF (tetrahydrofuran); DMEM
(Dulbecco's Modified Eagles Medium); FBS (fetal bovine serum); LB (Lucia
Broth); EDTA (ethelene diamine tetracidic acid); Phorbol Myristate Acetate
(PMA); phosphate buffered saline (PBS); AAMI (Association for the
Advancement of Medical Instruments); ISO (International Standards
Organization); EU (endotoxin units); LVI (large volume injectables); GC (gas
chromatography); M (mega-); kGy (1000 Gray = 0.1 MRad); M~ (Mohm); PAS
III (platelet additive solution III); dH20 (distilled water); IAD
(immobilization
adsorption device); SCD (sterile connection [connect] device).
One of the examples below refers to HEPES buffer. This buffer contains
8.0 g of 137 mM NaCI, 0.2 g of 2.7 mM KCI, 0.203 g of 1 mM MgCl2(6H20), 1.0
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g of 5.6 mM glucose, 1.0 g of 1 mg/ml Bovine Serum Albumin (BSA) (available
from Sigma, St. Louis, MO), and 4.8 g of 20 mM HEPES (available from Sigma,
St. Louis, MO).
EXAMPLE 1
Fiberized Resin With Amberlite~ XAD-16
This example compares both the kinetics of removal of aminopsoralens
from platelets and platelet function and morphology utilizing fiberized resin
and
devices containing non-immobilized adsorbent beads . More specifically,
fiberized resin comprising immobilized Amberlite~ XAD-16 was compared with
devices containing free (i.e., not immobilized) Amberlite~ XAD-16 HP and
Dowex XUS-43493.
Preparation Of Fiberized Resin And Adsorbent Beads
Immobilized adsorbent media containing Amberlite~ XAD-16 in a cleaned
and hydrated state (Rohm and Haas) was obtained from AQF. The fibers of
Hoechst Celanese's fiber network consisted of a polyethylene terephthalate
core
and a nylon sheath, the sheath having a lower melting temperature than the
core.
The fiberized resin was prepared by first evenly distributing the adsorbent
beads
in the fiber network. Next, the fiber network was rapidly heated causing the
polymer sheath of the fibers to melt and bond to the adsorbent beads and other
fibers, forming a cross-linked fiber network. The fiberized resin formed
contained the Amberlite~ XAD-16 at a loading of 130 g/m2 (i.e., each square
meter of fiber contained 130 g of adsorbent beads).
The fiberized resin was cut into squares (14 cm x 14 cm), and the resulting
sections contained approximately 2.5 g of dry Amberlite~ XAD-16. The
Amberlite~ XAD-16 beads were then pre-wet by soaking the fiberized resin in
30% ethanol for approximately 10 minutes. The residual ethanol was then
removed by rinsing twice in saline for 10 minutes. Alternative methods of
wetting the Amberlite~ XAD-16 and other adsorbents are also effective and are
contemplated by the present invention. It should be noted that fiberized resin
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containing other types of beads (e.g., bridged or hypercrosslinked resins like
Dowex~ XUS-43493) do not require a wetting step for effective psoralen
removal.
Amberlite~ XAD-16 HP (High Purity) beads were also obtained directly
from Rohm and Haas in a cleaned and hydrated state. No pre-wetting was
required for the loose (i. e., not immobilized) Amberlite~ XAD-16 HP beads
prior
to incorporation into a mesh pouch; however, the mass of adsorbent was
corrected
to account for the water content of the beads (2.5 g dry = 6.8 g with 62.8%
moisture). The Dowex~ XUS-43493 beads were obtained from Dow, and the dry
beads did not require wetting nor did the mass of the beads require correction
for
water. Polyester mesh pouches (7 cm x 7 cm square; 30 um openings) were then
filled with 2.5 g (dry weight) of either the loose Amberlite~ XAD-16 HP or
Dowex~ XUS-43493 beads.
The fiberized resin and adsorbent-containing pouches were sterilized by
autoclaving on "wet" cycle for 45 minutes at 121 °C. Thereafter, the
fiberized
resin and the adsorbent-containing pouches were inserted into separate,
sterile, 1-
liter PL 2410 Plastic containers (Baxter). Following insertion, the PL 2410
Plastic
containers were heat sealed in a laminar flow hood, using sterile scissors,
hemostats, and an impulse sealer.
Contacting Fiberized Resin And Adsorbent Beads With Psoralen-
Containing Platelet Concentrate (PC)
Pools of platelet concentrate were prepared by combining 2-3 units of
single donor apheresis platelets in 35% autologous plasma/65% Platelet
Additive
Solution (i.e., synthetic media). To this solution was added the aminopsoralen
4'-
(4-amino-2-oxa)butyl-4,5',8-trimethyl psoralen (S-59) in an amount to achieve
a
final concentration of 150 uM 4'-(4-amino-2-oxa)butyl-4,5',8-trimethyl
psoralen.
The resulting PC solution was divided into 300 mL units, and the units were
then
placed in PL 2410 Plastic containers (Baxter) and illuminated with 3 J/cm2 of
UVA. Following illumination, the treated PCs were transferred into the PL 2410
Plastic containers containing either fiberized resin with immobilized
Amberlite~
XAD-16, loose Amberlite~ XAD-16 HP or loose Dowex~ XUS-43493, or into an
empty PL 2410 Plastic container as a control. The PL 2410 Plastic containers
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(Baxter) were then placed on a Helmer platelet incubator at 22°C and
agitated at
approximately 70 cycles/minute.
Samples of each PC were removed at 1-hour intervals during the first 8
hours of storage for analysis by HPLC of residual 4'-{4-amino-2-oxa)butyl-
4,5',8-
trimethyl psoralen. Each sample of PC was diluted 5-fold with sample diluent
(final concentration = 35% methanol, 25 mM KH2P04, pH = 3.5) containing
trimethylpsoralen (TMP) as the internal standard. Proteins and other
macromolecules were precipitated by incubating the samples at 4°C far
30
minutes. The samples were then centrifuged and the supernatant was filtered
(0.2
IO umeter) and analyzed on a C-18 reversed phase column (YMC ODS-AM 4.6 mm
x 250 mm) by running a linear gradient from 65% solvent A (25 mM KH2P04, pH
= 3.5), 35% B (methanol) to 80% B in 20 minutes.
Platelet yield during a 5-day storage period with the fiberized resin or one
of the loose beads was monitored daily by counting platelets on a Baker System
9118 CP (Baker Instrument Co.; Allentown, PA). Blood gases and pH were
evaluated using a Ciba-Corning 238 pHBlood Gas Analyzer. In vitro platelet
function following 5 days of~contact with the fiberized resin or the device
containing free adsorbents was evaluated using assays for morphology, shape
change, hypotonic shock response, aggregation, and GMP-140 (p-selectin)
expression. Shape change, aggregation, and hypotonic shock response were
evaluated using a Lumi-Aggregometer {Chrono-Log ), while GMP-140 was
determined by flow cytometry using a Becton-Dickinson FACScan Fluorescence
Analyzer (Becton Dickinson).
Psoralen Removal and Platelet Yield and Function
FIG. 5 compares the adsorption kinetics for removal of 4'-(4-amino-2-
oxa)butyl-4,5',8-trimethyl psoralen from platelets in 35% plasma/65% synthetic
media (PAS III) with XUS-43493, XAD-16 HP, and fiberized resin containing
XAD-16. Specifically, the data indicated by the circles connected by the solid
line represents the device containing the non-immobilized adsorbent XUS-43493
(2.5 g beads; <5% moisture); the data indicated by the triangles connected by
the
dashed line represents the device containing non-immobilized XAD-16 HP {6.8 g
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WO 99/34914 PCTNS98/14134
beads; 62.8% moisture); and the data indicated by the squares connected by the
dashed line represents the fiberized resin (Hoechst fibers with XAD-16 beads
wet
in 30% ethanol; 14 cm x 14 cm). As the data in FIG. S indicate, the kinetics
of 4'-
(4-amino-2-oxa)butyl-4,S',8-trimethyl psoralen adsorption are very comparable
S for both the device containing non-immobilized adsorbents and device
containing
the fiberized resin. Thus, the fiberization process does not appear to have a
significant impact on the removal kinetics.
In addition, platelet yield and function of the fiberized resin compared to
the loose beads were studied. Specifically, the experiments of this study used
i)
6.8 g XAD-16 HP (62.8% moisture); ii) 14 cm x 14 cm fiberized XAD-16 (130 g
resin/cm2) wet in 30% ethanol; and iii) 2.S g XUS-43493 (< S% moisture).
Duplicate platelet units were prepared for the XAD-16 HP and the fiberized
resin
samples, but only a single platelet unit was prepared for the XUS-43493
sample.
The results are set forth in Table 1.
1 S As indicated in Table 1, the day-S pH and p02 values were slightly
elevated relative to the day S control for samples containing non-immobilized
beads (XAD-16 HP and XUS-43493). The experiment with the fiberized resin
had pH and p02 values which were more comparable to the control. Platelet
counts indicated a 9-22% platelet loss following S days of contact in the
control
with the fiberized media and the device containing non-immobilized adsorbents.
As set forth in Table 1, the fiberized resin gave better yields (9% loss on
day S)
and performed better in all in vitro assays when compared to device containing
non-immobilized XAD-16 or XUS-43493 adsorbent particles.
2S TABLE 1
w , ~,;.
MP::.
P'lateleiShape c 140
..
Sample' : pCUz Count''change ,sl~c~k'.14~~(%)..
pH P!px. .
.
SO
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WO 99/34914 PCT/US98/14134
sesp-: ;. :
>
~mL),.
Control6.9856 31 4.40
~
Day 0.18
0
Control6.8980 27 4.21 0.58 83 0.24 297 60.6
t t f f
3
Day 0.16 0.03 0.02
XAD-166.98110 22 3.65 0.38 70 0.29 278 53.8
t t * t
b
Hp 0.09 0.09 0.09
(-13.3%)
AD 6.9072 28 3.82 0.56 80 0.27 292 55.8
t t f t
4
AD-lb 0.15 0.12 0.04
(-9.3%)
XUS- 6.98115 20 3.27 0.37 51 0.64 271 67.0
t t ~ t
2
43493 0.05 0.28 0.10
-22.3%)
Though an understanding of the mechanism underlying the higher pH and
p02 values observed for the device containing non-immobilized XUS-43493 and
XAD-16 HP is not required in order to practice the present invention, the
higher
values are believed to be caused by a slight decrease in the metabolism of the
platelets in the presence of the device containing non-immobilized adsorbent.
By
comparison, the fiberized media consistently gave day-5 pH and p02 values
which
were more comparable to the control than the XUS-43493 or XAD-16 beads.
The day-5 platelet yields were also better for the fiberized media relative
to the XUS-43493 and XAD-16 HP adsorbent beads. The 22-28% loss which was
observed for the XUS-43493 media was observed on several occasions.
However, it should be noted that the current preferred embodiment for a device
containing non-immobilized adsorbent with XIJS-43493 involves transfer of the
platelets from this device after 8 hours of exposure; this procedure results
in < 5%
loss in platelets.
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WO 99/34914 PCTNS98/14134
The data presented in Table 1 indicates that the fiberized media gives a
higher platelet yield relative to devices containing non-immobilized
adsorbents.
Surprisingly, the fiberized media also results in better day-5 platelet
function as
indicated by pH/p02, shape change, aggregation, morphology and GMP-140.
S While an understanding of the rationale for the enhanced performance of the
fiberized media is not required to practice the present invention, several
hypotheses can be proposed. First, the fibers which are attached to the
surface of
the adsorbent beads may hinder interaction between platelets and the surface
of
the beads. Second, immobilizing the beads may prevent the beads from
interacting and eliminate mechanical effects that are detrimental to
platelets.
Third, immobilizing the beads may enhance fluid shear at the bead surface,
thereby decreasing interaction between platelets and the surface of the beads;
by
comparison, non-immobilized beads are free to flow with the fluid resulting in
low flow of fluid relative to the surface of the bead.
Platelet Loss
When reviewing the above data, it appears that there is some variability in
platelet loss from one study to the next. However, the platelet loss expressed
as a
percentage of the day-5 control count is smaller for studies where the initial
platelet count is higher. A study was performed in order to confirm whether
the
number of platelets that are lost is constant for a given area of material
available
for platelet adhesion. For this study, two platelet units were pooled and the
pool
was divided into two samples. One sample was diluted in half with 35%
autologous plasma/65% synthetic media (PAS) so that the platelet count was
half
of the other unit. The platelet mixtures were treated with 4'-(4-amino-2-
oxa)butyl-4,5',8-trimethyl psoralen + UVA and were contacted with a device
containing non-immobilized adsorbent (2.5 g XUS-43493) for 5 days under the
previously discussed standard platelet storage conditions.
The total number of platelets that was lost was virtually identical for the
two units, while the losses calculated as a percent differed greatly. Thus,
the
results indicate that total platelet loss appears to be~ essentially constant
after a
period of time; that is, while the percentage of platelet loss varies with the
initial
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CA 02318508 2000-07-OS
WO 99134914 PCT/US98114134
platelet count, the total number of platelets lost will be approximately
constant
when equilibrium is reached. Based on the results set forth in this example,
the
fiberized resin does not have a negative effect on in vitro platelet function.
EXAMPLE 2
Fiberized Resin With Activated Charcoal
This example compares the kinetics of removal of 4'-(4-amino-2-
oxa)butyl-4,5',8-trimethyl psoralen from platelets and platelet function and
morphology for fiberized resin comprising Amberlite~ XAD-16 and for fiberized
resin comprising immobilized activated charcoal.
Preparation Of Fiberized Resin
Hoechst Celanese prepared fiberized resin containing Amberlite~ XAD-16
HP (Rohm and Haas). The fiberized resin containing the Amberlite~ XAD-16
was prepared as described in the preceding example, including the 30% ethanol
wetting step. Hoechst Celanese also prepared fiberized resin containing
immobilized activated charcoal (Westvaco) at a loading of 375 g/m2 (AQF-375-
B) and 500 g/m2 (AQF-500-B). #In a preferred embodiment, the adsorbent
particles is a synthetic activated carbon, including for example, Ambersorb
and A-
Supra. Synthetic activated carbons are preferred due to their ability to
eliminate
the amount of particulate material shed from the immobilized adsorption
medium.
this fiberized resin was prepared in a method analogous to that for the
fiberized
resin containing Amberlite~ XAD-16. The composition of the fibers for each
fiberized resin was the same.
The fiberized resin was cut into squares (14 cm x 14 cm); the resulting
sections contained approximately 2.5 g of dry Amberlite~ XAD-16. Next, the
fiberized resin was sterilized by autoclaving on "wet" cycle for 45 minutes at
121°C. Thereafter, the fiberized resin were inserted into separate
sterile, 1-liter
PL 2410 Plastic containers (Baxter), and the containers were heat sealed in a
laminar flow hood, using sterile scissors, hemostats, and an impulse sealer.
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WO 99/34914 PCT/US98/14134
Contacting Fiberized Resin With Psoralen-Containing PC
Pools of platelet concentrate were prepared by combining 2-3 units of
single donor apheresis platelets in 35% autologous plasma/65% synthetic media
(PAS III). 4'-(4-amino-2-oxa)butyl-4,5',8-trimethyl psoralen was added in an
amount to achieve a final concentration of 154 uM 4'-(4-amino-2-oxa)butyl-
4,5',8-trimethyl psoralen. The resulting PC solution was divided into 300 mL
units, and the units were placed in PL 2410 Plastic containers (Baxter) and
illuminated with 3 Jlcm2 of UVA. Following illumination, the treated PCs were
transferred into the PL 2410 Plastic containers containing fiberized resin
with
either XAD-16, AQF-375-B, AQF-500-B or into an empty PL 2410 Plastic
container as a control. The PL 2410 Plastic containers were then placed on a
Helmer platelet incubator at 22°C and agitated at approximately 70
cycles/minute.
As performed in the preceding example, samples of each PC were
removed at 1-hour intervals during the first 8 hours of storage for analysis
of
residual 4'-(4-amino-2-oxa)butyl-4,5',8-trimethyl psoralen by HPLC. Each
sample of PC was diluted 5-fold with sample diluent (final concentration = 35%
methanol, 25 mM KH2POa, pH = 3.5) containing trimethylpsoralen (TMP) as the
internal standard. Proteins and other macromolecules were precipitated by
incubating the samples at 4°C for 30 minutes. The samples were then
centrifuged
and the supernatant was filtered (0.2 umeter) and analyzed on a C-18 reversed
phase column (YMC ODS-AM 4.6 mm x 250 mm) by running a linear gradient
from 65% solvent A (25 mM KH2P04, pH = 3.5), 35% B (methanol) to 80% in 20
minutes.
Platelet yield during a 5-day storage period with the fiberized resin or the
device containing non-immobilized adsorbent was monitored daily by counting
platelets on a Baker System 9118 CP. Blood gases and pH were evaluated using a
Ciba-Corning 238 pH/Blood Gas Analyzer. In vitro platelet function following 5
days of contact with the fiberized resin or the device containing non-
immobilized
adsorbent was evaluated using assays for morphology, shape change, hypotonic
shock response, aggregation, and GMP-140 (p-selectin) expression. Shape
change, aggregation, and hypotonic shock response were evaluated using a
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CA 02318508 2000-07-OS
WO 99/34914 PCT/US98/14134
Chrono-Log Lumi-Aggregometer, while GMP-140 was determined by flow
cytometry using a Becton-Dickinson FACScan Fluorescence Analyzer.
Psoralen Removal and Platelet Yield and Function
FIG. 6 compares the adsorption kinetics for removal of 4'-(4-amino-2-
oxa)butyl-4,5',8-trimethyl psoralen from platelets in 35% plasma/65% synthetic
media (PAS III) with fiberized resin containing XAD-16 and fiberized resin
with
the two different loadings of activated charcoal. Specifically, the data
indicated
by the circles represents 4'-(4-amino-2-oxa)butyl-4,5',8-trimethyl psoralen
removal with fiberized XAD-16 beads; the data indicated by the squares
represents removal with fiberized AQF-500-B; and the data indicated by the
triangles represents removal with fiberized AQF-375-B. As the data in FIG. 6
indicate, the kinetics of 4'-(4-amino-2-oxa)butyl-4,5',8-trimethyl psoralen
adsorption are very comparable for the different fiberized resin, and the
fiberized
resin all showed very good kinetics of removal (_< 0.5 uM residual 4'-(4-amino-
2-
oxa)butyl-4,5',8-trimethyl psoralen after 4 hours).
Platelet yield and in vitro platelet function for each of the fiberized resin
were also evaluated and the data are summarized in Table 2.
CA 02318508 2000-07-OS
WO 99/34914 PCT/US98/14134
TABLE 2
PlateletShape
Sample pH pOi pC0= Count ChangeAggregati
(10"/300 on
mL)
Control 7.07 59 23 3.34 1.17 90 f
t 0.13 t 5
Day 0 0.07
Control 6.88 112 21 3.09 0.52 72 t
t 0.22 t 8
Day 5 0.02
Fiberized 6.93 117 20 2.55 0.39 69 f
Resin t 0.16 t 6
(-17%) 0.13
XAD-16
Fiberized 7.07 100 20 2.83 0.74 61 f
Resin t 0.04 t 0
with (-8%) 0.05
AQF-500-B
Fiberized 6.99 107 20 2.82 0.46 65 t
Resin t 0.16 t 2
(-9%) 0.06
AQF-375-B
Refernng to Table 2, the charcoal-based fiberized resin gave good platelet
yields with losses of less than 10%; as in the studies of the preceding
example, the
fiberized resin containing XAD-16 had a slightly higher platelet loss (about
17%).
Regarding p02, the day 5 values for the charcoal fiberized resin are
comparable to
the control. Though an understanding of why the charcoal fiberized resin had
slightly elevated pH values is not required to practice the present invention,
it may
be an artifact caused by residual extractables (e.g., phosphate) from the
activation
process; the rapid rise in pH (pH = 7.3-7.4) observed after 8 hours of storage
of
the PC with the charcoal-based fiberized resin supports that idea. The use of
USP
charcoals, which are associated with fewer extractables, may eliminate the
observed initial rise in pH.
The charcoal-based fiberized resin provided good results in both the shape
change and aggregation assays. Although the shape change result for the AQF-
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500-B fiberized resin is better than that of the control, the platelets
associated with
AQF-500-B performed slightly poorer in the aggregation assay.
EXAMPLE 3
Effect of pHEMA Coating On Adsorbent Hemocompatibility
This example compares the kinetics of removal of 4'-(4-amino-2-
oxa)butyl-4,5',8-trimethyl psoralen from platelets and platelet function and
morphology for both Dowex~ XUS-43493 and fiberized resin containing
Amberlite~ XAD-16 coated with pHEMA.
Preparation Of pHEMA-Coated Adsorbent Beads And Fiberized Resin
Dowex~ XUS-43493 (commercially known as Optipore~ L493)
containing approximately 50% water by weight was obtained from Dow, and
polymerized HEMA with a viscosity average molecular weight of 300 kD was
obtained from Scientific Polymer Products. Prior to coating, the adsorbent
beads
were dried to a water content of < 5%. A stock solution of pHEMA was prepared
by dissolving the polymer in 95% denatured ethanol/5% water to achieve a
pHEMA concentration of SO mg/ml.
The coating process was performed by International Processing Corp. in a
9-inch Wurster fluidized bed coater with a charge of approximately 4 kg (dry)
of
adsorbent. The coating process involved a pHEMA flow rate of 60-70 g/min, an
inlet temperature of 50°C, and an air flow rate of approximately 200
ft3/min.
Samples (50 g) of coated adsorbent were removed during the coating process so
that coating levels ranging from 3-18% (w/w) pHEMA were obtained; adsorbent
beads coated with 3.7%, 7.3%, and 10.9% pHEMA (w/w) were used in the studies
described below.
A device containing non-immobilized dry (uncoated) Dowex~ XUS-43493
(2.5 g) and pHEMA-coated Dowex~ XUS-43493 (3.0 g or 5.0 g) were prepared
by placing the desired mass of adsorbent into a square 30 pm polyester mesh
pouch (7 cm x 7 cm). The adsorbent-filled pouches were inserted into separate
sterile, 1-liter PL 2410 Plastic containers (Baxter) and heat sealed with an
impulse
sealer. Thereafter, the adsorbent-filled pouches containing PL-2410 Plastic
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containers were sterilized by either E-beam (1VIS) or gamma irradiation
(SteriGenics) to 2.5 MRad; as previously alluded to, E-beam sterilization is
generally preferred.
Hoechst Celanese prepared fiberized resin containing Amberlite~ XAD-16
according to the method described in Example 1. The fiberized resin was cut
into
squares (14 cm x 14 cm); the resulting sections contained approximately 2.5 g
of
dry Amberlite~ XAD-16. The Amberlite~ XAD-16 of the fiberized resin was
simultaneously wet and coated with pHEMA by soaking in a solution containing
50 mg/mL pHEMA in 95% ethanol/5% distilled water. Residual ethanol was
removed by rinsing twice in saline for 10 minutes. This procedure resulted in
a
coating of approximately 6% (w/w) pHEMA. The fiberized resin was then
sterilized by autoclaving on "wet" cycle for 45 minutes at 121 °C.
Thereafter, the
fiberized resin was inserted into separate sterile, 1-liter PL 2410 Plastic
containers
(Baxter) and heat sealed in a laminar flow hood, using sterile scissors,
hemostats,
and an impulse sealer.
Contacting pHEMA-Coated Adsorbent Beads
And Fiberized Resin With Psoralen-Containing PC
Pools of platelet concentrate were prepared by combining units of single
donor apheresis platelets in 35% autologous plasma/65% synthetic media (PAS
III). 4'-(4-amino-2-oxa)butyl-4,5',8-trimethyl psoralen was added in an amount
to achieve a final concentration of 150 uM 4'-(4-amino-2-oxa)butyl-4,5',8-
trimethyl psoralen. The resulting PC solution was then divided into 300 mL
units,
and the units were placed in PL 2410 Plastic containers (Baxter) and
illuminated
with 3 J/cm2 of UVA. Following illumination, the treated PCs were transferred
into the PL 2410 Plastic containers containing the devices as indicated in the
following result sections. Control samples without an adsorption device were
also
prepared. The PL 2410 Plastic containers were then placed on a Helmer platelet
incubator at 22°C and agitated at approximately 70 cycles/minute.
Samples of each PC were removed at 1-hour intervals during the first 8
hours of storage for analysis of residual 4'-{4-amino-2-oxa)butyl-4,5',8-
trimethyl
psoralen by HPLC. Each sample of PC was diluted 5-fold with sample diluent
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(final concentration = 35% methanol, 25 mM KH2P04, pH = 3.5) containing
trimethylpsoralen (TMP) as the internal standard. Proteins and other
macromolecules were precipitated by incubating the samples at 4°C for
30
minutes. The samples were then centrifuged and the supernatant was filtered
(0.2
umeter) and analyzed on a C-18 reversed phase column (YMC ODS-AM 4.6 mm
x 250 mm) by running a linear gradient from 65% solvent A (25 mM KH2P04, pH
= 3.5), 35% B (methanol) to 80% in 20 minutes.
Platelet yield after a 5-day storage period with the fiberized resin or the
device containing non-immobilized adsorbent was determined by counting
platelets on a Baker System 9118 CP. Blood gases and pH were evaluated using a
Ciba-Corning 238 pH/Blood Gas Analyzer. In vitro platelet function following 5
days of contact with the fiberized resin or the control device containing non-
immobilized adsorbent was evaluated using assays for morphology, shape change,
hypotonic shock response, aggregation, and GMP-140 (p-selectin) expression.
Shape change, aggregation, and hypotonic shock response were evaluated using a
Chrono-Log Lumi-Aggregometer, while GMP-140 was determined by flow
cytometry using a Becton-Dickinson FACScan Fluorescence Analyzer.
Effect Of pHEMA Coating On Psoralen Removal
FIG. 7 compares the adsorption kinetics for removal of 4'-(4-amino-2-
oxa)butyl-4,5',8-trimethyl psoralen from platelets in 35% plasma/b5% synthetic
media (PAS III) with pHEMA-coated and uncoated Dowex~ XUS-43493 beads.
Specifically, 4'-(4-amino-2-oxa)butyl-4,5',8-trimethyl psoralen removal with
3.0
g Dowex~ XUS-43493 coated with 3.7% (wlw) pHEMA is represented by the
circles, with 7.3% (w/w) pHEMA is represented by the triangles, and with 10.9%
(w/w) pHEMA is represented by the diamonds; the squares represent 4'-(4-amino-
2-oxa)butyl-4,5',8-trimethyl psoralen removal with 2.5 g (dry) uncoated Dowex~
XUS-43493. As the data in FIG. 7 indicate, the kinetics of 4'-(4-amino-2-
oxa)butyl-4,5',8-trimethyl psoralen adsorption decreased as the level of pHEMA
coating was increased. While the mechanism need not be understood in order to
practice the invention, this decrease is believed to be due to an increase in
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resistance to diffusion of 4'-(4-amino-2-oxa)butyl-4,5',8-trimethyl psoralen
to the
interior of the adsorbent particles.
The adsorption kinetics of 4'-(4-amino-2-oxa)butyl-4,5',8-trimethyl
psoralen removal were also determined with 5.0 g Dowex~ XUS-43493 coated
with 3.7%, 7.3%, and 10.9% (w/w) pHEMA. The results (not shown) indicated
that the removal kinetics for the beads coated with 10.9% pHEMA were
comparable to that with the uncoated (control) beads.
Effect Of pHEMA Coating On Platelet Yield
The effect of pHEMA on platelet yield was determined in two studies
using different amounts of adsorbent (3.0 and 5.0 g) but the same levels of
pHEMA coating (3.7%, 7.3%, and 10.9% [w/w]) in each. Platelet yields were
calculated relative to the platelet count on day 5 for treated PC which was
not
contacted with a device containing non-immobilized adsorbents. As the results
in
Table 3 indicate, when 3.0 g of XUS-43493 were tested, there was a nominal
dose
response on day 5 platelet yield with increasing pHEMA coating levels; those
results suggest that a low level of pHEMA coating may be most effective since
it
has a smaller effect on 4'-(4-amino-2-oxa)butyl-4,5',8-trimethyl psoralen
removal
kinetics while still inhibiting platelet adhesion to the adsorbent surface. In
contrast to the nominal effect seen with 3.0 g of XUS-43493, a dose response
was
observed when 5.0 g were tested - increasing pHEMA coating levels did increase
the day 5 platelet yield. However, yields were still lower than those observed
when 3.0 g of adsorbent beads were used.
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TABLE 3
Polymer Coating Adsorbent Platelet
Coating Level (%) MASS (g) Yield,
Day
5 (%),
None 0 2.5 73.1
pHEMA 3.7 3.0 91.4
pHEMA 7.3 3.0 87.7
pHEMA 10.9 3.0 89.0
pHEMA 3.7 5.0 71.8
pHEMA 7.3 5.0 80.2
pHEMA 10.9 5.0 86.6
The results presented in Table 3 suggest that the use of a lower mass of
adsorbent (e.g., 2.5 - 3.0 g) along with a low level of pHEMA coating (e.g.,
<_
3.0% w/w) will provide the best platelet yield. As previously indicated, the
optimum level of pHEMA coating is the minimum coating at which a protective
effect on platelet yield and in vitro platelet function is observed.
Effect Of pHEMA Coating And Sterilization On Platelet Function
It was previously indicated that methods of sterilization may have a
substantial effect on adsorbent function since the pHEMA coating can be
crosslinked or cleaved by the radiation. In order to evaluate this effect, a
study
was performed with devices containing non-immobilized pHEMA-coated (3.7%
w/w} and uncoated XUS-43493 sterilized with either 2.5 MRad E-beam or 2.5
MRad gamma irradiation. Each device contained 2.5 g (dry) of non-immobilized
coated or uncoated XUS-43493 housed in a square 30 um polyester mesh pouch
(7 cm x 7 cm); the control comprised treated PC stored in a PL 2410 Plastic
container alone. The results are summarized in Table 4.
TABLE 4
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PlateletShape Aggre-HSR Morph GM-140
Sample pH p0~ Count Change gation oiogy Acti-
(10"/300 vatlon
I,) (%)
Control 7.0367 4.94 - - - - -
f
Day 0 0.24
Control 6.8784 4.58 0.96 76 0.35 295 70.0
f t t t
2
Day 5 0.08 0.05 0.03
)
Uncoated 6.95128 3.49 0.38 44 0.47 260 58.3
t t t t
0
XUS-43493 0.08 0.00 0.12
(-23.8%)
3.7% 6.93108 4.19 0.59 59 0.32 240 58.5
f t t t
5
pHEMA 0.09 0.03 0.04
XUS-43493 (-8.5%)
Gamma
3.7% 6.8892 4.35 0.71 69 0.40 264 54.8
f ~ t t
9
pHEMA 0.11 0.06 0.04
XUS-43493 (-5.0%)
E-beam
Referring to Table 4, the day-5 pH values appear to be very stable
compared to the control. The p02 value measured with the device containing non-
immobilized uncoated adsorbent is slightly elevated, suggesting a mild
decrease
in metabolism; coating with pHEMA appeared to reduce this effect, with the
results for the E-beam sterilized device containing non-immobilized adsorbent
closer to the control than those for the gamma sterilized device.
Platelet yields were very good for both of the pHEMA-coated samples, the
E-beam sterilized sample performing slightly better. Shape change and
aggregation exhibited a pattern similar to that for yield, with the device
containing
non-immobilized uncoated adsorbent giving the lowest values and the pHEMA-
coated/E-beam sterilized sample providing higher values similar to the
control.
The samples treated with a device containing non-immobilized adsorbents
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performed as well as or better than the control in the hypotonic shock
response
(HSR) assay. Samples that were treated with the devices containing non-
immobilized adsorbents gave lower morphology scores than the control, but
showed lower levels of activation as indicated by the GMP-140 assay.
Another hemocompatibility study was performed comparing a device
containing non-immobilized uncoated XUS-43493 (2.5 g beads; 30 um polyester
mesh pouch; 7 cm x 7 cm), uncoated fiberized resin (14 cm x 14 cm) containing
Amberlite~ XAD-16, and fiberized resin containing pHEMA-coated Amberlite~
XAD-16. The favorable effect of pHEMA on the fiberized resin is indicated by
the results presented in Table 5
TABLE 5
Platelet Shape GM-140
Sample pH p0= Count ChangeAggre HSR Morph Activ-
(10~t/300 gation ology ation
L)
(%)
Control7.11 50 3.01 t - - - - -
0.10
Day
0
Conuol 6.91 122 2.85 t 0.60 78 0.31 302 72.5
0.16 t ~ t
4
Day (-0%) 0.09 0.01
5
Uncoated7.00 151 2.11 t 0.35 55 0.57 267 68.7
0.07 f t t
4
XUS- (-26.0%) 0.03 0.03
43493
Uncoated6.94 126 2.23 t 0.52 82 0.45 297 65.3
0.13 t t f
4
Fiberized (-21.8%) 0.03 0.01
Resin
with
XAD-16
pHEMA- 6.95 120 2.44 f 0.59 86 0.46 305 63.0
0.11 t t f
5
Fiberized (-14.4%) 0.01 0.02
Resin
with
XAD-16
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As indicated by the data regarding in vitro platelet function and platelet
yield in Table 5, coating with pHEMA brought the p02 values for the fiberized
resin closer to that observed for the control. The pHEMA-coated fiberized
resin
also exhibited higher platelet yield than the uncoated fiberized resin. The
results
indicate that the pHEMA-coated fiberized resin performed better than the
uncoated XUS-43493 beads in all in vitro platelet function assays. Moreover,
the
uncoated fiberized resin performed better than the uncoated XUS-43493 beads in
most in vitro platelet function assays.
EXAMPLE 4
Effect Of Glycerol And Polyethylene Glycol On Adsorbent Capacity
This example examines the effect of glycerol and polyethylene glycol as
stabilizing agents on adsorbent capacity and kinetics of removal of
aminopsoralens from plasma. Free (i. e., not fiberized) Amberlite~ XAD-16 and
Dowex XUS-43493 adsorbent beads were used in the experiments of this
example.
Methodology
Amberlite~ XAD-16 HP (Rohm & Haas (Philadelphia, PA)) and Dowex~
XUS-43493 (Supelco, Bellefonte, PA) were dried to < 5% water in a
80°C oven.
Known masses of adsorbent were soaked in ethanol solutions containing 0-50%
glycerol, 50% PEG-200 or 50% PEG-400 (glycerol, PEG-200, and PEG-400 from
Sigma). Following a 15 minute incubation period at room temperature, the
excess
solvent was removed and the samples were dried overnight in a 80°C
oven; drying
the adsorbent at temperatures > 120°C was avoided since changes in
adsorbent
properties (e.g., pore melting) were previously observed at higher
temperatures.
After drying, adsorbent samples were weighed to determine the mass of
stabilizing agent per mass of adsorbent.
Several individual studies were performed. Control samples of "non-wet"
adsorbent and "optimally wet" adsorbent were included in the studies as
described
below. The non-wet samples of adsorbent were dried adsorbent which was not
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subjected to any pre-treatment, while the optimally wet samples of adsorbent
were
prepared by wetting the adsorbent with 30% ethanol/70% dH20. The optimally-
wet adsorbent was rinsed with dH20 to remove residual ethanol. The adsorbent
was prepared just prior to the adsorption study to assure that drying did not
occur.
Each of the adsorption studies was performed using 100% human plasma
containing 150 uM 4'-(4-amino-2-oxa)butyl-4,5',8-trimethyl psoralen spiked
with
3H-4'-(4-amino-2-oxa)butyl-4,5',8-trimethyl psoralen. Plasma (6.0 mL) was
added to vials containing adsorbent treated with different stabilizing agents.
Masses of adsorbent were corrected for glycerol or PEG content to give 0.2 g
of
adsorbent. The vials were placed on a rotator and agitated at room
temperature.
Plasma samples were removed at various times and levels of residual 3H-4'-(4-
amino-2-oxa)butyl-4,5',8-trimethyl psoralen were determined. Samples (200 uL)
were diluted in 5.0 mL of Optiphase HiSafe Liquid Scintillation Cocktail
(Wallac)
and were counted on a Wallac 1409 Liquid Scintillation Counter (Wallac).
Adsorption Capacities Of Amberlite~ XAD-16
And Dowex~ XUS-43493 Treated With Glycerol
FIG. 8 compares the effect of pre-treatment with ethanol solutions
containing various levels of glycerol on relative 4'-{4-amino-2-oxa)butyl-
4,5',8-
trimethyl psoralen adsorption capacity in 100% plasma for Amberlite~ XAD-16
and Dowex~ XUS-43493. Adsorbent samples were wet in the ethanol/glycerol
solutions for 1 S minutes prior to drying for 48 hours at 80°C. Single
measurements of adsorption capacity were made after 4 hours of contact.
Referring to FIG. 8, glycerol content shown on the x-axis is weight/volume
percent of glycerol in ethanol. Adsorption capacities shown on the y-axis are
percentages relative to the adsorption capacity of the optimally wet adsorbent
sample. The adsorption capacity of XUS-43493 is represented by the squares,
while that of XAD-16 is represented by the circles.
As the data in FIG. 8 indicate, the capacity of XAD-16 increased from
about 30% in the dry sample to over 90% in the sample wet in a 20% glycerol
solution. These results indicate that very low levels of glycerol are required
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maintaining high adsorbent capacity after drying. Control samples that were
wet
in 50% ethanol/50% dH20 (no glycerol) prior to drying demonstrated. adsorption
capacities which were similar to untreated samples that were dried. In
contrast,
the XUS-43493 samples did not show any effect of glycerol on adsorption
capacity; adsorption capacity approached 100% at all levels of glycerol. While
not critical to the practice of the present invention, this observation
supports the
hypothesis that glycerol acts to prevent the adsorbent pores from collapsing
during drying; because XUS-43493 has a highly crosslinked structure, it is not
subject to pore collapse upon drying.
Samples that were treated with glycerol appeared to be very stable to
drying. No changes were observed in adsorption capacity for samples that were
stored for 7 days in a laminar flow hood (data not shown).
In a preferred embodiment of the present invention, 2.5 g dry of adsorbent
are used for the removal of psoralen and psoralen photoproducts from each unit
of
platelets. Soaking the adsorbent in 30% glycerol/70% ethanol, followed by
drying, results in adsorbent which contains approximately 50% glycerol. A S.0
g
sample of adsorbent would therefore contain 2.5 g dry adsorbent and 2.5 g of
glycerol. Thus, a typical 300 mL unit of platelets would contain 0.8%
glycerol, a
level thought to be acceptable for transfusion.
Adsorption Capacities Of Amberlite~ XAD-16
And Dowex~ XUS-43493 Treated With Glycerol Or PEG
Additional studies were performed with the low molecular weight
polyethylene glycols PEG-200 and PEG-400, low-toxicity agents that are
nonvolatile and are soluble in ethanol and water. Samples of adsorbent were
treated for 15 minutes in 50% solutions of PEG-400, PEG-200 or glycerol in
ethanol. FIG. 9 compares the effect of the stabilizing agents on 4'-(4-amino-2-
oxa)butyl-4,5',8-trimethyl psoralen adsorption capacities with dried adsorbent
in
100% plasma for Amberlite~ XAD-16 (bottom) and Dowex~ XUS-43493 (top);
the samples that were not wet are labeled "No Tx". Adsorbent capacities are
reported as percentages relative to the capacity of optimally wet adsorbent.
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As indicated by the data in FIG. 9 and predictable based on the its
"macronet" structure, the capacity of Dowex~ XL1S-43493 was not affected by
drying ("No Tx" sample). Conversely, the Amberlite~ XAD-16 had
approximately 35% of the maximum capacity when dried. Treating XAD-16 with
glycerol, PEG-200, and PEG-400 all improved the capacity of the dried
adsorbent; the adsorbent capacities with each were all greater than 90%, with
glycerol>PEG-200>PEG-400. Though an understanding of the precise
mechanism of action is not required to practice the present invention,
differences
in capacity between the glycerol and the two PEG solutions may be caused by
decreasing penetration of the stabilizing agent with increasing molecular
weight.
That is, during the 15 minute application procedure, the glycerol (MW = 92.1)
may be able to penetrate the adsorbent pores more completely than either PEG-
200 (MW = 190-210) or PEG-400 (MW = 380-420), which diffuse more slowly
because of their larger size.
Adsorption Kinetics Of Amberlite~ XAD-16 Treated With Glycerol Or
PEG
A study was also performed to determine whether filling the pores of the
adsorbent with glycerol or PEG results in reduced adsorption kinetics. FIG. 10
compares adsorption of 4'-(4-amino-2-oxa)butyl-4,5',8-trimethyl psoralen over
a
3-hour period from 100% plasma using Amberlite~ XAD-16 wet in several
different solutions. Specifically, the data in FIG. 10 represents XAD-16 l)
wet
prior to drying with a 50% solution of glycerol (open squares connected by
solid
lines), ii) wet prior to drying with a 50% solution of PEG-400 (shaded circles
connected with dashed lines), iii) pre-wet, i.e., just prior to initiating the
study,
with 50% ethano1/50% dH20 (shaded triangles connected by dashes), and iv) not
subjected to any treatment (shaded squares connected by solid lines; "No Tx").
The data in FIG. 10 demonstrate that Amberlite~ XAD-16 samples that were wet
in 50% glycerol/SO% ethanol or SO% PEG-400/50% ethanol solutions had
adsorption kinetics which were very close to the sample that was optimally wet
in
ethanol (i.e., the sample pre-wet with ethanol). The XAD-16 sample that was
dried but not treated (No Tx) achieved only about 30% removal by 3 hours.
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The data presented in this example indicate that treating Amberlite~ XAD-
16 with stabilizing agents in the form of solutions containing 50% ethanol and
50% glycerol, PEG-200, or PEG-400 can prevent loss of adsorption capacity
associated with drying. The results obtained with these stabilizing agents
suggest
that low molecular weight wetting agents represent viable methods for
enhancing
adsorbent function.
EXAMPLE 5
Removal Of Methylene Blue From FFP
This example is directed at the ability of a variety of different polymeric
adsorbent materials to remove methylene blue from fresh frozen plasma.
The experiments of this example evaluated "free" adsorbent resin (i.e., not
incorporated into device containing non-immobilized adsorbents) and fiberized
resin. The free adsorbent resins tested were Amberlite~ XAD-16 HP (Rohm and
Haas), MN-200 (Purolite), and Dowex~ XUS-43493 (Dow Chemical Co.). The
XAD-16 HP came in a hydrated state so that no pre-treatment (i. e., no
wetting)
was necessary, and the MN-200 was also supplied in a fully hydrated state; the
XUS-43493 was dry.
Fiberized resin containing XAD-16 was prepared as generally described in
Example 1. Briefly, a 2 cm x 7 cm (i.e., 14 cm2) strip of fiberized resin
containing
130 g/m2 XAD-16 was first wet in 70% ethanol and then rinsed exhaustively in
distilled water.
A stock solution of methylene blue ( 10 mM) was prepared by dissolving
U.S.P. methylene blue (Spectrum) in distilled water. The stock solution of
methylene blue was added to a sample of 100% plasma to give a final
concentration of 10 uM. Samples of the "free" adsorbent resin (i.e., XAD-16
HP,
MN-200, and XUS-43493) were weighed into 50 mL polypropylene tubes for
adsorption studies. The water content of each adsorbent was determined by
measuring mass loss upon drying. The mass of each adsorbent was corrected for
water content so that the equivalent of 0.25 g dry adsorbent was used for
each.
A 30 mL sample of the 100% plasma containing 10 uM methylene blue
was added to each vial. The vials were placed on a rotator at room
temperature.
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Samples (200 uL) were removed from each vial at 15 minute intervals and
assayed for residual methylene blue by HPLC. Each sample of plasma was
diluted 5-fold with sample diluent (final concentration = 35% methanol, 25 mM
KH2PO4, pH = 3.5). Proteins and other macromolecules were precipitated by
incubating the samples at 4°C for 30 minutes. Samples were centrifuged
and the
supernatant was filtered (0.2 um) and analyzed on a C-18 reversed phase column
(YMC ODS-AM, 4.6 mm x 250 mm) by running a linear gradient from 65%
solvent A (25 mM KH2P04, pH = 3.5), 35% B (Methanol) to 80% B in 20
minutes. The limit of detection for the HPLC assay was approximately 0.5 uM
methylene blue.
FIG. 11 compares the kinetics of adsorption of methylene blue over a 2-
hour period from 100% plasma. Referring to FIG. 11, XAD-16 HP data is
represented by open diamonds connected by dashed lines, the MN-200 data is
represented by shaded triangles connected by solid lines, the XUS-43493 data
is
represented by open circles connected by dashed lines, and the fiberized resin
containing XAD-16 is represented by shaded squares connected by solid lines.
As
the data indicate, the XAD-16 HP and MN-200 gave the fastest adsorption
kinetics, followed by XUS-43493. The slightly slower kinetics of the XIJS-
43493
may be a result of slower wetting, as it was used in the dry state. Finally,
the
fiberized resin containing XAD-16 had the slowest adsorption kinetics. This
may
have resulted from poor contacting between the fiberized resin and plasma
during
the batch incubation, as a portion of the 14 cm2 strip of fiberized resin was
not
completely submersed in the plasma throughout the adsorption study, thereby
reducing the effective contact area between the adsorbent and plasma.
The data indicate that non-psoralen pathogen-inactivating compounds like
the phenothiazine dyes can be removed from blood products using the resins and
fiberized resin contemplated for use with the present invention.
EXAMPLE 6
Removal Of Acridine Compounds From Packed Red Blood Cells
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This example is directed at the ability of a variety of different resin
materials to remove acridine compounds from packed red blood cells (PRBCs).
More specifically, the experiments of this example evaluate the removal of the
acridine compound, 5-[([i-carboxyethyl)amino]acridine, from PRBCs.
The chemical structures of several acridines are depicted in FIG. 12. As
indicated in FIG. 12, 9-amino acridine and S-[([i-carboxyethyl)amino]acridine
are
aminoacridines.
Resin Selectivity
Equilibrium adsorption of compound 5-[((3-carboxyethyl)amino]acridine
was studied with several types of resins. The polymeric adsorbent resins
evaluated were Amberlite~ XAD-2, XAD-4, XAD-7, and XAD-16 HP (Rohm and
Haas); Purolite~ MN-150, MN-170, MN-200, MN-300, MN-400, MN-500, and
MN-600; and Dowex~ XUS-43493 and XUS-40285 (Dow Chemical Co.). In
addition, several Amberlite~ anion exchange resins (IRA-958, IRA-900, IRA-35,
IRA-410 and IRA-120; Rohm and Haas) and an Amberlite~ weak cation
exchange resin (DP-1; Rohm and Haas) were tested. Moreover, several charcoals
were evaluated, including Hemosorba~ AC (Asahi), PICA 6277 and Norit A
Supra (both commercially available from American Norit). Finally, Porapak~
RDx (Waters), a styrene vinyl pyrrolidone copolymer which has affinity for
vitro
aromatic compounds, was also tested.
Initially, an equilibrium adsorption study was performed with samples of
each resin to evaluate capacity for 5-[([i-carboxyethyl)amino]acridine and
adenine
(6-aminopurine). Approximately 0.1 g of resin was weighed and transferred into
a 6 mL polypropylene tube. A 5.0 mL aliquot of 25% plasma/75% Adsol~
(Baxter) containing 100 uM of 5-[([i-carboxyethyl)amino]acridine in distilled
water was then added to each tube. Cellular products such as red blood cells
are
typically stored in a medium containing a low percentage of plasma (10-35%)
with a balance of synthetic media; Adsol~ is one example of a synthetic media
that consists of adenine, dextrose, and mannitol in a saline solution. Of
course,
the present invention contemplates the use of concentrations of acridines
other
CA 02318508 2000-07-OS
WO 99/34914 PCT/US98/14134
than 100 uM in mixtures of plasma and other synthetic media. Next, the tubes
were placed on a tumbling agitator and incubated for 3 hours at room
temperature.
Following incubation, aliquots of each sample were removed for analysis of
residual 5-[([i-carboxyethyl)amino]acridine and adenine by HPLC.
For the HPLC procedure, each sample was diluted 2-fold with sample
diluent (50% methanol, 25 mM KH2P04, pH = 3.5), and proteins and other
macromolecules were precipitated by incubating the samples at 4°C for
30
minutes. The samples were then centrifuged and the supernatant was filtered
(0.2
umeter) and analyzed on a C-18 reversed phase column (YMC ODS-AM 4:6 mm
x 250 mm) by running a linear gradient from 75% solvent A (25 mM KH2P04, pH
= 3.5), 25% B (methanol) to 80% B ~n 20 minutes. For 5-[((3-
carboxyethyl)amino]acridine removal, estimated capacities (umole/g) at Cf= 1
uM were determined from single adsorption measurements with C° = 100
uM; the
results are set forth in the second column of Table 6 (ND = not detectable).
For
adenine removal, estimated capacities (mmole/g) at Cf= 1 mM were estimated
from single adsorption measurements with C° = i .5 mM. The results are
set forth
in the third column of Table 6 (ND = not detectable).
TABLE 6
Estimated 5-[((3- Adenine Capacity
carboxyethyl)amino]acridi(mmole/g)
esin ne Capacity (umole/g)at Cf = 1 mM
at Cf = 1 uM
XAD-2 0.1 0.00
XAD-4 3.2 0.02
XAD-7 0.1 0.01
XAD-16HP 4.3 0.03
XUS-43493 17.5 0.46
XUS-40285 6.8 0.27
MN-1 SO I 1.6 I 0.24
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WO 99/34914 PCT/US98/14134
MN-170 4.2 0.43
MN-200 8.9 0.46
MN-300 5.1 0.33
MN-400 4.4 0.22
MN-500 8.0 1.17
MN-600 5.8 0,36
IRA-958 0.0 0.00
IRA-900 0.0 0.01
DP-1 0.0 0.00
IRA-35 0.0 0.01
IRA-120 4.7 0.41
Hemosorba AC ND ND
PICA 6277 5.0 ND
Norit A Supra ND
Porapak RDx 0.1 0.01
IRA-410-D 0.0 0.00
While the use of any resin capable of adsorbing acridine compounds is
contemplated, preferred resins selectively adsorb 5-[((3-
carboxyethyl)amino]acridine over adenine and exhibit low hemolysis. FIG. 13
plots the data for adenine capacity (y-axis) and 5-[([3-
carboxyethyl)amino]acridine
capacity (x-axis) for various resins. As indicated by the data in Table 6 and
FIG.
13, the Dowex°~ XUS-43493 and Purolite~ MN-200 resins had the highest S-
[([i-
carboxyethyl)amino]acridine capacity; moreover, when both high 5-[((3-
carboxyethyl)amino]acridine capacity and low adenine capacity were considered,
the Amberlite~ XAD-16 HP performed well.
The results from the in vitro experiments described in this example
suggest that Dowex~ XUS-43493 and the related resin Purolite~' MN-200 are
preferred resins for the removal of acridine compounds from PRBCs.
72
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EXAMPLE 7
Removal Of Acridine Compounds From Packed Red
Blood Cells With Styrene-divinylbenzene Adsorbents
This example is directed at the ability of a variety of different styrene-
divinylbenzene (styrene-DVB) adsorbents to remove aminoacridine compounds
from blood preparations. More specifically, the experiments of this example
evaluate the removal of acridine orange and 9-amino acridine (depicted in FIG.
12) from a plasma/Adsol~ solution.
A. Experimental Procedures
For the experiments of this example, stock solutions ( 10 mM) of 5-[([3-
carboxyethyl)amino]acridine (Cerus) and acridine orange (Aldrich) were
prepared
in distilled water, and a stock solution (10 mM) of 9-amino acridine (Aldrich)
was
prepared in ethanol. The 5-[([i-carboxyethyl)amino]acridine was added to a
solution containing 25% plasma/75% saline to achieve a final concentration of
100 uM, while the acridine orange and the 9-amino acridine compounds were
added to solutions containing 25% plasma/75% Adsol'~ (Baxter) Red Cell
Preservation Solution to achieve a final acridine concentration of 100 uM.
The adsorbents utilized were Amberlite~ XAD-16 HP (Rohm and Haas);
Purolite~ MN-200, and Dowex~ XUS-43493 (Supelco). The water content of
each adsorbent was determined by measuring the mass loss upon drying; the
water
content was corrected for so that the equivalent of 0.25 g dry of each
adsorbent
was used. Adsorbents were accurately weighed into 50 mL Falcon tubes. Thirty
(30) mL of the 25% plasma/75% Adsol~ solution containing 100 uM acridine was
added to each tube containing adsorbent. The tubes were then placed on a
rotator
at room temperature, and 500 uL samples of solution were removed at various
times and stored for later analysis.
Samples were analyzed by HPLC for levels of residual acridine. Each
sample was diluted 2-fold with sample diluent (50% methanol, 25 mM KH2P04,
pH = 3.5), and proteins and other macromolecules were precipitated by
incubating
73
CA 02318508 2000-07-OS
WO 99/34914 PC'r/US98/14134
the samples at 4°C for 30 minutes. The samples were then centrifuged
and the
supernatant was filtered (0.2 umeter) and analyzed on a C-18 reversed phase
column (YMC ODS-AM 4.6 mm x 2S0 mm) by running a linear gradient from
7S% solvent A (2S mM KH2P04, pH = 3.S), 2S% B (methanol) to 80% B in 20
S minutes. Detection was by visible absorbance using a diode array detector
set at
400 nm for 9-amino acridine, 490 nm for acridine orange, and 410 nm for S-[((3-
carboxyethyl)amino]acridine.
B. Adsorption Kinetics
The kinetics of adsorption of S-[([i-carboxyethyl)amino]acridine over a 3-
hour period from 2S% plasma/7S% saline were compared for Dowex~ XUS-
43493, Purolite~ MN-200, and Amberlite~ XAD-16 HP. FIG. 14A and FIG. 14B
both represent residual S-[([i-carboxyethyl)amino]acridine as a function of
time,
FIG. 14B presenting the data on a logarithmic scale. In FIG. 14A and FIG. 14B,
the XAD-16 HP data is represented by shaded circles connected by dashed lines,
1 S the MN-200 data is represented by shaded squares connected by dashed
lines, and
the XUS-43493 data is represented by shaded triangles connected by solid
lines.
As the data indicate, the XUS-43493 and MN-200 gave the fastest adsorption
kinetics and were nearly equivalent. The XAD-16 HP appears to have a lower
capacity for S-[((3-carboxyethyl)amino]acridine.
FIG. 1 S compares the adsorption kinetics for removal of 9-amino acridine
and acridine orange from 2S% plasma/7S% Adsol~ with Dowex~ XUS-43493.
Referring to FIG. 1 S, the shaded squares with the dashed lines represent 9-
amino
acridine, and the shaded circles with the solid lines represent acridine
orange. As
the data indicate, levels of 9-amino acridine were undetectable beyond 3
hours.
2S By comparison, the capacity of the Dowex~ XUS-43493 for acridine orange was
lower, which may be related to the presence of two tertiary amino groups on
acridine orange.
74
CA 02318508 2000-07-OS
WO 99134914 PCT/US98/14134
EXAMPLE 8
Preparation of PRBCs Treated With a 5-((~carboxyethyl)aminoJacridine
derivative and Glutathione. Pools of PRBC were prepared from fresh ABO-
matched whole blood. The units of whole blood were centrifuged at 3800 rpm for
5 minutes using a Beckman Sorvall RC3B centrifuge. The plasma was expressed
into another clean bag using an expresser. The units of PRBC were pooled into
a
3.0 L size Clintec Viaflex bag if more than one unit was needed. For each unit
of
PRBC, 94 mL of Erythrosol was added. The percentage of Hematocrit (HCT)
was measured by filling a capillary tubing with the blood sample and spinning
it
for 5 minutes. The hematocrit was determined to ensure that it did not
decrease
below 55%. For each 100 mL of PRBC, 3.3 mL of 12% glucose was added. The
final percentage hematocrit was determined. PRBC (300 mL) was refilled into
plastic containers PL 146 (Baxter Healthcare). To the blood bags was added 6.0
mL of 150 mM glutathione to reach a 3.0 mM final concentration and 3.0 mL of
30 mM a 5-[((3-carboxyethyl)amino]acridine derivative for a final
concentration
of 300 p.M. The PRBC mixture was agitated for 1 minute using a wrist action
shaker (manufacturer). The S-[([3-carboxyethyl)amino]acridine derivative and
glutathione treated PRBCs were allowed to incubate at room temperature
overnight to allow break down of the 5-[((3-carboxyethyl)amino]acridine
derivative into 5-[((3-carboxyethyl)amino]acridine.
EXAMPT.R 9
HPLC Assay for 5-((J3-carboxyethyl)aminoJacridine in PRBC and PRBC
Supernatant. The sample (100 p,L) was diluted with 100 pL of saline and the
resulting mixture was vortexed. To the solution was added 300 pL of 20 mM
H3P04 in CH3CN, and the mixture was vortexed for 15 sec. The sample was
centrifuged at 13,200 rpm for 5 minutes. The supernatant (200 p,L) was diluted
into 800 pL of cold 0.1 M HCl and vortexed. The sample was filtered into an
autosampler vial using a 0.2 ~Cm Gelman Acrodisc filter. The HPLC conditions
CA 02318508 2000-07-OS
WO 99/34914 PCT/US98/14134
were as follows: (Manufacturer and part no. for column and general column.)
Column = Zorbax SB-CN, 4.6 mm x 1 SO mm, 3.5 pm particles; guard column =
(4.6 mm x. I2.5 mm, 5 ~.m particles (Mac Mod Analytical, lnc. (Chadds Ford,
PA)); the mobile phase for A was 10 mM H3P04 in HPLC water; the mobile
phase for C was 10 mM H3P04 in acetonitrile; temperature was 20 °C;
sample
volume was 100 ~,L; gradient conditions were as follows:
Time A (%) C (%) Flow Rate
(mL /
min)
0.00 90.0 10.0 1.0
5.28 77.0 23.0 1.0
10.00 40.0 60.0 1.0
11.00 90.0 10.0 1.0
16.00 STOP STOP STOP
the DAD settings were as follows:
Detection DetectionReference Reference
Signal WavelengthBandwidthWavelengthBandwidth
A 410 5 580 20
B 260 5 580 20
EXAMPLE 10
HPLC Assay for Glutathione in PRBC and PRBC Supernatant. The
sample was prepared as for the HPLC assay described above. The HPLC
conditions were as follows: analytical column = YMC ODS-AM-303, 250 mm x
1 S 4.6 mm, 5 ~m particle; guard column = Brownlee, 15 x 3.2 mm, 7 pm
particle;
the mobile phase for A was 10 mM H3P04 in HPLC water; the mobile phase for C
was 10 mM H3P04 in acetonitrile; the temperature was 15°C; the sample
volume
was 75 ~.L; the gradient conditions were as follows:
Flow Rate
Time A (%) C (%) (mL / min)
0.00 95.0 5.0 0.5~
6.00 95.0 5.0 0.5
76
CA 02318508 2000-07-OS
WO 99/34914 PGT/US98/14134
8.00 10.0 90.0 1.0
~ ~
9.00 95.0 S.0 1.0
'
20.00 STOP STOP STOP
the DAD settings were as follows:
Detection Detection Reference Reference
Signal Wavelength Bandwidth WavelengthBandwidth
A 205 10 600 100
S EXAMPLE 11
Method of'ScreeningAdsorbents. A test solution containing 25% plasma
and 75% Erythrosol was used to represent the supernatant from PRBCs.
Erythrosol was prepared as two separate stock solution parts (solution C and
solution D) that were sterilized separately. The final solutions were prepared
by
mixing equal volumes of solution C and solution D.
Solution C Solution D
3.2 mM Adenine 53.2 mM Na citrate 2 H20
85 mM mannitol 5.4 mM NaH2P0, 2 Hz0
100 mM glucose 38 mM NazHPO, 2 HZO
6.2 mM HCl
The plasma-Erythrosol solution was spiked with 5-[(~i-
carboxyethyl)amino)acridine to a final concentration of 300 p.M. Glutathione
(reduced form, Sigma Chemical Co.) was added to the mixture to obtain a final
concentration of 3 mM. Samples of adsorbents (0.2-0.8 g) were accurately
weighed (~ 0.001 g) into fared 7 mL polypropylene vials with screw tops.
Samples of adsorbent that required pre-wetting were suspended in 70% ethanol.
The adsorbent was allowed to settle and the supernatant was removed. Residual
ethanol was removed by resuspending the adsorbent in distilled water, allowing
the adsorbent to settle, and decanting the supernatant. Adsorbent masses were
77
CA 02318508 2000-07-OS
WO 99/34914 PCT/U598/14134
corrected for water content when adsorption capacities were calculated. A 5.0
mL
aliquot of plasma/Erythrosol was added to each tube. The tubes were placed on
a
rotator and tumbled at room temperature for 24 hours. The vials were removed
from the rotator and the adsorbent was allowed to settle. A sample of the
supernatant was removed from each tube. Samples were centrifuged to remove
residual adsorbent. Samples were analyzed by HPLC to determine residual levels
of 5-[((3-carboxyethyl)-amino]acridine. The reversed phase assay described
above
was used to determine residual levels of glutathione. The results from the
adsorbent screen are shown in Tables 7 and 8.
Table 7.
Adsorbent Screen - Removal of 5-[((3-carboxyethyl)amino)acridine from 25%
Plasma/75% Erythrosol. ("LOD" is limit of detection.)
Purolite Weak base,0.108 No 1.4 13.5 9.7
MN-150 PS-
DVB
Purolite Weak base,0.105 No 0.4 14.0 38.4
MN-170 PS-
DVB
Purolite Nonfunctional,0.077 No 0.9 19.1 21.5
MN-200
PS-DVB
Purolite Weak Base,0.111 No 1.0 13.2 13.4
MN-300 PS-
DVB
Purolite Strong 0.102 No 1.1 14.3 13.0
MN-400 base,
PS-
DVB
Purolite Strong 0.098 No 0.6 15.0 24.2
MN-500 acid,
PS-
DVB
Purolite Weak acid,0.112 No 0.8 13.1 16.7
MN-600 PS-
DVB
Dowex OptiporeNonfunctional0.189 No 0.3 7.8 2 2.5
L-
493 PS-DVB
46 A, 1100
m'/g
78
CA 02318508 2000-07-OS
WO 99/34914 PCT/US98/14134
Dowex Optipore L- Weak base, No 0.5 6.0 11.2
PS- 0.245
285 DVB
25 A, 800 m=/g
Amberlite XAD-2 NonfunctionalYes 117.5 4.7 0.0
0.190
PS-DVB
90 A, 300 ms/g
Amberlite XAD-4 Nonfunctional
0.118 Yes 1.6 12.4 7.7
PS-DVB
40 A, 725 m=/g
Amberlite XAD-7 Polyacrylic
ester
90 A, 450 m'/g 0.097 Yes 117.4 9.1 0.1
Amberlite XAD-16 Nonfunct.
0,101 Yes 44.1 12.4 0.3
polystyrene
100 A, 800 m~/g
Hemosorba AC Activated
0,149 No LOD > 9.8 >
charcoal 196.8
pHBMA-coated
Duolite GT-73 Thiol-containing
0.144 No 1.0 10.2 10.2
macroreticular
Kieselguhr Diatomaceous
0.131 No 317.7 -0.9 0.0
earth
Graver GL-711 Ground XUS-
0,134 No 1.9 10.9 5.8
43493 attached to
fiber
Amberlite IRA 900 Quat. Am.
p,067 No 239.1 4.1 0.0
macroreticular
PS, CI-form
Amberlite IRA 35 Weak base
O,p76 No 223.7 4.6 0.0
modified PS
Amberlite Strong base gel
0_086 No 127.2 9.7 0.1
IRA 410 D PS
CI-form
Amberlite IRA 958 Quat. Am.
0.084 No 304.9 -0.6 0.0
macroreticular
Polystyrene, CI-
form
Amberlite DP-1
Carboxylic
- ~ No 292.8 0.1 0.0
Ip,066 ~
macroreticular PS
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CA 02318508 2000-07-OS
WO 99/34914 PCT/US98/14134
Duolite S-761 Phenol- -
0,093 No 1.8 15.8 8.6
formaldehyde
modified PS
Duolite A-7 Polyamine
0.079 No 189.0 6.6 0.0
macroreticular PS,
free base
Amberlite IRA-68 Polyamine
gel PS
0,083 No 203.4 5.5 0.0
Amberlite IRA-458 Quat. Am.
gel PS,
0.148 No 292.8 0.0 0.0
CI-form
Amberlite IRA-958 Quat. Am.
0,139 No 296.3 -0.1 0.0
macroreticular PS,
Cl-form
Ambersorb 563 Synthetic AC,
550 N
0
228
. o 0.4 6.4 17.5
mx/g, high
hydrophobicity
Ambersorb 572 Synthetic AC,
1100 m'/g, low 0.267 No LOD > 5.5 > 55.0
hydrophobicity
Ambersorb 575 Synthetic AC,
800 N
0
258
. o LOD > 5.7 > 56.9
m'/g, mid
hydrophobicity
PICA 6277 AC Activated charcoal
0.283 No LOD > 5.2 > 51.9
PICA NC506 AC Activated charcoal
0
266
_ No LOD > S.5 > 55.3
PICAtif Med. AC Activated charcoal
0.280 No LOD > 5.3 > 52.5
West VACO CX-S Activated charcoal
0.228 No LOD > 6.4 > 64.4
Norit ASupra Activated charcoalN
0
268
, o LOD > 5.5 > 54.$
Norti B Supra Activated charcoalN
0
258
. o LOD > 5.7 > 56.9
Norit Supra E Activated charcoalN
0
266
. o LOD > 5.5 > 55.2
Norit S51 AC Activated charcoal
0.237 No LOD > 6.2 > 61.9
Norit SX Ultra Activated charcoal
0
203
. No LOD > 7.2 > 72.2
Chemviron Activated charcoal N
0
233
. o LOD > 6.3 > 63.1
Norit CN 1 Activated charcoal N
0
261
. o LOD > 5.6 > 56.4
Norit G60 Activated charcoal N
0
230
. o LOD > 6.4 > 64.0
Norit ROX,0,8 Activated charcoalN
0
246
. o LOD > 6.0 > 59.8
Norti Darco Activated charcoalN
0
219
. o LOD > 6.7 > 67.3
(20x50)
PICAtif Medicinal Activated
charcoal
0.129 No LOD > 11.4> 113.8
CA 02318508 2000-07-OS
WO 99/34914 PCT/US98/14134
Davison SilicaUnmodified
silica
0,245No 77.0 4.4 0.1
Grade 15
Davison SilicaUnmodified
silica
0,255No 140.1 3.0 0.0
Grade 636 -
BioRad AG501-Mixed bed
ion
X8(D) exchange 0.083No 119.4 10.5 0.1
Amberlite Sulfonic
200 acid
p,080No 46.5 15.5 0.3
macroret.
PS, Na-
form
MP-3 (C-18) Sulfonated
SPE C-18
0,239Yes 1.7 6. 3.7
media resin I
Alltech C-18C-18 modified
SPE
p_234Yes 64.4 4.9 0.1
silica
BioRad t-ButylC-4 modified
0,213Yes 163.9 3.1 0.0
HIC polymethacrylate
Baker C-18 C-18 modified
SPE
p.263Yes 94.9 3.8 0.0
silica
Waters Sep C-18 modified
Pak C-
p,190Yes 81.3 5.6 0.1
18 silica
Baker C-4 C-4 modified
SPE
0,232Yes 178.3 2.5 0.0
silica
Waters BondapakC-8 modified3
0,21 Yes 156.9 3.2 0.0
C-8 silica
Waters BondapakC-4 modified
p,228Yes 251.0 0.9 0.0
C-4 silica
Amberchrom Polystyrene
cg-
161 xcd 150 A, 900 0.192Yes 79.3 5.6 0.1
m~/g
Amberchrom Polystyrene
cg-
1000 sd 1000 A, 250 0-176Yes 43.7 1.4 0.0
m'/g 2
Amberchrom Polystyrene
cg-
300 and 300 A, 700 0.135Yes 172.1 4.5 0.0
m=/g
Amberchrom Polymethacrylate
cg-71
and 250 A, 500 0.189Yes 56.4 .6 0.0
m~/g 1 3
Waters PorapakPS-vinyl
0-125Yes 07.8 0.6 0.0
RDx pyrrolidone 3 -
porous adsorbent
CUNO DelipidResin-modified
0 ,663 No 2 45.8 .4 0.0
Media cellulose 0
CUNO Weak base
0 .361 No 2 80.8 .2 0.0
DEAF media modified 0
cellulose
Sigma DE Diatomaceous
0 ,179 No 3 17.6 0.7 0.0
earth -
Diaion SP-850Polystyrene
38 A, 1000 .236 Yes .4 6 .2 .4
m'/g 0 1 4
Diaion SP-207Brominated
PS
105 A, 650 .191 No 2 11.1 .2 0.0
m'/g 0 2
Diaion HP-2MGPolymethacrylate
170 A, 500 .258 Yes 82.6 .2 .0
m~/g 0 1 2 0
Table 7. (continued)
81
CA 02318508 2000-07-OS
WO 99/34914 PCT/US98/14134
i
i
Diaion IMP=20 Polystyrene
260 A, 500 m'/g 0. i 75 Yes 107.4 5.3 0.0
Amberlite 1180 Polystyrene-DVB
300 A, 600 m~/g 0.094 Yes 9.9 15.1 1.5
Amberilte 1600 Polystyrene-17VB
0.208 Yes 73.0 5.3 0.1
Amberlite XAD- Polystyrene-DVB
2000 42 A, 580 m'/g 0.172 Yes 61.0 6.8 0.1
Amberlite XAD- Polystyrene-DVB
0_203 Yes 173.0 3.0 0.0
2010 280 A, 660 m~/g
Dowex XUS- Polystyrene-DVB
40323 100 A, 650 m'/g 0.213 Yes 102.6 4.5 0.0
Whatman DE-52 Weak base
p.284 No 177.5 2.0 0.0
modified
cellulose
Whatman CM-32 Weak acid
p.294 No 268.7 0.4 0.0
modified
cellulose
Whatman QA-52 Strong base
p.2~ No 285.2 0.2 0.0
modified
cellulose
Whatman SE-53 Strong acid
p,238 No 294.3 0.0 0.0
modified
cellulose
Pharmacia Q Seph Strong base
p.1 11 No 267.8 1.2 0.0
FF modified agarose
Pharmacia S Seph Strong acid
p,112 No 267.0 1.2 0.0
FF modified agarose
Toyopearl QAE- Wead base
p, l 18 No 66.8 1.2 0.0
550 C modified agarose 2
Toyopearl Butyl Hydrophobic
(C-
0.112 Yes 32.8 2.7 0.0
650-M 4) modified 2
methacrylate
Toyopearl SP- Strong acid
p.1 1 I No 30.2 .9 0.0
SSOC modified 2 2
methacrylate
Toyopearl CM- Weak acid
p_ 118 No 49.0 .9 0.0
650M modified 2 1
methacrylate
Toyopearl Weak base
p,112 No 60.4 .5 0.0
DEAE-650M modified 2 1
methacrylate
Toyopearl Super Q Strong base
p, l l l N o 2 63.2 .4 .0
650C modified 1 0
methacrylate
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CA 02318508 2000-07-OS
WO 99/34914 PCT/US98/14134
Table 8
Adsorbent Screen - Removal of Glutathione from 25% Plasma/75% Erythrosol
y,
Purolite MN-150336.1 ~~~~ . 2 . .
Purolite MN-170386.7 2732 12.8 0.1
Purolite MN-200445.5 3147 -9.6 -0.1
-
Purolite MN-300356.7 2520 21.7 0.3
Purolite MN-400297.0 2099 44.1 0.6
Purolite MN-500392.8 2775 11.5 0.1
Purolite MN-600392.4 2773 10.1 0.1
Dowex XUS-43493428.5 3027 -0.7 0.0
Dowex XUS-40285251.6 1778 24.9 0.4
Amberlite XAD-2420.8 2973 0.7 0.0
Amberlite XAD-4449.0 3172 -7.3 -0.1
-
Amberlite XAD-7448.0 3165 -8.5 -0.1
-
Amberlite XAD-16412.2 2912 4.4 0.0
Hemosorba AC 7.9 56 98.6 53.3
Duolite GT-73 379.3 2680 11.2 0.1
Kieselguhr 471.3 3330 - 12.6 - 0.1
Graver GL-711 466.2 3294 - 11.0 - 0.1
Amberlite IRA 441.1 3 116 - 8.6 - 0.1
900 434.4 - 069 - 4.5 0.0
Amberlite IRA 3
35
Amberlite 1RA 413.0 2 918 4 ,8 0,0
410 D
Amberlite IRA 446.2 3 152 - 9.1 - 0.1
958
Amberlite DP-1433.3 3 061 - 4.6 0 .0
-
Amberlite IRA-12088.6 745 1 6.2 0 .2
3 2
Diaion HPA 47.3 3 160 - 8.8 - 0.1
75 4
Duolite S-761 12.9 2 211 4 2.6 0 .6
3
Table 8. lcontinuedl
83
CA 02318508 2000-07-OS
WO 99/34914 PCT/US98/14134
I,
i
MP-3 28.0 3024 8.~ .-.....
. _
Alltech C-18 411.3 2906 2.0 0.0
SPE
Duolite A-7 444.2 3138 -8.7 -0.1
Amb IRA-68 443.5 3133 -8.1 -0.1
Amb IRA-458 460.6 3254 -8.6 -0.1
Amb IRA-958 426.3 3012 -0.4 0.0
Ambersorb 563 435.2 3075 -1.6 0.0
Ambersorb 572 0.0 LOD >5b. l > I 67.9
Ambersorb 575 0.0 LOD >58.1 >173.8
PICA 6277 AC 0.0 LOD >53.0 >158.4
PICA NC506 AC 0.0 LOD >56.4 >168.7
PICAtif Med. 0.0 LOD >53.6 > 160.4
AC
West VACO CX-S 0.0 LOD >65.7 >196.5
Norit A Supra 0.0 LOD >55.9 - >167.2
Norti B Supra 0.0 LOD >58.1 >173.7
Norit Supra 0.0 LOD >56.3 > 168.5
E
Norit S51 AC 0.0 LOD >63.2 > 189.0
Norit SX Ultra 0.0 LOD >73.7 >220.5
Chemviron 0.0 LOD >64.4 >192.6
Norit CNl 0.0 LOD >57.5 >172.1
Norit G60 0.0 LOD >65.3 >195.4
Norit ROX,0,8 0.0 LOD >61.0 > 182.4
Norti Darco 0.0 LOD >68.6 - >205.3
(20x50)
PICAtif Medicinal0.0 LOD > 116.2 >347.4
Whatman 150A 448.8 3171 - 3.6 0.0
Silica
Davison Silica 443.5 3133 - 2.7 0.0
Grade
15
Davison Silica 423.9 2995 0.1 0.0
Grade
636
1 able 1S. (contlnuea)
84
CA 02318508 2000-07-OS
WO 99/34914 PG"T/IJS98/14134
i
I _
io d' AG 0 - 45.6 148 .
(D)
Amberlite 200 458.1 3237 -14.8 -0.1
BioRad t-Butyl 484.0 3419 -9.8 -0.1
HIC
Baker C-18 SPE 427.7 3022 -0.4 0.0
Waters Sep Pak 422.2 2983 0.4 0.0
C-18
Baker C-4 SPE 429.8 3036 -0.8 0.0
Waters Bondapak 405.5 2865 3.2 0.0
C-8
Waters Bondapak 390.8 2761 5.2 0.1
C-4
Amberchrom cg-161414.8 2931 1.8 0.0
xcd
Amberchrom cg-1000373.5 2639 10.2 0.1
sd
Amberchrom cg-300396.2 2799 7.4 0.1
and
Amberchrom cg-71396.4 2800 5.3 0.1
and
Waters Porapak 449.9 3179 - 7.1 - 0.1
RDx
CUNO Delipid 452.5 3197 - 1.5 0.0
Media
CUNO DEAE media 213.7 1510 20.6 0.4
Sigma Diatomaceous473.9 3348 - 9.7 - 0.1
Earth
Dision SP-850 424.1 2996 0.1 0.0
Diaion SP-207 471.6 3332 - 8.7 - 0.1
Diaion HP-2MG 429.7 3036 - 0.7 0.0
Diaion HP-20 422.3 2984 0.5 0.0
Amb 1180 441.7 3120 - 6.4 - 0.1
Amberilte 1600 426.6 3014 - 0.3 0.0
Amberlite XAD-2000407.4 2879 3.5 0.0
Arnberlite XAD-201023.0 2988 0.3 0.0
4
Dowex XUS-40323 72.7 2633 8.6 0.1
3
Whatman DE-52 384.7 2718 5.0 0.1
Whatman CM-32 514.8 3637 - 10.8 - 0.1
CA 02318508 2000-07-OS
WO 99/34914 PCT/US98/14134
Table 8. (continued)
t, ~ i
~a~~ ~~-~~ .. 398.1-...--_. ____.__
Whatman SE-53 458.0 3236 -4.9 0.0
Pharmacia Q 377.6 2668 15.0 0.2
Seph FF
Pharmacia S 390.5 2759 10.8 0.1
Seph FF
-
Toyopearl QAE-550395.1 2792 8.8 0,1
C
-
Toyopearl Butyl372.9 2635 16.2 0.2
650-M
Toyopearl SP-SSOC404.2 2856 6.5 0.1
Toyopearl CM-650M394.0 2784 9.2 0.1
-
Toyopearl DEAF-650M392.3 2772 10.2 0.1
Toyopearl Super384.6 2717 12.8 0.1
Q 650C
EXAMPLE 12
Adsorption Capacities for Adsorbents. Adsorption isotherm experiments
were carried out to determine the adsorptive capacities (mole 5-[((3-
carboxyethyl)-amino]acridine/g adsorbent) for various types of adsorbents.
Figure 17 shows adsorption isotherms obtained for several Ambersorbs as
compared to the adsorption isotherm for Purolite MN-200. Adsorption studies
were performed in 25% plasma/75% Erythrosol solutions containing 0.2-3 mM 5-
[([i-carboxyethyl)amino]acridine and 0.6-10 mM GSH. Samples were agitated for
24 hours at room temperature.Calculations using the adsorption capacity from
Table 7 (22 p,mole/g) determined that approximately 4 g of Purolite MN-200
would be required to reduce the level of 5-[((3-carboxyethyl)-aminoJacridine
in a
300 mL unit of PRBC from 300 ~M to a final level of 1 ~,M. Less than 1 g of
Ambersorb 572 (130 ~,mole/g) would be required to achieve comparable removal.
A similar calculation estimated that less than 1 g of Ambersorb 572 would be
required to reduce the level of GSH in a 300 mL unit of PRBC from 6 mM to a
final level of SOO 1xM in the 150 mL of supernatant (50% HCT).
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EXAMPLE 13
Long Term Removal of Breakdown Products.
This experiment examined S-[([i-carboxyethyl)-amino)acridine and GSH
levels in PRBCs from which a fiberized PICA G-277 activated carbon device
(AQF, 7.3g, 500 m2/g) was removed after 24 hours of exposure. This study was
conducted in parallel with studies where the PRBCs had continued device
exposure. Figure 18 shows that the concentrations of S-[([3-carboxyethyl)-
amino]acridine and GSH in the supernatant samples were considerably higher in
the absence of a device over storage times of 1 to 4 weeks.
The concentration of 5-[((3-carboxyethyl)-amino]acridine was reduced to
5 pM in initially shaken PRBCs after 35 days of storage in the presence of a
1 S removal device (MN-200). 'This indicates that 5-[((3-carboxyethyl)-
amino]acridine removal does occur in static storage conditions at 4 °C.
EXAMPLE 14
Effect of Enclosure Material (membrane, woven, non-woven) on an IAD
The use of an enclosure material surrounding the adsorbent media was
investigated for the primary purpose of particle retention. The primary
purpose is
particle retention. However, membranes can enhance hemocompatibility of the
devices by preventing contact between the RBCs and membranes. Membranes
can easily be modified with hydrophilic polymers (PEO, PEG, HPL) to enhance
hemocompatibility without altering function. Approximately 10 g of fiberized
Pica 6277 activated carbon media (AQF 500 g/m2) was surrounded by a heat-
sealed membrane, woven polyester, or non-woven polyester material. PRBC
units (300 mL) were dosed with 300 pM of a degradable 5-[((3-
carboxyethyl)amino]acridine derivative and 3 mM GSH, held at room temperature
for 20 hours on a platelet shaker, and then transferred to IADs. Concentration
of
5-[((3-carboxyethyl)-amino]acridine was monitored over 24 hours. Figure 19
87
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WO 99/34914 PCT/US98/14134
shows 5-[([3-carboxyethyl)-amino]acridine levels in the supernatant of 300 mL
PRBC units exposed to IADs consisting of fiberized Pica 6277 activated carbon
(500 g/m2) and enclosed by a membrane, woven, or non-woven material. PRBCs
(Erythrosol, glucose, 62% HCT) were dosed with 300 ~M of a degradable 5-[([i-
carboxyethyl)amino]acridine derivative and 3 mM GSH, and agitated on a
platelet
shaker at room temperature prior to transfer to the IADs. PRBC-containing IADs
were agitated at room temperature for 24 hours.
These studies indicate that the Tetko woven enclosure shows the fastest
removal kinetics for 5-[((3-carboxyethyl)-amino]acridine over 24 hours. Final
levels achieved for all enclosure materials after 2 weeks were similar, with 5-
[([3-
carboxyethyl)-amino]acridine concentrations decreasing to approximately 2 pM
after 1 day, but rising back to 10 pM near day 8.
EXAMPLE 15
Effect of the Compound Adsorption Device on Red Blood Cell Function.
Indicators of red blood cell function were monitored over the course of 5-[([i-
carboxyethyl)-amino]acridine and GSH removal experiments for various device
configurations. Parameters measured included percentage lysis, ATP and K+
concentration. Table 9 shows that ATP concentrations were generally not
affected by the presence of a compound removal device: The decrease in ATP
concentrations was approximately the same for Control (no IAD) as for the MN-
200 or Pica 6277 devices. Levels of K+ in PRBCs were found to increase with
time. The temperature at which removal occurred did not influence K+ levels in
PRBC units exposed to compound removal devices over 20 days. Where the time
period of exposure was extended to 35 days, however, the rate of increase of
K+ in
PRBCs varied with the type of adsorbent, with final levels achieved of 40 and
45
mmol/Lfor MN-200 and PICA G-277 devices, respectively, compared to the no
device control at 39 mmol/L. The percentage of red blood cells lysed in device-
exposed and no-device control PRBC units has generally been found to be
between 0. l and 1 % after 24 hours. As shown in Table 10 and Table 11 and
88
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WO 99/34914 PCT/US98/14134
graphed in Figures 20 and 21, the % lysis varied significantly with the type
of
adsorbent used. Table 10 shows lysis values obtained for PRBCs exposed to two
types of immobilized adsorption devices over 35 days. Table 11 shows lysis
values obtained for PRBCs exposed to loose adsorbent particles enclosed in a
woven polyester mesh over 42 days. A wrist action shaker was used in dosing
all
PRBC units for 1 minute, after which the PRBCs were in a static condition for
4
hours at room temperature. The devices were held at room temperature for 24
hours on a platelet shaker, after which they were in a static condition at
4°C for
the duration of the study. The immobilization MN-200 showed lower hemolysis
levels than immobilized PICA G-277, while the Ambersorb synthetic
carbonaceous adsorbent showed one of the lowest hemolysis levels upon
comparison of the loose particle adsordents. The MN-200 IAD showed lower
hemolysis than the same non-immobilized adsorbent. Similar observations have
been observed for other IADs as compared to the same non-immobilized
adsorbent.
Table 9. ATP Concentration (~mol/dL) in PRBCs over time
Table 10. Percent Lysis in PRBCs (56% HCT) exposed to different IADs over
time
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4 hr 0.11 0.14 0.37
8 hr 0.12 0.18 0.53
24 hr 0.13 0.21 0.62
8 days 0.18 0.22 0.67
22 days 0.22 0.24 0.77
35 days 0.34 0.33 0.89
Table 11 Percent lysis in PRBCs (55% HCT) exposed to different loose particle
adsorbents enclosed in woven mesh.
.. _ ______ ~ _ _""",~"~"
0 hr 0.10 . 0.08 0.07 0.06 0.07
0.07
4 hr 0.07 0.09 0.38 0.10 0.08 0.13
8 hr 0.09 0.17 0.54 0.12 0.12 0.18
24 hr 0.1 I 0.39 1.06 0.16 0.13 0.92
Day 0.12 0.48 1.11 0.22 0.13 0.40
14
Day 0.15 0.59 1.24 0.29 0.17 0.57
21
Day 0.22 0.60 1.52 0.40 0.26 0.74
28
Day 0.30 0.78 1.85 0.55 0.41 1.05
35
Day 0.53 1.08 2.72 0.86 0.79 1.60
42
The critical nature of the adsorbent particle with respect to the
maintenance of red blood cell function is illustrated. A comparative study of
the
effects of five different adsorbents on red blood cell hemolysis is presented.
Ambersorb 572 produced only 0.16% lysis in PRBCs (55%) after a 24 hour
exposure, while Darco AC (Norit Americas, Inc. (Atlanta, GA) produced 0.13%
Iysis. Those adsorbents were significantly better at minimizing hemolysis of
red
blood cells than the PuroIite MN-200, Hemosorba CH-350 and Pica 6277
adsorbents.
Table 12 shows a comparative study where supernatants from red blood
cell samples containing glutathione and 5-[((3-carboxyethyl)amino)acridine
were
CA 02318508 2000-07-OS
WO 99/34914 PCT/US98/14134
contacted with a number of different adsorbents. Activated carbon adsorbents
were the only type of adsorbent that was capable of substantially reducing the
concentrations of both S-[((3-carboxyethyl)-amino)acridine and glutathione.
Both
natural activated carbons (Norit and PICA) and synthetic activated carbons
(Ambersorb) proved to be effective at compound reduction.
Table 12. Properties of Several Different Adsorbents
"~, ,, ; ;
I I
ypercrosslinked 0 7
N-200 urolite 0*
macroreticular .
PS-DVB, 200 -
1200 ~,m
particles
1100 m'/g
Hypercrosslinked
Optipore Dow Chemical 0 22*
L-493 0*
macroreticular .
PS-DVB, 300 -
840 pm
particles
1100 m'/g, 46
A avg.
pore diameter
Macroporous adsorbent
Duolite Rohm & Haas 1 10*
GT-73 7*
W,i~ viol functional.
groups
Norit A Norit AmericasSteam 1=gnite > 2790* 560+
Supra AC,
' 2000 m /g, 97%
< 150
Inc. pm particles
Picatiff PICA Powdered AC from> 2670* > 53*
Med.
coconuthusk,
2000
m=/g, 8-35 um
particles
Norit ROX Norit Steam-activate > 3p,10* > 60*
0.8 peat AC,
extruded
900 m'/g, 840-1000
ltm
cylinders
Synthetic AC
Ambersorb Rohm & Haasfrom > 2800* 134
572
sulfonated PS,
1100
m'/g, 300 - 840
pm
particles
Microporous (ca.
50%
pores < 20A)
Granular activated
G-277 PICA > 2~0* > 52*
c~~n from coconut
husk,
@ PS-DVB = polystyrene-divinyl benzene, AC = activated carbon
~Values listed as ">" were single measurements with residual levels below the
assay LOD
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WO 99/34914 PCTNS98/14134
* Estimated from single-point adsorption studies in 25% plasma, 75%
Erythrosoi.
+ Estimated from mufti-point adsorption isotherms in 25% plasma, 75%
Erythrosol
EXAMPLE 16
Fiberized media consisting of Dowex Optipore L-493 attached to a
nonwoven polyester fiber matrix (Hoechst-L493) has been manufactured by the
AQF division of Hoechst Celanese (Charlotte, NC). The performance of this
adsorbent media in a batch removal device for platelets was evaluated.
Platelet Preparation
Single donor apheresis platelet units containing 3.5 - 4.5 x 1 O1 ~ platelets
in
300 mL of 35% autologous plasma, 65% PAS III were obtained from the
Sacramento Blood Bank Center. 4'-(4-Amino-2-oxa)butyl-4,5',8-trimethyl
psoralen (Baxter Healthcare) was added to each platelet unit to achieve a
final
1 S concentration of 150 p,M. Platelet units (4-5) were pooled in a single PL-
2410
plastic container and thoroughly mixed. The platelet pool was evenly divided
into
4-5 1 L PL2410 plastic containers each containing approximately 300 mL of the
platelet mixture. Units were photochemically treated with 3.0 J/cm2 UV-A and
transferred into the appropriate removal device for the study. All experiments
included a control platelet unit which was photochemically treated ( 150 pM
psoralen, 3.0 J/cm2 UVA) but was not contacted with a removal device.
Device Preparation
Standard removal devices containing 2.5 g of Dowex XUS 43493 were
prepared by Baxter Healthcare Corporation (Lot FX1032 D96F20042R).
Experimental IADs were prepared with Hoechst-L493 media (Hoechst Celanese
Corp.) that was supplied as roll stock. Media was measured and cut to give the
appropriate adsorbent mass for each IAD (5.0 g, and 7.5 g). The cut media was
placed in pouches constructed by impulse welding 30 pm polyester mesh (Tetko).
Mesh pouches containing the media were autoclaved ( 121 °C, 20 min) and
placed
in sterile PL 2410 plastic containers. Alternatively, mesh pouches were placed
in
PL 2410 containers, the containers were sealed, and the entire assembly was
92
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WO 99/34914 PCT/US98/14134
sterilized by gamma-irradiation to 2.5 MRad (SteriGenics). Excess air was
manually evacuated from devices using a syringe prior to transfer of the
photochemically treated platelets.
Adsorption Kinetics
S Following photochemical treatment with 4'-(4-amino-2-oxa)butyl-4,5',8-
trimethyl psoralen + UVA, the platelet mixtures were transferred to PL2410
plastic containers with removal devices. The devices were placed on a standard
platelet incubator (Helmer) and agitated at 70 cycles/min at 22 °C.
Samples of the
platelet mixture were removed at 1 hour intervals for the first 8 hours of
storage.
These samples were stored at 4 °C and later analyzed for residual 4'-(4-
amino-2-
oxa)butyl-4,5',8-trimethyl psoralen by HPLC analysis. The assay involves an
initial sample preparation which lyses the platelets and solubilizes the 4'-(4-
amino-2-oxa)butyl-4,5',8-trimethyl psoralen and free photoproducts. The
supernatant from the sample preparation is analyzed on a C-18 reverse phase
column with a gradient of increasing methanol in KH2PO4 buffer.
In vitro Platelet Function
Platelet mixtures were agitated in contact with the removal devices for 7
days. In one study, the platelet mixture was contacted with the IAD for 24
hours
and transferred to sterile 1 L PL2410 plastic containers using a sterile
tubing
welder (Terumo SCD 312). Platelets were placed back in the platelet incubator
and stored for the remainder of the 7 day storage period.
Following 5 or 7 days of storage, the platelet count and pH were
determined for each platelet unit. The pH and p02/pC02 was measured using a
CIBA-Corning model 238 Blood Gas Analyzer. The platelet count of each
sample was determined using a Baker 9118+ Hematology analyzer.
Several assays were performed to evaluate in vitro platelet function. The
shape change of platelet samples was monitored using a Chrono-Log model 500
VS whole blood aggregometer. Shape change was quantified as the ratio of
maximum change in light transmission following addition of ADP relative to the
change in light transmission following addition of EDTA.
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The response of platelets to hypotonic stress was evaluated by the
Hypotonic Shock Response (HSR) assay. The change in light transmission
following addition of a hypotonic solution was measured using a Chrono-Log
model 500 VS whole blood aggregometer. Data is reported as percent recovery
from the hypotonic stress two minutes after absorbance reached its minimum
value.
The ability of platelets to aggregate in response to ADP/collagen agonist
was indicated by change in optical transmission as measured by a Chrono-Log
model 500 V S whole blood aggregometer.
The status of the platelets was evaluated by scoring the platelet. Samples
were blinded and morphology scores of 100 platelets were totaled for each
sample. The highest possible score is 400 (Disc = 4, intermediate = 3, sphere
= 2,
dendrite = 1, balloon = 0).
Platelet activation as indicated by expression of p-selectin (Granular
I S Membrane Protein, GMP-140) was measured. CD62 antibody was used for the
test sample, mouse control IgGl was used for the negative control, and CD62
antibody with phorbal myristate acetate (PMA) was used for the positive
control.
Samples were analyzed on a FACScan (Becton Dickinson). The percent of
activation that is reported is relative to the positive control and is the
difference
between the test value and negative control value.
Adsorption Kinetics
The impact of incorporating the adsorbent beads into a fiber matrix was
investigated. Platelet units containing 4.0 x 101 platelets in 300 mL of 35%
plasma, 65% PAS III were treated with 150 pM 4'-(4-amino-2-oxa)butyl-4,5',8-
trimethyl psoralen + 3.0 J/cm2 LJVA. IADs were prepared from Hoechst-L493
with an adsorbent loading of 450 g/m2. The IADs were sterilized by gamma
irradiation. Following treatment with psoralen + UVA, the platelets were
transferred to the removal devices. Samples were removed at 1 hour intervals
and
analyzed for residual 4'-(4-amino-2-oxa)butyl-4,5',8-trimethyl psoraien by
HPLC. Figure 22 compares the kinetics for removal of 4'-(4-amino-2-oxa)butyl-
4,5',8-trimethyl psoralen for IADs containing 5.0 g and 7.5 g Hoechst-L493
94
CA 02318508 2000-07-OS
WO 99/34914 PCT/US98/14134
media to that of a standard removal device containing 2.5 g of loose beads.
IADs
contained either 5.0 g Hoechst-L493 (triangles), 7.5 g of Hoechst-L493 media
{squares), or 2.5 g of loose Dowex L493 adsorbent beads (circles). The Hoechst-
L493 media contained adsorbent beads at a loading of 450 g/m2.
The kinetics of 4'-(4-amino-2-oxa)butyl-4,5',8-trimethyl psoralen
adsorption were slower for the Hoechst media when compared to an equal mass of
loose adsorbent beads. 'fhe removal device containing 2.5 g of loose beads
achieved the lowest levels of residual 4'-(4-amino-2-oxa)butyl-4,5',8-
trimethyl
psoralen at short times ( 1 hr). However, at longer times, the IADs containing
7.5
g and 5.0 g of Hoechst-L493 media performed better. In this study the
adsorption
kinetics were relatively rapid with all three removal devices achieving the
target
of < 0.5 pM residual 4'-(4-amino-2-oxa)butyl-4,5',8-trimethyl psoralen in
under 4
hours of contact.
Note that at long times {6-8 hr) the Hoechst-L493 IADs which contained a
higher mass of adsorbent achieved lower levels of residual 4'-(4-amino-2-
oxa)butyl-4,5',8-trimethyl psoralen. This observation suggests that the slower
adsorption kinetics for the Hoechst-L493 IADs is a result of mass transport
limitations (fluid flow vs. diffusion) and is not a result of loss of
functional
adsorption area due to fiber attachment. Additional studies indicate that
Hoechst-
L493 media with lower levels of adsorbent loading (200-300 g/m2) allows fluid
to
penetrate the media more readily resulting in faster kinetics of adsorption.
Previous studies (data not shown) with Hoechst media containing Amberlite
XAD-16 adsorbent at a loading of 160 g/m2 indicated that adsorption kinetics
were not affected by incorporation into a fiber matrix at a low level of
loading.
Platelet Yield and In Vitro Platelet Function
Data from two separate studies are presented in this section. The platelet
units within each study were derived from a single pool so that the effect of
the
IAD media format, adsorbent mass, and contact time could be clearly evaluated.
The first study evaluated the effect of fiberization (Hoechst media) on
yield and function of the platelets following extended contact (5 and 7 days).
A
total of four platelet units that were derived from a single pool were used in
this
CA 02318508 2000-07-OS
WO 99/34914 PCf/US98/14134
study. The no-IAD control unit was treated with psoralen + UVA. Two of the
platelet units were contacted with 5.0 g Hoechst-L493. One unit was contacted
with the IAD for 24 hours before being transferred to an empty PL 2410 storage
container. The other unit remained in contact with the IAD for the duration of
the
study. The Hoechst-L493 IADs were sterilized with steam ( 120 °C, 20
min). The
standard removal device (2.5 g loose Dowex XUS-43493), which was obtained
from Baxter, was sterilized by gamma-irradiation. Note that the platelets were
not
transferred away from the standard removal device following 8-16 hour contact
as
is typically the practice with the device that utilizes loose adsorbent
particles.
Results from platelet counts and in vitro function following 5 and 7 days of
storage are summarized in Table 13A and Table 13B respectively.
Table 13A. (Day 5)
Comparison of Hoechst-L493 Fiberized Media to Standard Removal Device
Platelet Yield and In Vitro Function following 5-Day Storage
Platelet
Count
Sample yield pH pC02 p02
(x10"/300
(%)
mL)
Control (+ PCT 3.50 + 100 6.91 18 120
- RD) 0.11
5.0 g Hoechst
/L-493
3.41 t 97 t 6.95 21 95
0.06 4
Transfer at 24
hr
5.0 g Hoechst/L-493
3.33 f 95 t 6.93 24 87
0.08 4
No Transfer
2.5 g Dowex Optipore
L-493 2.60 + 74 t 7.04 13 146
0.07 3
Baxter Lot FX
1032
D96F20042R
No Transfer
Shape
HSR AggregationMorphologyMP-140
Sam Ch
le
p ange
Control (+ 0.83 t 0.31 69t 4 273 67.2
PCT - 0.26 f 0.08
IAD)
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WO 99/34914 PCT/US98/14134
5.0 g HoechstO,gO t 0.43 83 t 0 268 61.5
/L-493 0.02 t 0.04
Transfer at
24 hr
5.0 g Hoechst/L-493
p,g3 t 0.40 80 t 2 280 58
0.12 t 0.01 9
No Transfer .
2.5 g Dowex
Optipore L-4930.24 t 0.38 47 t 3 240 71.3
0.04 f 0.02
Baxter Lot
FX1032
D96F20042R
No Transfer
97
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Table 13B. (Day 7)
Comparison of Hoechst-L493 Fiberized Media to Standard Removal Device
Platelet Yield and In Vitro Function following 7-Day Storage
~.
Platelet CountYield
Sam 1e H pC0 O
P P z P z
(x10~~/300 (%)
mL)
Control {+ 3.45 t 0.06 100 6.97 13 126
PCT -
IAD)
5.0 g Hoeehst
/L-
3.28 f 0.22 95 f 6.90 18 105
493 7
Transfer
at 24 hr
5.0 g Hoechst/L-
3.11 t 0.15 90 t 6.88 21 100
493 5
No Transferred
2.5 g Dowex
Optipore 2.29 t 0.07 66 t 7.04 9 161
L-493 2
Baxter Lot
FX1032
D96F20042R
No Transfer
Shape
HSR AggregationMorphologyGMP-140
Sam Ch
le
p ange
Control (+ 0.54 0.25 48 ~ 284 80.
PCT - t 0.05 t 0.03 0 I
IAD)
5.0 g Hoechst
/L-
0,76 0.64 86 t 267 72.7
493 t 0.02 f 0.16 11
Transfer
at 24 hr
5.0 g HoechstlL-
0,67 0.29 79 t 280 66.1
493 t 0.00 t 0.04 7
No Transfer
2.5 g Dowex
Optipore 0.31 0.28 29 t 261 77.1
L-493 f 0.09 t 0.06 11
Baxter Lot
FX 1032
D96F20042R
No Transfer
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The platelet yields for the Hoechst-L493 IADs (5.0 g) were substantially
better than the yield for the standard removal device (2.5 g). Losses of < 10%
were achieved for 7 days of storage in continuous contact with the Hoechst-
L493
IAD (5.0 g). Both platelet units that were treated with the Hoechst-L493 IADs
displayed better performance in the shape change, aggregation, and GMP-140
assays than the no-IAD control. The platelets that remained in contact with
the
Hoechst-L493 IAD (5.0 g) for the entire 5 days showed comparable in vitro
function to the platelets that were transferred after 24 hours of contact.
Interestingly, the platelets that remained in contact with the Hoechst-L493
IAD
performed better in the GMP-140 assay. The difference in performance between
the two was even larger after 7 days. This observation suggests that contact
with
the IAD decreases the rate of p-selectin expression by platelets during
storage.
The second study looked at IADs containing 5.0 g and 7.5 g of Hoechst-
L493 media to determine if there was a significant decrease in platelet yield
or in
vitro function with a higher mass of media. In this study, the Hoechst-L493
IADs
were sterilized by gamma irradiation. A standard removal device (2.5 g Dowex
XUS-43493) was included in the study. Platelets remained in contact with the
removal devices for the entire duration of the study. Results from platelet
counts
and in vitro function following 5 and 7 days of storage are summarized in
Table
14A and Table 14B respectively.
Table 14A. (Day 5)
Evaluation of Gamma Sterilized Hoechst-L493 Fiberized Media
Platelet Yield and In Vitro Function following 5-Day Storage (No Transfer)
Sam 1e Platelet Yield H pC0 O
p Count P z P z
(x101/300 (%)
mL)
Control (+ 3.96 f 100 6.89 25 88
PCT - 0.19
IAD)
5.0 g Hoechst3.681 0.1693 t 6.89 27 70
/L-493 6
7.5 g Hoechst/L-4933.44 t 87 t 6.87 26 84
0.11 S
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2.5 g Dowex
2,g5 f 72 t 7.04 13 145
Optipore L-4930.13 5
Baxter Lot
FX1032
D96F20042R
Shape
SR ggregationMorph- GMP-140
Sam Ch
le
p ange
ology
Control (+ 0.56 f .26t 73t 1 289 65.1
PCT - 0.12 0.04
IAD)
5.0 g Hoechst 0.45 t .40 76 t 278 54.5
/L-493 0.04 t 0.00 2
7.5 g Hoechst/L-4930.48 t .25 73 f 290 55.4
0.06 t 0.01 2
2.5 g Dowex
0~~ t .30 38 t 232 62
0.06 t 0.01 3 8
Optipore L-493 .
Baxter Lot
FX 1032
D96F20042R
Table 14B. (Day 'n
Evaluation of Gamma Sterilized Hoechst-L493 Fiberized Media
Platelet Yield and In Vitro Function following 7-Day Storage (No
Transfer)
Sam 1e Platelet Yield H pC0 O
p Count p z p z
(x10'/300 (%)
mL)
Control 3.88 t 100 6.99 18 93
(+ 0.18
PCT - IAD)
5.0 g Hoechst3.67f 0.0895 t 6.92 22 81
5
/L-493
7.5 g 3.48 t 90 t 6.91 20 91
0.07 4
Hoechst/L-493
2.5 g Dowex2.65 f 68 f 7 9 159
0.22 6 04
Optipore .
L-
493
Baxter
Lot
FX 1032
D96F20042R
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Shape
HSR AggregatioMorphology
le
Sam
p Change
n
Control 0.53 t 0.23f 65 t 260
0.12 0.02 5
5.0 g Hoechst 0.54 f 0.30 83 t 266
/L-493 0.00 f 0.01 8
7.5 g HoechstlL-4930.53 t 0.29 81 f 280
0.09 t 0.01 13
2.5 g Dowex
Optipore
p,23 t 0.25 33 t 228
L-493 0.04 t 0.03 9
Baxter Lot
FX 1032
D96F20042R
The results that are summarized in Tables 14A and 14B are similar to the
results that were observed in the first study. Platelets that were contacted
with the
Hoechst-L493 IAD had significantly higher yields than platelets that were
contacted with the standard removal device. Platelet yield was slightly lower
for
the 7.5 g Hoechst-L493 IAD relative to the S.0 g IAD. Platelets that were
treated
with the Hoechst-L493 IADs performed comparably to the control platelets in
all
in vitro assays. Platelets that were contacted with the Hoechst-L493 IADs
showed improved performance in the aggregation assay relative to the no-IAD
control on day 7. The GMP-140 assay was not performed on day 7.
IADs that were prepared with Hoechst-L493 media (5.0, 7.0 g) resulted in
superior in vitro function when compared to standard removal devices (2.5 g
loose
XUS-43493) stored in contact with the platelets for 5 days. Moreover,
platelets
that were treated with 150 ~M 4'-(4-amino-2-oxa)butyl-4,5',8-trimethyl
psoralen
+ 3.0 J/cm2 UVA showed improved in vitro function as indicated by shape
change, aggregation, and GMP-140 assays when contacted with Hoechst-L493
IADs (5.0 g) for 5 and 7 days. An additional study that compared 5-7 day
storage
for platelets (no psoralen/CJVA) with and without IAD (50.g Hoechst-L493)
demonstrated that storage with an IAD may improve platelet performance as
indicated by in vitro function assays.
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EXAMPLE 17
Comparison of AQF Fiberized Beads vs. Free Beads in PRBCs. This
study compared the removal of S-300 (N-(9-acridinyl)-(3-alanine) and
glutathione
from PRBC using AQF fiberized vs. free beads of Ambersorb 572.
PRBCs were prepared by centrifuging whole blood at 2100 rpm for 5
minutes and expressing off the plasma, then adding 84 mL of Erythrosol per
unit.
Six ABO matched units were pooled into a 3.0 L Clintec Viaflex bag.
Approximately 300 mL was transferred back to each original PL 146 bag and
dosed with 6.0 mL of 150 mM glutathione for a final concentration of 3.0 mM
and 3.0 mL of 30 mM S-300 derivative for a final concentration of 300 ~.M.
This
was mixed manually and allowed to incubate at room temperature for 4 hours.
The PRBCs were transferred to 1 liter PL 1813 bags containing one of two
adsorption devices consisting of either 4.8 g of Ambersorb beads in AQF
fiberized media (400 g/m2) or 4.8 g of loose Ambersorb beads. The fiberized
media or loose beads are enclosed in a Tekto woven mesh pouch. Duplicates
were run for each adsorption device and a unit in a PL 1813 bag without an
adsorption device was used as a control. These were stored on a platelet
shaker at
room temperature for 24 hours, then transferred to storage under static
conditions
at 4 °C. Units were sampled prior to dosing with the S-300 derivative
and
glutathione, after treatment but prior to transfer to the adsorption devices,
and at
2, 4, 6, 8, 20, and 24 hours and 1, 2 and 5 weeks after transfer to the
adsorption
devices. These samples were prepared for HPLC analysis of S-300 and
glutathione and results are shown in table 15. Samples were also analyzed for
percentage hemolysis, ATP concentration, K+ concentration, and pH (see table
16).
The results of this experiment suggest that the fiberized resin is preferred
over the free beads. The fiberized resin shows equivalent removal of S-300 and
glutathione with the benefit of improved red cell function as compared to the
free
beads. Table 15 shows that the removal of S-300 is similar for the two
compound
adsorption devices. The removal kinetics for glutathione appear to be slightly
better for the fiberized beads but is at acceptable levels for either device
after 24
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hours. Table 16 shows that for red cell functional assays the fiberized resin
treated samples are comparable to the control unit. Comparing the two devices,
the ATP and pH are essentially the same. The % hemolysis and K+ levels are
much higher after 24 hours for the free beads, indicating substantial loss or
red
blood cell function.
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Table 15. Removal of S-300 and glutathione from PRBC using Ambersorb 572 beads
in a
fiberized media vs. free beads. Residual concentration of compounds in PRBC (S-
300) or
supernatant (glutathione) after treatment.
Ambersorb Ambersorb
572 beads 572
in AQF free beads
ime fiberized ontrol
media Exp.l
Exp.l Exp.2
Exp.2
Residual0 33.5 33.7 31.1 32.1 32.9
S-300 2 hr 4.3 6.5 _ 5.5 6.9 42.6
~tM 4 hr 2.4 2.8 3.1 2.5 _
54.6
6 hr 1.9 1.9 2.2 2.0 _
66.8
8 hr 2.0 2.2 2.1 1.8 74.3
20 hr 2.4 2.5 2.4 2.1 I 13.8
24 hr 2.4 2.4 2.0 2.0 120.9
1 week 13.5 13.9 14.8 15.5 150.6
2 weeks 17.3 18.7 17.I 17.4 174.7
S weeks 16.7 19 12.5 15.4 171.2
Residual0 _ 6.55 6.59 6.40 6.50 6.40
gluts- 2 hr 0.59 0.87 1.03 1.50 6.35
thione 4 hr 0.11 0.16 0.54 0.67 6.46
mM b hr 0.04 0.22 0.21 0.53 6.60
8 hr 0.33 0.33 0.27 0.29 6.40
20 hr 0.31 0.09 0.28 0.44 6.60
24 hr 0.09 0.21 0.3 5 0.49 6.40
Table 16 % Hemolysis, K+, ATP, and pH comparison of PRBC treated with
Ambersorb
572 as beads in a fiberized media vs. free beads.
Ambersorb Ambersorb
572 beads 572
in AQF free beads
ime fiberized ontrol
media Exp.l
Exp.l Exp.2
Exp.2
0 0.13 0.14 0.13_ 0.11 0.15
~
Hemolysis2 hr 0.14 0.16 0.61 0.81 0.14
4 hr 0.19 0.13 0.98 1.73 0.11
6 hr 0.24 0.17 1.49 2.73 0.12
8 hr 0.29 0.19 2.18 3.37 0.12
20 hr 0.29 0.28 6.66 9.56 0.14
24 hr 0.37 0.35 9.57 14.67 0.20
1 week 0.47 0.36 9.39 13.79 0.14
2 weeks0.45 0.39 9.94 15.55 0.25
S weeks0.47 0.36 10.19 19.30 0.28
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Ambersorb Ambersorb
572 beads 572
in AQF free beads
ime fiberized ontrol
media Exp.l
Exp.l Exp.2
Exp.2
K+ 0 3.51 3.52 3.52 3.49 ~ 3.57
~
mmol/L 4 hr 3.50 3.44 4.59 5.52 3.89
8 hr 3.88 3.76 6.43 7.83 4.27
20 hr 4.94 4.88 12.68 16.14 5.54
24 hr 5.29 5.23 15.36 19.22 5.96
1 week 11.36 11.14 19.38 22.47 12.46
2 weeks 17.70 17.76 23.92 26.52 19.35
5 weeks 32.68 31.96 34.28 35.16 35.12
ATP 0 71.37 71.18 71.18 70.40 68.84
pmol/dL 1 week 63.38 62.40 60.45 58.11 69.81
2 weeks 50.90 51.48 50.27 47.00 56.55
5 weeks 24.77 23.79 23.01 24. 2
18 3.40
pI-I 0 6.74 6.74 6.74 _ _
~~ _ _
6.75 ~ 6.75
4 hr 6.82 6.81 6.81 ~~ 6.80 6.73
8 hr 6.80 6.80 6.81 6.80 6.71
20 hr 6.74 6.74 6.76 6.76 6.64
24 hr 6.73 6.72 6.75 6.76 6.62
2 weeks 6.53 6.52 6.57 6.59 6.44
~ 5 weeks 6.40 ~ 6.39 ~ 6.46 6.48 _
~ 6.26
EXAMPLE 18
Effect of Mode of Agitation on the Removal of S-300 and glutathione and on Red
Blood Cell Function using Fiberized Ambersorb 572. This study looked at the
effect of the
mode of agitation used during IAD treatment on removal of S-300 (N-(9-
acridinyl)-(3-
alanine) and glutathione and on percentage hematocrit, percentage hemolysis,
ATP
concentration, K+ concentration, and pH.
PRBCs were prepared as one pool and treated as per Example 17. After treatment
at
room temperature for 4 hours, six units were transferred to IADs consisting of
4.8 g of
Ambersorb 572 (AQF manufactured, 400 g/m2) enclosed in a Tekto woven mesh
pouch.
The IADs were contained in 1 liter PL 1813 bags. A control unit was
transferred to a PL
1813 bag without an IAD. These were then treated as follows:
105
SUBSTITUTE SHEET (RULE 26)
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Test Agitation Type 4C Storage
Article during first 24 hours at room Condition
temperature
1 latelet shaker 72 c cles/min) static
2 latelet shaker 72 c cles/min orbital shaker intermittent
3 orbital shaker 72 c cles/min static
4 latelet rotator 6 c cles/min static
S Nutator 3-D rotation, 24 c cles/minstatic
6 static no a itation static
7 controllatelet shaker (no media or enclosure)static
Units were sampled similarly to Example 17. The results indicate that some
method
of agitation is preferable for the removal of S-300 and glutathione. All modes
result in
equivalent removal while the orbital shaker shows lower levels of hemolysis
than the other
methods. Table 17 shows the S-300 and glutathione levels. Table 18 shows the
red cell
function.
Table 17 S-300 and glutathione levels in units treated with various agitiation
modes with
AQF fiberized Ambersorb 572.
Test 1 ~ 2 3 4 _ S 6 7
article
RT agitation PS PS OS PR N static Control
mode ~
4 C static OS static static static static static
condition
Residual0 34.7 33.8 34.3 34.2 34.7 34.8 33.9
S-300 2 hr 4.4 4.7 5.0 4.0 3.6 32.5 ~ 43.9
~.M 4 hr 2.9 2.5 2.2 2.7 2.1 35.9 57.4
6 hr 2.3 2.1 2.3 2.1 1.8 34.7 65.1
8 hr 2.5 2.0 2.0 2.0 1.7 33.6 74.4
20 hr 2.0 2.0 1.9 2.0 1.8 39.0 109.2
24 hr 2.9 2.5 2.6 2.6 2.5 35.5 114.6
1 week 13.3 8.0 15.0 15.0 16.9 26.2 141.0
2 weeks16.3 8.6 17.7 17.1 18.0 22.6 160.6
5 weeks13.7 4.0 14 12.9 14.4 14.4 166.8
Residual0 5.97 6.02 5.98 5.96 5.92 5.92 6.93
gluta- 2 hr 0.52 0.71 0.84 0.65 0.55 4.39 5.99
thione 4 hr 0.74 0.82 0.73 0.67 0.64 4.33 6.93
mM 6 hr 0.59 0.67 0.59 0.6 0.55 3.79 6.41
8 hr 0.55 0.62 0.61 0.58 0.55 3.38 6.56
20 hr 0.10 0.10 0.10 0.10 0.10 1.82 5.70
24 hr 0.10 0.10 0.10 0.10 0.10 1.1 5.03
TA 1 S
~ N - 1.7101G1G1 JrIC4AGr, v~ - urururr amcr, rtt -_ ~.71imc1cr rVtil~Ur, iv =
lVilIaiOr
106
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Table 18 % hemolysis, K+, ATP, and pH results with various agitation modes
using
fiberized AQF.Ambersorb 572.
Test 1 2 3 4 5 6 7
article
~
RT agitation PS PS OS PR N static Control
mode
4 C static OS static static static static static
condition
0 0.10 0.09 0.09 0.09 0.10 0.08 0.08
Hemo- 2 hr 0.14 0.13 0.11 0.19 0.28 0.08 0.08
lysis 4 hr 0.20 0.17 0.15 0.29 0.38 0.09 0.09
6 hr 0.28 0.20 0.16 0.42 0.47 0.09 0.10
8 hr 0.32 0.26 0.19 0.46 0.56 0.12 0.12
-
20 hr 0.63 4.43 0.27 0.97 0.99 0.10 0.13
-
24 hr 0.72 0.48 0.28 1.08 1.14 0.10 0.14
-
1 week 0.73 0.53 0.30 1.11 1.16 0.15 0.15
2 weeks0.76 0.53 0.33 1.18 1.26 0.20 0.19
-
r 5 1.18 0.75 0.40 1.18 1.34 0.34 0.24
weeks ~ j ~ ~
Test 1 2 3 4 5 6 7
article
-
RT agitation PS PS OS PR N static Control
mode
4 C static OS static static static static static
condition
K+ 0 3.64 3.65 3.62 3.62 3.64 3.64 3.63
mmol/L 4 hr 3.84 3.76 3.80 4.01 4.15 3.67 4.02
8 hr 4.30 4.14 4.10 4.51 4.58 3.91 4.47
20 hr 5.46 5.14 4.93 5.89 5.78 4.57 5.44
24 hr 5.92 5.59 5.20 6.30 6.14 4.83 5.85
1 week 12.88 12.80 12.38 13.12 12.88 11.76 13.12
2 weeks18.82 19.08 18.54 19.32 19.22 18.74 20.40
S weeks31.65 31.50 31.40 31.25 30.65 30.65 24.80
pH 0 7.35 7.32 7.35 7.35 7.34 7.35 7.35
8 hr 7.47 7.44 7.44 7.43 7.46 7.39 7.32
24 hr 7.41 7.41 7.45 7.44 7.40 7.39 7.29
1 week 7.33 7.22 7.27 7.24 7.25 7.24 7.08
ATP 0 75.08 76.83 77.22 75.86 75.86 77.22 76.25
~mol/dL1 week 62.40 62.01 65.72 63.18 62.60 66.11 _
66.50
2 weeks50.31 48.36 51.29 50.12 49.14 53.43 51.29
5 weeks23.40 22.43 23.99 23.79 24.18 26.13 23.99
EXAMPLE 19
Removal ofActivated Complement by ftberized Ambersorb 572 treated PRBCs.
This study looked at the formation of complement fragments C3a and SCSb-9 in
PRBCs
107
SUBSTITUTE SHEET (RULE 26)
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after treatment with an S-300 derivative and glutathione followed by removal
using AQF
fiberized media.
PRBCs were prepared as one pool and treated as per Example 17. After treatment
at
room temperature for 4 hours, units were transferred to 1 liter PL 1813 bags
containing the
following:
Test Article Description
(PRBC unit _
#) ~
1 no IAD
2 no IAD-ice incubated
3 4.8 g Ambersorb IAD (400 g/m2) (no enclosure)
4 7 sheets cellulose acetate membrane (47 mm dia.)
The cellulose acetate membrane is known to cause complement activation and is
used as a positive control. All but unit 2 were treated for 24 hours at room
temperature
prior to storage under static conditions at 4 °C. Unit 2 was stored at
4 °C continuously.
Three 1.5 mL samples were taken from each test article prior to IAD treatment,
during treatment after 4, 8, and 24 hours, and 5 days. Each sample was
centrifuged at 2000
x g for 15 minutes and 450 pL of each supernatant was mixed with 50 p,L of
cold 200 mM
EDTA and vortexed. These were frozen rapidly on dry ice and stored at -70
°C.
Enzyme immunoassays (Quidel) were used to detect the formation of complement
fragments C3a and SCSb-9. Presence of these fragments are an indication of
activation of
the complement system. The assay involves binding of the target fragment by a
mouse
antibody which is conjugated to Horse Radish Peroxidase (HRP) and detection
using a
chromogenic substrate of the HRP. Sample absorbance was measured against a
standard
curve to calculate the fragment concentration in the sample. Samples were also
assessed
for S-300, glutathione and hemolysis similarly to Example 17.
The results indicate that complement activation is reduced in samples treated
with
the Ambersorb 572 beads in AQF media relative to controls. Table 19 shows that
for the
controls, complement activation is lower in the sample stored continuously at
4 °C. The
sample treated with the IAD showed lower levels of C3a and SCSb-9 than the
control at 5
days, with C3a near the detection limit after 24 hours. Table 20 indicates
that S-300 and
glutathione removal was as expected with the AQF media.
108
SUBSTITUTE SHEET (RULE 21~)
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Table 19 Complement fragment C3a and SCSb-9 levels with various treatments.
Test 1 2 3 4
article
Treatment no IAD no IAD AQF cellulose
4C no acetate
pouch
C3a 0 411 354 391 388
ng/mL 24 hr 609 383 -1 701
5 days 665 404 26 N/A
SCSb-9 0 38 37 18 _ 25
ng/mL 24 hr 120 52 22 87
5 days 226 N/A 34 N/A
Table 20 S-300 concentration (PRBC), glutathione concentration (supernatant),
and
hemolysis with various treatments.
Test 1 2 3 4
article
Treatment no IAD no IAD AQF cellulose
4C no acetate
pouch
Residual0 30.55 30.18 29.38 29.6
~
S-300 24 hr 121.39 61.93 2.51 118.39
pM 5 days 142.36 136.33 12.35 ~ 153.58
gluts- 5 days 7.1 7.26 0.72 7.07
thione S
mM
0 0.11 0.11 0.10 0.10
Hemo- 24 hr 0.42 0.11 0.13 0.61
lysis 5 days 0.44 0.17 0.14 0.64
EXAMPLE 20
Hemocompatibility enhancement of adsorbent by an inert particulate matrix.
This
example demonstrates that immobilization of adsorbent particles in an inert
particulate
matrix enhances the hemocompatibility of the adsorbent without substantially
impacting
removal of low molecular weight compounds. In addition, this example supports
the
contention that immobilizing adsorbent particles in an inert matrix (fiber or
particulate) is a
general method for enhancing the hemocompatibility of the adsorbent. Results
for IADs
comprised of adsorbent particles immobilized in a fiber matrix and a
particulate matrix are
presented below.
The media that was studied in this example is comprised of Purolite MN-200
adsorbent particles (200-1200 pm) immobilized in ultra high molecular weight
109
SUBSTITUTE SHEET (RULE 26)
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polyethylene (UHMWPE) particulate matrix. Representative media is manufactured
by
Porex (Fairburn, GA). Disks of immobilized adsorbent media were formed by
mixing
approximately 50% (w/w) Purolite MN-200 (200-1200 p,m) with 50% (w/w) UHMWPE
particles having a similar particle size. The mixture was placed in a
cylindrical cavity and
heated under pressure at conditions sufficient to cause the UHMWPE particles
to fuse and
entrap the adsorbent particles. The resulting disks had a diameter of 3.50 in.
and were
approximately 0.250 in. thick. The disks weighed approximately 24 g
corresponding to
approximately 12 g MN-200 in each disk. The IAD was prepared by placing the
disk of
media in a plastic storage container (PL2410, Baxter Healthcare Corp.) and the
entire
assembly was sterilized by irradiating with 25-40 kGy of gamma irradiation
(Sterigenics,
Hayward, CA).
The fiber matrix IAD was comprised of Purolite MN-200 immobilized in a non-
woven polyester matrix at loading of 300 g adsorbent/m~. Approximately 2.5 g
(adsorbent
mass) of immobilized Purolite MN-200 was placed in a pouch constructed from 30
~,m
woven polyester material (Tetko, DePew, NY). The pouch assembly was placed in
a
plastic storage container (PL2410, Baxter Healthcare Corp.) and the entire
assembly was
sterilized by irradiating with 25-40 kGy of gamma irradiation (Sterigenics,
Hayward, CA).
Units of ABO-matched platelet concentrates comprised of platelets (3-5 x 101'
cells)
suspended in approximately 300 mL of 35% autologous plasma, 65% platelet
additive
solution were obtained from Sacramento Blood Bank (Sacramento, CA). The
platelet units
were pooled and divided before dosing each unit with 3 mL of 15 mM aminated
psoralen
(4'-(4-amino-2-oxa)butyl-4,5',8-trimethyl psoraleri). Each unit was subjected
to
photochemical treatment by illuminating with 3.0 J/cm2 of UVA in a UVA
illumination
device (Baxter Healthcare Corp.). Two photochemically treated platelet units
were
transferred to the PL2410 plastic storage containers with the two test SRDs.
One treated
unit was kept as a control and was not contacted with an IAD. All units were
placed on a
platelet shaker (Helmer, Noblesville, IN) at room temperature (22 °C)
for the duration of
the experiment.
The kinetics of psoralen adsorption by each of the IAD embodiments was
determined. Samples of each platelet unit (ca. 1 mL) were removed at 2 hour
intervals
during the first 8 hours of storage. These samples were later analyzed for
levels of residual
psoralen by High Pressure Liquid Chromatography. Following 5 days of storage
at room
110
SUBSTITUTE SHEET (RULE 26~
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temperature, the platelet counts, pH, and dissolved gases for each of the
units was
measured. Platelet function was also assessed by performing in vitro tests
which included:
shape change, aggregation, hypotonic shock response, GMP-140 (p-selectin
expression),
and morpohology score.
The kinetics for psoralen adsorption are shown in Figure 23. Both IADs removed
the psoralen to the limit of quantitation for the HPLC assay during the eight
hour incubation
period. The particulate IAD had much faster adsorption kinetics due to the
higher mass of
adsorbent (about 12 g) relative to the fiber IAD (2.5 g).
The platelet counts, pH measurements, and in vitro platelet function assay
results
are summarized in Table 21. The fiber and particulate matrix IADs both gave
greater than
90% recovery of platelets. This observation is particularly impressive for the
particulate
IAD considering that it contains about 12 g of MN-200. Note that a removal
device that
contains 2.5 g of non-immobilized adsorbent particles will typically result in
a loss of 25-
35% platelets by day 5. A device containing 12 g of non-immobilized particles
would
therefore by expected to remove > 50% of the platelets. It is obvious that
immobilizing the
adsorbent in the particulate matrix has drasticall-y reduced platelet loss
while the kinetics of
psoralen removal are still very rapid.
Results from the in vitro platelet function studies are summarized in the
second half
of Table 21. Once again, both IADs demonstrated satisfactory performance.
Hypotonic
shock response was slightly higher for the control due to a single high
measurement as
indicated by the large standard deviation. The IADs did perform better in the
aggregation
assay with all other assays demostrating essentially identical performance.
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Table 21. Comparison of Day 5 Platelet Yield and In Vitro Platelet Function
for Fiber
Matrix and Particulate Matrix IADs
Sample Platelet % Yield pH p pp2
p
(x10"/300
mL)
Control 2.89 t 0.06 100 6.95 22 116
AQF Fiber Matrix 2.71 t 0.11 94 t 6.96 23 112
IAD 4
(300 g/mz MN-200)
Porex Particulate 2.61 0.10 90 t 6.90 22 123
Matrix 4
IAD (50% MN-200)
Sample Shape Change HSR AggregationMorphologyGMP-140
Control 1.03 0.33 0.71 44 3 308 67.0
0.28
AQF Fiber0.96 t 0.14 0.53 60 t 0 311 63.9
Matrix 0.06
IAD
(300 g/m~
MN-200)
Porex 1.00 t 0.05 0.42 64 t 1 304 66.2
Particulate 0.01
Matrix
IAD
(50% MN-
200)
The particulate matrix IAD may be further optimized from the present
configuration by
changing the geometry of the disk to allow more complete penetration of the
media disk
with liquid. A thinner disk would probably result in equivalent removal
kinetics with a
lower mass of adsorbent.
In addition to optimizing the geometry of the disk, the wetting of the device
and
therefore the adsorption kinetics could be further enhanced by increasing the
wetting of the
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media. The inherent hydrophobic nature of the UHMWPE matrix make the device
wet
slowly during the initial phase of removal. Strategies that could be used to
enhance wetting
include the use of wetting agents (e.g., glycerol, polyethylene oxide,
polyethylene glycol,
hydrophilic polymers) or treatment with gas plasma glow discharge. Unlike
wetting agents,
treatment with glow discharge can be used to directly alter the chemistry of
the surface of
the UHMWPE binder matrix.
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